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https://openalex.org/W4384025192	https://www.frontiersin.org/articles/10.3389/fevo.2023.1171358/pdf	English	null	Extraction and monitoring of vegetation coverage based on uncrewed aerial vehicle visible image in a post gold mining area	Frontiers in ecology and evolution	2,023	cc-by	7,948	OPEN ACCESS OPEN ACCESS EDITED BY Jie Lou, Zhejiang Lab, China REVIEWED BY Jinman Wang, China University of Geosciences, China Vishal Mishra, Indian Institute of Technology Roorkee, India CORRESPONDENCE Lei Han hanshuanglei@chd.edu.cn RECEIVED 22 February 2023 ACCEPTED 20 June 2023 PUBLISHED 12 July 2023 CITATION Chen R, Han L, Zhao Y, Zhao Z, Liu Z, Li R, Xia L and Zhai Y (2023) Extraction and monitoring of vegetation coverage based on uncrewed aerial vehicle visible image in a post gold mining area. Front. Ecol. Evol. 11:1171358. doi: 10.3389/fevo.2023.1171358 OPEN ACCESS EDITED BY Jie Lou, Zhejiang Lab, China REVIEWED BY Jinman Wang, China University of Geosciences, China Vishal Mishra, Indian Institute of Technology Roorkee, India CORRESPONDENCE Lei Han hanshuanglei@chd.edu.cn RECEIVED 22 February 2023 ACCEPTED 20 June 2023 PUBLISHED 12 July 2023 CITATION Chen R, Han L, Zhao Y, Zhao Z, Liu Z, Li R, Xia L and Zhai Y (2023) Extraction and monitoring of vegetation coverage based on uncrewed aerial vehicle visible image in a post gold mining area. Front. Ecol. Evol. 11:1171358. doi: 10.3389/fevo.2023.1171358 Rui Chen 1, Lei Han 2,3*, Yonghua Zhao 2,3, Zilin Zhao 1, Zhao Liu 2,3, Risheng Li 4, Longfei Xia 4 and Yunmeng Zhai 2,3 1School of Earth Science and Resources, Chang’an University, Xi’an, China, 2School of Land Engineering, Shaanxi Key Laboratory of Land Consolidation, Chang’an University, Xi’an, China, 3Key Laboratory of Degraded and Unused Land Consolidation Engineering, Ministry of Natural Resources of the People’s Republic of China, Chang’an University, Xi’an, China, 4Institute of Land Engineering and Technology, Shaanxi Provincial Land Engineering Construction Group, Xi’an, China Vegetation coverage reﬂects the degree of environmental degradation. Timely and effective monitoring of vegetation conditions is the basis for promoting vegetation protection and improving the ecological environment of mining areas. Exploring vegetation coverage extraction methods and selecting the optimal vegetation index in mining areas can provide scientiﬁc reference for estimating vegetation coverage based on vegetation index in mining areas. Uncrewed aerial vehicles (UAVs) are widely used because of their fast real-time performance, high spatial resolution, and easy accessibility. In this study, the performances of nine visible vegetation indices and two threshold segmentation methods for extracting vegetation coverage in a post-gold mining area in the Qinling Mountains were comprehensively compared using visible spectrum UAV images. Of the nine indices, the excess green index (EXG) and visible-band difference vegetation index (VDVI) were the most effective in discriminating between vegetation and non-vegetation by visual interpretation. remote sensing, uncrewed aerial vehicle, vegetation coverage, eco-monitoring, post- mining area TYPE Original Research PUBLISHED 12 July 2023 DOI 10.3389/fevo.2023.1171358 © 2023 Chen, Han, Zhao, Zhao, Liu, Li, Xia and Zhai. 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. Frontiers in Ecology and Evolution 1 Introduction index (Zhou et al., 2021). An increasing number of studies have shown that vegetation coverage can be extracted using a vegetation index derived from UAV-visible images. Therefore, the limited wavelength information of UAV visible-light images must be used to construct a universally applicable and highly accurate vegetation index and effectively extract green vegetation information. Another key aspect of vegetation coverage extraction by vegetation indices is the determination of a suitable threshold, which can be based on threshold segmentation methods (Akash et al., 2019). However, few studies have used this method to determine the thresholds in mining areas, and the effectiveness of vegetation indices in mountainous mining areas has not yet been evaluated. Vegetation restoration and reconstruction are key components of ecological restoration in mining areas and are effective ways to improve the quality of the local ecological environment (Li et al., 2019). Therefore, obtaining vegetation information quickly and accurately in mining areas to evaluate the status of local ecological restoration is an urgent issue. As an important indicator of vegetation status, fractional vegetation coverage (FVC) is deﬁned as the percentage ratio of the vertical projection area of vegetation (including leaves, stems, and branches) on the ground in a statistical area (Jia et al., 2015). FVC is not only the main indicator of regional environmental status and quality assessment but also an important part of terrestrial ecosystem research. Thus, accurate and rapid extraction of vegetation coverage requires timely monitoring of vegetation change, which is crucial for protecting biodiversity and promoting economic development. The Qinling Mountains are an important ecological security barrier in China and provide many ecosystem services, such as climate regulation, water yield, carbon sequestration, and biodiversity preservation (Fu et al., 2022). Rich gold mineral resources in the Qinling Mountains provide a good foundation for mining activities; however, long-term mining has resulted in serious vegetation destruction (Li et al., 2022), which has plagued sustainable local development (Huo et al., 2022). Therefore, a rapid and accurate method for acquisition of mine vegetation cover is required. Currently, research on vegetation coverage extraction based on visible vegetation index focuses mostly on cities, forests, grasslands, and farmlands with well-growing plants (Geng et al., 2022). However, an optimal vegetation index for extracting vegetation coverage suitable for Qinling gold mining areas with sparse vegetation and complex terrain has not yet been determined. 1 Introduction Furthermore, previous studies focused on extraction methods for the current vegetation situation and lacked long-term monitoring. Therefore, an abandoned gold mining area in the Qinling Mountains was selected as the research area, and high spatial resolution visible spectrum images obtained by a UAV were used as the data source. The objectives of this study were to (1) compare the performances of nine visible light vegetation indices (RGRI, BGRI, EXG, EXGR, NGRDI, NGBDI, RGBVI, VDVI, and VEG) and two threshold segmentation methods (bimodal histogram method and Otsu’s threshold method) in the extraction of vegetation coverage information; (2) select the optimal combination of the vegetation index and threshold segmentation method with high extraction accuracy and wide applicability; and (3) analyze the interannual variation of FVC in the study area using results obtained by the optimal combination. This study provides scientiﬁc guidance for rapidly and accurately extracting vegetation coverage and offers technical support for evaluating vegetation restoration in mining areas. Currently, remote sensing observations and land surface measurements are primarily used to monitor FVC (Lu et al., 2020). As a low-cost and highly efﬁcient monitoring technology, remote sensing can provide objective and accurate environmental monitoring for large-scale mining areas. With the rapid development of satellite remote sensing technologies, many vegetation products, such as those derived from NOAA/AVHRR (Boyd et al., 2002), TM/Landsat (Voorde et al., 2008; Leng et al., 2019), and Terra & Aqua/MODIS (Song et al., 2017), have facilitated large-scale monitoring of vegetation coverage. However, for small-scale areas, such as mining areas with complex topography and heterogeneous habitats, monitoring FVC using satellite remote sensing technologies is challenging because of their relatively coarse spatial resolution and long revisit period. Furthermore, although in situ measurements have high accuracy, they are usually time consuming and labor intensive, rendering them unsuitable for real-time and long-term monitoring. Notably, uncrewed aerial vehicles (UAVs) have the advantages of strong real-time performance, high spatial resolution, and easy access; thus, they have attracted wide attention as a novel and improved method to extract vegetation coverage with high efﬁciency and precision on small spatial scales in agriculture, forestry, surveying, mapping, and other related ﬁelds (Watanabe et al., 2017; Schoﬁeld et al., 2019; Ana et al., 2021; Guo et al., 2021; Park et al., 2022; Mishra et al., 2023). OPEN ACCESS In addition, the accuracy of the bimodal histogram threshold method in extracting vegetation coverage was higher than that of Otsu’s threshold method. The bimodal histogram threshold method combined with EXG yielded optimal extraction results. Based on optimal methods, the total percentages of fractional vegetation coverage in 2019, 2020, and 2021 were 31.47%, 34.08%, and 42.77%, respectively, indicating that the vegetation in the mining area improved. These results provide valuable guidance for extracting vegetation information and evaluating vegetation restoration in mining areas. COPYRIGHT © 2023 Chen, Han, Zhao, Zhao, Liu, Li, Xia and Zhai. 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. 01 Frontiers in Ecology and Evolution frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 10.3389/fevo.2023.1171358 1 Introduction Compared with multispectral, hyperspectral, and other sensors, visible light sensors are better options for extracting vegetation coverage via UAV technology owing to their outstanding advantages, such as low cost and are less affected by weather and light (Coy et al., 2016; Jay et al., 2019; Ren et al., 2021). The vegetation index can effectively reﬂect vegetation vitality and information and is a commonly used method for extracting vegetation coverage (Woebbecke et al., 1995; Hague et al., 2006; Rasmussen et al., 2016; Kim et al., 2018; Geng et al., 2022). Various vegetation indices have been developed based on the spectral characteristics of green vegetation in the visible light band, such as the green leaf index (Shane et al., 2021), green-red vegetation index (Zhang et al., 2019), and difference-enhanced vegetation Frontiers in Ecology and Evolution 2.2 UAV image acquisition and processing Field and UAV aerial surveys were conducted in August 2019, 2020, and 2021 to monitor the vegetation coverage at the research site in the post-mining area. The UAV ﬂight test was conducted using a DJI Phantom 4 Pro on clear and cloudless days, and RGB- visible images were acquired. The ﬂight parameters are listed in Table 1. The automatic cruise mode was used for route planning during the ﬂight. The ﬂight area and route were designed prior to conducting the experiment. The ﬂight was 0.68 km2. Orthoimages of the study area are shown in Figure 1B. 2.1 Study area The study area is located in the southeastern part of Shangluo City, Shaanxi Province, China (Figure 1). It is between 108°34′20′′– 111°1′25′′ E and 33°2′30′′–34°24′40′′ N. The study area is located in the Qinling Mountains and has a warm, temperate climate. The mean annual temperature is 12.2°C, the mean annual precipitation is 804.8 mm, and the mean annual sunshine duration is 1947.4 h. 02 frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 B A FIGURE 1 (A) Geographical location and (B) UAV image with a spatial resolution of 0.0436 m of the study area. B B FIGURE 1 (A) Geographical location and (B) UAV image with a spatial resolution of 0.0436 m of the study area. 2.3 Calculation of visible light vegetation index The soil type is yellow cinnamon. It is high in the northwest and low in the southeast. A gold production company in the research area began operations in 1999 and ceased production after a dam failure in 2006. Even after several years, bare slag still poses a serious threat to human health, and this research area has been listed as a key area for heavy metal prevention and control (Chen et al., 2022). The basic principle behind the construction of a vegetation index is that vegetation absorbs and reﬂects light of different wavelengths. The corresponding vegetation index can be obtained by combining different bands of remote sensing images to enhance vegetation (Guilherme et al., 2018). The visible vegetation index is mainly constructed based on the red, green, and blue bands of the image because healthy green vegetation has a strong reﬂection in the green band and weak reﬂections in the red and blue bands. The nine commonly used visible light vegetation indices are listed in Table 2. 2.4 Vegetation information extraction based on threshold The vegetation index threshold method is effective for discriminating between vegetation and non-vegetation Frontiers in Ecology and Evolution TABLE 1 Flight setting of the UAV and image parameters. TABLE 1 Flight setting of the UAV and image parameters. TABLE 1 Flight setting of the UAV and image parameters. Flight setting Parameter Acquired image content Parameter Flight speed 14.1 m/s Number of original images 300+ Photo interval 2 s Picture resolution 72 dpi Number of routes 13 Graphic form JPEG Number of waypoints 26 Shutter speed 1/1600 Course overlap rate 80% ISO 800 Side overlap rate 60% Flight altitude 140 m 03 03 Frontiers in Ecology and Evolution frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 TABLE 2 Nine commonly used visible light vegetation indices considered in this study and the calculation formulas based on the visible spectrum. TABLE 2 Nine commonly used visible light vegetation indices considered in this study and the calculation formulas based on the visible spectrum. only used visible light vegetation indices considered in this study and the calculation formulas based on the visible spectrum. TABLE 2 Nine commonly used visible light vegetation indices considered in this study and the calculation formulas based on the Visible vegetation index Full name Calculation formula Reference RGRI Red–green ratio index R/G (Verrelst et al., 2008) BGRI Blue–green ratio index B/G (Romina et al., 2010) EXG Excess green index 2g−r−b (Kim et al., 2018) EXGR Excess green minus red index EXG−1.4r−g (Sun et al., 2014) NGRDI Normalized green–red difference index (G−R)/(G+R) (Gitelson et al., 2002) NGBDI Normalized green–blue difference index (G−B)/(G+B) (Hunt et al., 2005) RGBVI Red–green–blue vegetation index (G2−B×R)/(G2+B×R) (Juliane et al., 2015) VDVI Visible-band difference vegetation index (2G−R−B)/(2G+R+B) (Wang et al., 2015) VEG Vegetative index g/r0.67b0.33 (Geng et al., 2022) background and target and the difference between the two parts of the image are the largest; that is, the optimal threshold is determined based on the maximum between-cluster variance. The calculation process of Otsu’s method is as follows. (1) Identify the highest gray level in the image. (2) Take each gray level as a threshold. (3) Calculate the number of pixels and the average value of the two categories segmented by the threshold. (4) Calculate the variance between the two clusters. (5) Determine the threshold of the maximum variance (Xu et al., 2022). information in an image. Three steps are required to extract the vegetation coverage using the visible light vegetation index. The ﬁrst step is to calculate the vegetation index, the second is to set an appropriate threshold, and the ﬁnal is to separate the vegetation and non-vegetation parts. 2.5 Extraction accuracy evaluation A bimodal histogram is an image with two obvious peaks in a gray histogram (Zhou et al., 2021). These two wave peaks correspond to the internal and external target points. The wave trough between the two wave peaks corresponded to the target point near the edge of the object. Typically, the value at the wave trough is selected as the threshold. The calculation process of the bimodal histogram used in this study is as follows. (1) Calculate the average gray value (avg) and standard deviation of the pixels. (2) Considering the average pixel value as the dividing point, ﬁnd the positions of the maximum values of the left (small peak) and right (large peak) parts. (3) If the two peak positions are close (within the standard deviation range), then one of the two peaks of the histogram is very low; hence, another low peak position must be found; otherwise, proceed to step (7). (4) Determine the position of the pixel gray median point (midpoint). (5) If midpoints>avg, then the small peak is on the left side of the large peak (lower gray level); otherwise, the small peak is on the right side of the large peak (higher gray level), and the position of the dividing point should be adjusted accordingly. (6) Re-ﬁnd the positions of the large and small peaks. (7) The wave trough of the two peak positions is considered the required threshold (Liang, 2002). Accuracy, Precision, and Recall were calculated as follows to evaluate the classiﬁcation accuracy (Shukla and Jain, 2020): Accuracy = TP + TN TP + TN + FP + FN = TP + TN P + N Presicion = TP TP + FP Recall = TP TP + FN where TP, which stands for “true positive,” is the object that is correctly classiﬁed as vegetation among all the extracted objects; TN, which stands for “true negative,” is the object that is correctly classiﬁed as non- vegetation among all the extracted objects; FP, which stands for “false positive,” is the object that is misclassiﬁed as vegetation among all extracted objects; and FN, which stands for “false negative,” is the object that is misclassiﬁed as non-vegetation among all the extracted objects. TABLE 1 Flight setting of the UAV and image parameters. The accuracy of vegetation coverage extraction largely depends on threshold selection (Wang et al., 2015). Two commonly used methods, the bimodal histogram threshold method and Otsu’s threshold method, were applied to determine the threshold for each vegetation index. Frontiers in Ecology and Evolution 3.1.1 Visual interpretation and supervision classiﬁcation The performance of the visible vegetation indices in extracting vegetation was evaluated by comparison with the results of the maximum likelihood classiﬁcation method (Figure 2). Fifty regions of interest (ROIs) with non-vegetation and ﬁfty ROIs with vegetation were uniformly selected to verify the classiﬁcation accuracy. The overall accuracies of the typical and validation quadrats were 99.99% and 99.39%, respectively. 3 Results 3 Results non-vegetation in the gray image of each band and vegetation index, 75 representative ROIs were randomly selected to count the pixel eigenvalues of each visible band and vegetation index (Table 3). The results indicated that the reﬂectance in the green band of the vegetation was signiﬁcantly higher than that of the non- vegetation. In the BGRI, RGRI, and NGRDI gray images, the pixel values of vegetation and non-vegetation overlapped over a large range; therefore, vegetation and non-vegetation areas overlapped. In addition, the calculation formulas for RGRI, NGBDI, and NGRDI only used blue + green or red + green bands, indicating that the red, green, and blue bands should be combined when calculating the visible light vegetation index. 2.4.2 Otsu’s threshold method The UAV images were converted into orthoimages using DJI Terra v.3.3 software developed by DJI (Shenzhen, China). Supervised classiﬁcation, calculation of vegetation indices, threshold segmentation, and extraction of vegetation coverage were performed using ENVI 5.3 software. Otsu’s threshold method, also known as the maximum between-cluster variance method, is a global threshold selection method (Otsu, 2007). This method divides an image into background and target images based on a threshold. When the optimal threshold is considered, the variance between the 04 frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 10.3389/fevo.2023.1171358 3.1.2 Vegetation index calculation results The vegetation indices derived from the gray image of a typical quadrat are shown in Figure 3. Most visible light vegetation indices can be used to effectively distinguish vegetation from non- vegetation information; however, the extraction effects are different. Some vegetation indices, such as EXG, EXGR, RGBVI, VEG, and VDVI, can clearly discriminate between vegetation and non-vegetation areas; however, BGRI, RGRI, NGBDI, and NGRDI cannot clearly distinguish between the two and resulted in some misclassiﬁcations, indicating poor extraction performance. Furthermore, to analyze the pixel value ranges of vegetation and The bimodal histogram threshold and Otsu’s threshold methods were employed to determine the threshold of each visible light vegetation index grey image. The vegetation and non- vegetation areas were discriminated based on the thresholds, and the extraction accuracy was veriﬁed by comparison with the supervised classiﬁcation results. The threshold segmentation results are shown in Figures 4, 5. By jointly viewing the orthoimages and supervised classiﬁcation results (Figure 2A1, A2), we found that the extraction results of the bimodal histogram threshold method had fewer misclassiﬁcations, and the B2 A1 B1 A2 FIGURE 2 Original images of (A1) typical and (B1) validation quadrats and classiﬁcation results of the (A2) typical and (B2) veriﬁcation quadrats. B1 A1 B1 A2 Frontiers in Ecology and Evolution 3.3 Vegetation coverage assessment According to the above results, EXG combined with the bimodal histogram threshold method was used to estimate vegetation coverage in 2019, 2020, and 2021 (the thresholds were 0.07848, 0.122353, and 0.125108, respectively). The extraction results were statistically classiﬁed as follows: vegetation coverage of 0–0.05 was considered a zero-coverage area, 0.05–0.2 was a low vegetation coverage area, 0.2–0.4 was a low–moderate vegetation coverage area, 0.4–0.6 was a moderate vegetation coverage area, 0.6–0.8 was a moderate–high vegetation coverage area, and 0.8–1 was a high vegetation coverage area (Zhao et al., 2022). Figure 7 shows that EXG can clearly discriminate between vegetation and non-vegetation areas. From 2019 to 2021, the non-vegetation area FIGURE 2 GURE 2 riginal images of (A1) typical and (B1) validation quadrats and classiﬁcation results of the (A2) typical and (B2) veriﬁcation quadrats. FIGURE 2 Original images of (A1) typical and (B1) validation quadrats and classiﬁcation results of the (A2) typical and (B2) veriﬁcation quadrat 05 Frontiers in Ecology and Evolution frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 FIGURE 3 Calculation results of nine vegetation indices for the typical quadrat. FIGURE 3 FIGURE 3 Calculation results of nine vegetation indices for the typical quadrat. histogram threshold method (the thresholds were 0.047603, 0.041258, and 0.075669, respectively) are shown in Figure 6. The results of vegetation coverage extraction were compared with those of the maximum likelihood classiﬁcation (Table 5). EXG combined with the bimodal histogram method still had the highest accuracy in extracting vegetation coverage, followed by VDVI and RGBVI, suggesting that EXG had the highest precision in extracting vegetation information and could be used to estimate vegetation coverage in mining areas. segmentation effect was obviously better than that of Otsu’s threshold method. In the segmentation results of the bimodal histogram threshold method, RGRI, EXGR, NGRDI, and VEG misclassiﬁed vegetation as non-vegetation, whereas BGRI and EXG misclassiﬁed non-vegetation as non-vegetation, indicating relatively poor extraction accuracy. The quantitative accuracy must be evaluated to accurately evaluate the effects of the segmentation results. The Accuracy, Precision and Recall of the threshold segmentation results were calculated based on the maximum likelihood classiﬁcation results (Table 4). Overall, the classiﬁcation accuracy of the bimodal histogram method was higher than that of Otsu’s threshold method. Among the visible light vegetation indices, EXG, based on the bimodal histogram method, had the highest classiﬁcation accuracy, with the Accuracy was 98.264%, Precision was 99. 811% and 97.572% in vegetation and non-vegetation, and Recall was 99.913% and 94.847% in vegetation and non-vegetation. frontiersin.org 3.2 Suitability performance test The reliability and applicability of EXG, VDVI, and RGBVI for extracting vegetation coverage were veriﬁed based on the supervised classiﬁcation results of the veriﬁcation quadrat. The vegetation extracted based on EXG, VDVI, RGBVI, and the bimodal 06 frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 FIGURE 4 Segmentation results of the bimodal histogram threshold method. TABLE 3 Differences in pixel values of visible bands and vegetation indices of the typical quadrat. Indicators Vegetation Non-vegetation ANOVA P value Min Max Mean Standard deviation Min Max Mean Standard deviation Red band 30.00 194.00 96.57 23.89 68.00 254.00 177.23 43.07 0.054 Green band 66.00 211.00 132.26 20.03 78.00 249.00 176.73 38.20 0.033 Blue band 27.00 163.00 84.02 17.51 86.00 249.00 174.07 29.24 0.048 BGRI 0.32 0.85 0.63 0.08 0.85 1.20 1.00 0.07 0.007 RGRI 0.43 0.96 0.73 0.10 0.86 1.13 1.00 0.04 0.031 EXG 0.07 0.70 0.28 0.10 −0.03 0.05 0.00 0.01 0.001 EXGR −0.76 −0.22 −0.57 0.09 −0.86 −0.73 −0.80 0.02 0.012 NGRDI 0.02 0.40 0.16 0.07 −0.06 0.08 0.00 0.02 0.151 NGBDI 0.08 0.51 0.23 0.06 −0.09 0.08 0.00 0.04 0.023 RGBVI 0.11 0.75 0.37 0.11 −0.05 0.07 0.00 0.02 0.017 VDVI 0.05 0.45 0.19 0.06 −0.03 0.03 0.00 0.01 0.001 VEG 1.10 2.51 1.47 0.20 0.95 1.06 1.01 0.01 0.002 TABLE 3 Differences in pixel values of visible bands and vegetation indices of the typical quadrat. Indicators Vegetation Non-vegetation ANOVA P value Min Max Mean Standard deviation Min Max Mean Standard deviation Red band 30.00 194.00 96.57 23.89 68.00 254.00 177.23 43.07 0.054 Green band 66.00 211.00 132.26 20.03 78.00 249.00 176.73 38.20 0.033 Blue band 27.00 163.00 84.02 17.51 86.00 249.00 174.07 29.24 0.048 BGRI 0.32 0.85 0.63 0.08 0.85 1.20 1.00 0.07 0.007 RGRI 0.43 0.96 0.73 0.10 0.86 1.13 1.00 0.04 0.031 EXG 0.07 0.70 0.28 0.10 −0.03 0.05 0.00 0.01 0.001 EXGR −0.76 −0.22 −0.57 0.09 −0.86 −0.73 −0.80 0.02 0.012 NGRDI 0.02 0.40 0.16 0.07 −0.06 0.08 0.00 0.02 0.151 NGBDI 0.08 0.51 0.23 0.06 −0.09 0.08 0.00 0.04 0.023 RGBVI 0.11 0.75 0.37 0.11 −0.05 0.07 0.00 0.02 0.017 VDVI 0.05 0.45 0.19 0.06 −0.03 0.03 0.00 0.01 0.001 VEG 1.10 2.51 1.47 0.20 0.95 1.06 1.01 0.01 0.002 FIGURE 4 Segmentation results of the bimodal histogram threshold method. FIGURE 4 Segmentation results of the bimodal histogram threshold method. FIGURE 4 Segmentation results of the bimodal histogram threshold method. 3.2 Suitability performance test 07 07 Frontiers in Ecology and Evolution frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 FIGURE 5 Segmentation results of Otsu’s threshold method. FIGURE 5 Segmentation results of Otsu’s threshold method. FIGURE 5 Segmentation results of Otsu’s threshold method. FIGURE 5 Segmentation results of Otsu’s threshold method. TABLE 4 Accuracy evaluation of the typical quadrat. Segmentation Accuracy (%) Precision (%) Recall (%) Vegetation Non-vegetation Vegetation Non-vegetation BGRI Otsu’s 92.741 99.330 89.790 99.667 81.332 Bimodal histogram 96.275 96.233 96.294 98.278 0.920 EXG Otsu’s 81.656 100.000 73.441 99.999 62.772 Bimodal histogram 98.264 99.811 97.572 99.913 94.847 EXGR Otsu’s 75.787 99.998 64.956 99.998 56.086 Bimodal histogram 82.903 99.999 75.315 99.998 66.471 MGRVI Otsu’s 75.024 99.996 64.967 99.997 56.106 Bimodal histogram 82.524 99.811 74.389 99.886 63.573 NGBDI Otsu’s 91.216 99.627 87.449 99.809 78.045 Bimodal histogram 96.421 95.192 96.979 97.828 93.384 NGRDI Otsu’s 75.184 99.997 64.006 99.998 55.439 Bimodal histogram 80.634 99.924 71.996 99.953 61.507 (Continued) 08 Frontiers in Ecology and Evolution frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 TABLE 4 Continued Segmentation Accuracy (%) Precision (%) Recall (%) Vegetation Non-vegetation Vegetation Non-vegetation RGBVI Otsu’s 84.722 99.999 77.880 99.999 66.936 Bimodal histogram 97.824 99.795 96.941 99.905 93.595 RGRI Otsu’s 76.536 99.993 66.031 99.995 56.864 Bimodal histogram 81.725 99.866 73.602 99.918 62.882 VDVI Otsu’s 83.005 99.999 75.395 99.999 64.539 Bimodal histogram 97.993 99.895 97.141 99.952 93.994 VEG Otsu’s 77.195 99.997 66.982 99.996 57.560 Bimodal histogram 91.142 99.999 87.175 99.999 77.737 FIGURE 6 Veriﬁcation results of the veriﬁcation quadrat. TABLE 5 Accuracy evaluation of the veriﬁcation quadrat. Segmentation Accuracy (%) Precision (%) Recall (%) Vegetation Non-vegetation Vegetation Vegetation EXG 95.073 99.967 91.114 99.989 77.046 RGBVI 91.421 99.999 88.861 99.999 72.816 VDVI 93.107 99.928 91.072 99.976 76.957 B C D A FIGURE 7 Estimated results of vegetation coverage based on EXG combined with the bimodal histogram threshold method in (A) 2019, (B) 2020, and (C) 2021. (D) Inter-annual variation of vegetation coverage from 2019 to 2021. FIGURE 6 Veriﬁcation results of the veriﬁcation quadrat. FIGURE 6 Veriﬁcation results of the veriﬁcation quadrat. TABLE 5 Accuracy evaluation of the veriﬁcation quadrat. TABLE 5 Accuracy evaluation of the veriﬁcation quadrat. 3.2 Suitability performance test Segmentation Accuracy (%) Precision (%) Recall (%) Vegetation Non-vegetation Vegetation Vegetation EXG 95.073 99.967 91.114 99.989 77.046 RGBVI 91.421 99.999 88.861 99.999 72.816 VDVI 93.107 99.928 91.072 99.976 76.957 Segmentation Accuracy (%) Precision (%) Recall (%) Vegetation Non-vegetation Vegetation Vegetation EXG 95.073 99.967 91.114 99.989 77.046 RGBVI 91.421 99.999 88.861 99.999 72.816 VDVI 93.107 99.928 91.072 99.976 76.957 B C D A FIGURE 7 Estimated results of vegetation coverage based on EXG combined with the bimodal histogram threshold method in (A) 2019, (B) 2020, and (C) 2021. (D) Inter-annual variation of vegetation coverage from 2019 to 2021. B C D A FIGURE 7 Estimated results of vegetation coverage based on EXG combined with the bimodal histogram threshold method in (A) 2019, (B) 2020, and (C) 2021. (D) Inter-annual variation of vegetation coverage from 2019 to 2021. C D B A C B FIGURE 7 Estimated results of vegetation coverage based on EXG combined with the bimodal histogram threshold method in (A) 2019, (B) 2020, and (C) 2021. (D) Inter-annual variation of vegetation coverage from 2019 to 2021. ts of vegetation coverage based on EXG combined with the bimodal histogram threshold method in (A) 2019, (B) 2020, and (C) 2021 l variation of vegetation coverage from 2019 to 2021. 09 Frontiers in Ecology and Evolution frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 10.3389/fevo.2023.1171358 NGRDI were relatively low, which may be related to the histogram characteristics. As shown in the histogram of each vegetation index (Figure 8), EXG, VDVI, and RGBVI showed similar changes and obvious bimodal characteristics, whereas the histograms of RGRI, EXGR, and NGRDI had no obvious bimodal characteristics. Therefore, the accuracy of vegetation coverage extraction varied greatly. decreased, and the proportion of moderate–high and high vegetation coverage areas increased signiﬁcantly. The average FVC values in 2019, 2020, and 2021 were 31.47%, 34.08%, and 42.77%, respectively, indicating that the FVC in the mining area increased. The results suggest that the effect of vegetation restoration was remarkable, and the quality of the ecological environment improved. However, most areas in the post-mining area had low, low–moderate, and moderate vegetation coverage, and vegetation restoration requires further strengthening. 4 Discussion 4 Discussion Satellite remote sensing images have advantages, such as large image areas and multiple bands (Xu et al., 2020; Guo and Guo, 2021). However, owing to the relatively coarse spatial resolution, the interpretation accuracy is relatively limited, and the temporal resolution often cannot meet the real-time requirements of vegetation monitoring on a small spatial scale, such as in mining areas. With the popularization of UAV technology, UAV images have compensated for the deﬁciencies in satellite remote sensing images in terms of spatial and temporal resolution. UAVs provide a new data source for the acquisition of vegetation coverage information in mining areas and offer new approaches for monitoring vegetation growth and recovery in mining areas (Sun et al., 2021). The results of this study indicate that vegetation coverage data can be accurately extracted from UAV images. As an unsupervised classiﬁcation method, the visible light vegetation index can be used to extract vegetation coverage quickly and accurately without manual visual discrimination of vegetation areas or non-vegetation areas. Two major advantages are commonly associated with using visible spectrum images for extracting vegetation coverage. One is that RGB images are low 4.1 Extraction accuracy of vegetation coverage The accuracy of vegetation coverage extraction was related to both the vegetation index and threshold segmentation method. In this study, EXG exhibited the highest extraction accuracy, followed by VDVI and RGBVI, which is consistent with the results of Wang et al. (2015) and Chen and Deng (2019). The calculation formulas for EXG, VDVI, and RGNVI show the reﬂectance characteristics of vegetation in the visible bands, which effectively increase the sensitivity of vegetation to green bands and make full use of the information in the red, green, and blue bands. Currently, the bimodal histogram threshold and Otsu’s threshold methods are widely used for threshold segmentation. In this study, the results of the threshold methods for vegetation coverage extraction suggested that the accuracy of the bimodal histogram method was signiﬁcantly better than that of Otsu’s threshold method (Figure 5), reafﬁrming the results of Zhao et al. (2019). Using the bimodal histogram method, the accuracies of RGRI, EXGR, and FIGURE 8 Statistical histogram of the nine vegetation indices considered in this study. FIGURE 8 Statistical histogram of the nine vegetation indices considered in this study. 10 10 Frontiers in Ecology and Evolution frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 10.3389/fevo.2023.1171358 cost, convenient to process, and less affected by weather and light. The other is that RGB images have a relatively high spatial and temporal resolution, which is more suitable for local studies. For example, Marcial-Pablo et al. (2019) indicated that the accuracy of visible vegetation indices is higher than that of visible NIR vegetation indices for early crop cover. Furukawa et al. (2021) reported that RGB images provide reliable information for vegetation monitoring. For the mining areas, the land-use type was relatively single, and vegetation coverage could be quickly obtained via UAV images. Moreover, the UAV-visible images were acquired in summer, when vegetation growth was the best. For most vegetation, summer is the most vigorous period for plant growth, during which the vegetation exhibits the strongest reﬂected spectral features. Thus, vegetation coverage can be accurately estimated using the vegetation index. threshold method had the highest accuracy for vegetation identiﬁcation, which was the closest to the results of the monitored and actual situations. (4) The spatiotemporal analysis of vegetation coverage in 2019, 2020, and 2021 showed that most mining areas had low, low–moderate, and moderate vegetation coverage, whereas the overall vegetation coverage was low. Data availability statement The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author. 4.1 Extraction accuracy of vegetation coverage The average FVC for the three years were 31.47%, 34.08%, and 42.77%, respectively, indicating an increasing trend. Future studies should continue monitoring vegetation coverage changes to provide technical support for land reclamation and ecological restoration in mining areas. Funding This work was supported by the Key Laboratory of Degraded and Unused Land Consolidation Engineering, Ministry of Natural Resources of the People’s Republic of China (Program No. SXDJ2017-9), and the Shaanxi Key Laboratory of Land Reclamation Engineering: (Program No. 2018-ZZ03). 5 Conclusions We are grateful to the reviewers whose comments have helped to clarify and improve the manuscript. In this study, a disused gold mining area in the Qinling Mountains was selected as the research area, and UAVs were deployed to obtain image data with high spatial resolution in the visible light. The performance of different visible light vegetation indices combined with two threshold segmentation methods for extracting vegetation coverage was evaluated. The main conclusions are as follows. (1) Except for RGRI, NGRDI, and NGBDI, the other visible light vegetation indices effectively discriminated between vegetation and non-vegetation in the study area. (2) EXG, VDVI, and RGBVI combined with the bimodal histogram threshold method had higher extraction accuracy in distinguishing between vegetation and non-vegetation areas. (3) EXG and the bimodal histogram Author contributions According to previous investigation and research results, the soil arsenic contamination in the gold mining area is serious (the average soil arsenic content was 93.96 mg/kg) (Chen et al., 2022). Vegetation types are scarce (mostly herbaceous plants), and vegetation coverage is low. The results of the vegetation coverage change from 2019 to 2021 indicated that most natural vegetation restoration sites had low to low–moderate vegetation coverage. This was mainly because soil As contamination limited the normal growth and development of plants in the early stages of vegetation restoration (Yang et al., 2020), and community succession was relatively slow. Increased vegetation coverage improves the quality of regional ecological environments. With the progress of ecological restoration, the soil arsenic content has decreased, and plants have developed their own unique physiological and ecological characteristics after a period of adaptation. The number of pixels with zero vegetation and low and low-moderate vegetation coverage decreased, those with moderate–high and high vegetation coverage increased, and the overall vegetation coverage increased. RC: conceptualization, methodology, analysis, and writing – original manuscript. LH: conceptualization, reviewing and editing, and funding acquisition. YHZ: reviewing and editing, supervision. ZZ: methodology, statistical analysis, and reviewing and editing. ZL: reviewing and editing, supervision. RL: methodology, reviewing and editing. LX: reviewing and editing, providing revisions and comments, and supervision. YMZ: methodology, providing revisions and comments, and reviewing and editing. All authors contributed to the article and approved the submitted version. Frontiers in Ecology and Evolution References The potential for RGB images obtained using unmanned aerial vehicle to assess and predict yield in sugarcane ﬁelds. Int. J. Remote Sens. 39 (15-16), 5402–5414. doi: 10.1080/01431161.2018.1448484 Ren, L., Liu, Y., Zhang, S., Cheng, L., Guo, Y., and Ding, A. (2021). Vegetation properties in human-impacted riparian zones based on unmanned aerial vehicle (UAV) imagery: an analysis of river reaches in the yongding river basin. Forests 12, 22. doi: 10.3390/f12010022 Guo, X., and Guo, Q. (2021). And feng z detecting the vegetation change related to the creep of 2018 baige landslide in jinsha river, SE Tibet using SPOT data. Front. Earth Sci. 9. doi: 10.3389/feart.2021.706998 Guo, Z. C., Wang, T., Liu, S. L., Kang, W. P., Chen, X., Feng, K., et al. (2021). Biomass and vegetation coverage survey in the mu us sandy land - based on unmanned aerial vehicle RGB images. Int. J. Appl. Earth Obs. 94, 102239. doi: 10.1016/j.jag.2020.102239 Romina, S., Marı́a, C., Santiago, A. T., Elizabeth, K., Ana, S. B., Constanza, R., et al. (2010). Cryptochrome as a sensor of the blue/green ratio of natural radiation in arabidopsis. Plant Physiol. 154 (1), 401–409. doi: 10.1104/pp.110.160820 Hague, T., Tillett, N. D., and Wheeler, H. (2006). Automated crop and weed monitoring in widely spaced cereals. Precis. Agric. 7 (1), 21–32. doi: 10.1007/s11119- 005-6787-1 Schoﬁeld, G., Esteban, N., Katselidis, K. A., and Graeme, C. H. (2019). Drones for research on sea turtles and other marine vertebrates – a review. Biol. Conserv. 238, 108214. doi: 10.1016/j.biocon.2019.108214 Hunt, E. R., Cavigelli, M., Daughtry, C., Mcmurtrey, J., and Walthall, C. (2005). Evaluation of digital photography from model aircraft for remote sensing of crop biomass and nitrogen status. Precis. Agric. 6 (4), 359–378. doi: 10.1007/s11119-005- 2324-5 Shane, G., Kevin, L., John, J., and Kenny, S. (2021). Use of an unmanned aerial vehicle (UAV) to document vegetation coverage rate in managed grasslands following a historic river ﬂood. J. Anim. Sci. 99 (2), 9–10. doi: 10.1093/jas/skab096.015 Shukla, A., and Jain, K. (2020). Automatic extraction of urban land information from unmanned aerial vehicle (UAV) data. Earth Sci. Inform 13, 1225–1236. doi: 10.1007/ s12145-020-00498-x Huo, A. D., Wang, X., Zhao, Z. X., Yang, L. Y., Zhong, F. Q., Zheng, C. L., et al. (2022). Risk assessment of heavy metal pollution in farmland soils at the northern foot of the qinling mountains, China. Int. J. Environ. Res. Public Health 19 (22), 14962. doi: 10.3390/ijerph192214962 Song, W. J., Mu, X. References Y., Huang, Y. H., Ye, H. P., Xu, C. C., et al. (2020). Optimizing distribution of drone ports for emergency monitoring of ﬂood disasters in China. J. Flood Risk Manag. 13 (1), e12593. doi: 10.1111/jfr3.12593 Coy, A., Rankine, D., Taylor, M., and Nielsen, D. C. (2016). Increasing the accuracy and automation of fractional vegetation cover estimation from digital photographs. Remote Sens. 8 (7), 474. doi: 10.3390/rs8070474 Marcial-Pablo, M. D. J., Gonzalez-Sanchez, A., Jimenez-Jimenez, S. I., Ontiveros- Capurata, R. E., and Ojeda-Bustamante, W. (2019). Estimation of vegetation fraction using RGB and multispectral images from UAV. Int. J. Remote Sens. 40 (2), 420–438. doi: 10.1080/01431161.2018.1528017 Fu, Y., Song, Y., Yang, C., Liu, X., Liu, Y., and Huang, Y. (2022). Relationship between brain size and digestive tract length support the expensive-tissue hypothesis in feirana quadranus. Front. Ecol. Evol. 10. doi: 10.3389/fevo.2022.982590 Mishra, V., Avtar, R., Prathiba, A. P., Mishra, P. K., Tiwari, A., and Sharma, S. K. (2023). Uncrewed aerial systems in water resource management and monitoring: a review of sensors, applications, software, and issues. Adv. Civ. Eng. 28, 3544724. doi: 10.1155/2023/3544724 Furukawa, F., Laneng, L. A., Ando, H., Yoshimura, N., Kaneko, M., and Morimoto, J. (2021). Comparison of RGB and multispectral unmanned aerial vehicle for monitoring vegetation coverage changes on a landslide area. Drones 5, 97. doi: 10.3390/ drones5030097 Geng, X., Wang, X. M., Fang, H. L., Ye, J. S., Han, L. K., Gong, Y., et al. (2022). Vegetation coverage of desert ecosystems in the qinghai-Tibet plateau is underestimated. Ecol. Indic 137, 108780. doi: 10.1016/j.ecolind.2022.108780 Otsu, N. (2007). A threshold selection method from gray-level histograms. IEEE T Syst. Man Cy-S 9 (1), 62–66. doi: 10.1109/TSMC.1979.4310076 Park, G., Park, K., Song, B., and Lee, H. (2022). Analyzing impact of types of UAV- derived images on the object-based classiﬁcation of land cover in an urban area. Drones 6, 71. doi: 10.3390/drones6030071 Gitelson, A. A., Kaufman, Y. J., Stark, R., and Rundquist, D. (2002). Novel algorithms for remote estimation of vegetation fraction. Remote Sens. Environ. 80 (1), 76–87. doi: 10.1016/S0034-4257(01)00289-9 Rasmussen, J., Ntakos, G., Nielsen, J., Svensgaard, J., Poulsen, R. N., and Christensen, S. (2016). Are vegetation indices derived from consumer-grade cameras mounted on UAVs sufﬁciently reliable for assessing experimental plots? Eur. J. Agron. 74, 75–92. doi: 10.1016/j.eja.2015.11.026 Guilherme, M. S., Daniel, G. D., Oriel, T. K., Ana, C. S. L., Sérgio, G., Fábio, M. O., et al. (2018). References Akash, A., Jinha, J., Anjin, C., Sungchan, O., Murilo, M., and Juan, L. (2019). A comparative study of RGB and multispectral sensor-based cotton canopy cover modelling using multi-temporal UAS data. Remote Sens. 11 (23), 2757. doi: 10.3390/ rs11232757 Akash, A., Jinha, J., Anjin, C., Sungchan, O., Murilo, M., and Juan, L. (2019). A comparative study of RGB and multispectral sensor-based cotton canopy cover modelling using multi-temporal UAS data. Remote Sens. 11 (23), 2757. doi: 10.3390/ rs11232757 Kim, D. W., Yun, H. S., Jeong, S. J., Kwon, Y. S., Kim, S. G., Lee, W. S., et al. (2018). Modeling and testing of growth status for Chinese cabbage and white radish with UAV- based RGB imagery. Remote Sens. 10 (4), 563. doi: 10.3390/rs10040563 Leng, R. L., Zhang, Y. Y., Xie, J. Q., Li, F. N., Xu, G., and Cui, X. (2019). An analysis of fractional vegetation cover of the gannan grassland in the non-growing season based on multispectral data and small UVAs. Pratacultural Sci. 36 (11), 2742–2751. doi: 10.11829/j.issn.1001-0629.2019-0013 Ana, D. C., Shi, Y. Y., Maja, J. M., and Peña, J. M. (2021). UAVs for vegetation monitoring: overview and recent scientiﬁc contributions. Remote Sens-Basel 13, 2139. doi: 10.3390/rs13112139 Li, X. L., Gao, J., Zhang, J., Wang, R., Jin, L. Q., and Zhou, H. K. (2019). Adaptive strategies to overcome challenges in vegetation restoration to coalmine wasteland in a frigid alpine setting. Catena 182, 104142. doi: 10.1016/j.catena.2019.104142 Boyd, D. S., Foody, G. M., and Ripple, W. J. (2002). Evaluation of approaches for forest cover estimation in the paciﬁc Northwest, USA, using remote sensing. Appl. Geogr. 22 (4), 375–393. doi: 10.1016/S0143-6228(02)00048-6 Chen, X. D., and Deng, J. H. (2019). Study on extraction method of vegetation coverage of summer maize based on visible image. Exp. Technol. Manage. 36 (12), 131– 136. doi: 10.6041/j.issn.1000-1298.2019.05.027 Li, T., Wu, M. H., Duan, C. G., Li, S. Y., and Liu, C. E. (2022). The effect of different restoration approaches on vegetation development in metal mines. Sci. Total Environ. 806 (2), 150626. doi: 10.1016/J.Scitotenv.2021.150626 Chen, R., Han, L., Liu, Z., Zhao, Y. H., Li, R. S., Xia, L. F., et al. (2022). Assessment of soil-heavy metal pollution and the health risks in a mining area from southern shaanxi province, China. Toxics 10, 385. doi: 10.3390/toxics10070385 Liang, H. W. (2002). Direct determination of threshold from bimodal histogram. Pattern Recognition Artiﬁcial Intell. 15 (2), 253–256. Lu, M., Liao, X. H., Yue, H. Conﬂict of interest RL and LX were employed by the Institute of Land Engineering and Technology, Shaanxi Provincial Land Engineering Construction Group, Xi’an, China. The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. frontiersin.org 11 Chen et al. 10.3389/fevo.2023.1171358 10.3389/fevo.2023.1171358 Publisher’s note 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. 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. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated References H., Ruan, G. Y., Gao, Z., Li, L. Y., and Yan, G. Y. (2017). Estimating fractional vegetation cover and the vegetation index of bare soil and highly dense vegetation with a physically based method. Int. J. Appl. Earth Obs. 58, 168–176. doi: 10.1016/j.jag.2017.01.015 Jay, S., Baret, F., Dutartre, D., Malatesta, G., Héno, S., Comar, A., et al. (2019). Exploiting the centimeter resolution of UAV multispectral imagery to improve remote- sensing estimates of canopy structure and biochemistry in sugar beet crops. Remote Sens. Environ. 231, 110898. doi: 10.1016/j.rse.2018.09.011 Sun, Z. Y., Wang, X. N., Wang, Z. H., Yang, L., Xie, Y. C., and Huang, Y. H. (2021). UAVs as remote sensing platforms in plant ecology: review of applications and challenges. J. Plant Ecol. 14 (6), 1003–1023. doi: 10.1093/jpe/rtab089 Jia, K., Liang, S. L., Liu, S. H., Li, Y. W., Xiao, Z. Q., Yao, Y. J., et al. (2015). Global land surface fractional vegetation cover estimation using general regression neural networks from MODIS surface reﬂectance. IEEE T Geosci Remote 53 (9), 4787–4796. doi: 10.1109/TGRS.2015.2409563 Sun, G. X., Wang, X. C., Yan, T. T., Li, X., Chen, M., Shi, Y. Y., et al. (2014). Inversion method of ﬂora growth parameters based on machine vision. Trans. Chin. Soc. Agric. Eng. 30 (20), 187–195. Juliane, Be, Kang, Y., Helge, A., Andreas, B., Simon, B., Janis, B., et al. (2015). Combining UAV-based plant height from crop surface models, visible, and near infrared vegetation indices for biomass monitoring in barley. Int. J. Appl. Earth Obs. 39, 79–87. doi: 10.1016/j.jag.2015.02.012 Verrelst, J., Schaepman, M. E., Koetz, B., and Kneubühlerb, M. (2008). Angular sensitivity analysis of vegetation indices derived from CHRIS/PROBA data. Remote Sens. Environ. 112 (5), 2341–2353. doi: 10.1016/j.rse.2007.11.001 12 Frontiers in Ecology and Evolution frontiersin.org Chen et al. 10.3389/fevo.2023.1171358 Voorde, T. V., Vlaeminck, J., and Canters, F. (2008). Comparing different approaches for mapping urban vegetation cover from landsat ETM+ data: a case study on Brussels. Sensors 8, 3880–3902. doi: 10.3390/s8063880 from terrestrial laser scanning data. Ecol. Indic. 108 (C), 105747. doi: 10.1016/ j.ecolind.2019.105747 Yang,G.Y.,Zhong,H.,Liu,X.,Liu,C.E.,Li,S.Y.,Hou,L.,etal.(2020).Arsenicdistribution, accumulation and tolerance mechanisms of typha angustifolia in different phenological growth stages. B Environ. Contam. Tox. 104 (3), 358–365. doi: 10.1007/s00128-020-02796-y Wang, X. Q., Wang, M. M., Wang, S. Q., and Wu, D. P. (2015). Extraction of vegetation information from visible unmanned aerial vehicle images. Trans. Chin. Soc. Agric. Eng. 31 (05), 152–159. Zhang, X. L., Zhang, F., Qi, Y. X., Deng, L. References F., Wang, X. L., and Yang, S. T. (2019). New research methods for vegetation information extraction based on visible light remote sensing images from an unmanned aerial vehicle (UAV). Int. J. Appl. Earth Obs. 78, 215–226. doi: 10.1016/j.jag.2019.01.001 Watanabe, K., Guo, W., Arai, K., Takanashi, H., Kajiya-Kanegae, H., Kobayashi, M., et al. (2017). High-throughput phenotyping of sorghum plant height using an unmanned aerial vehicle and its application to genomic prediction modeling. Front. Plant Sci. 8. doi: 10.3389/fpls.2017.00421 Zhao, D., Wang, Z. W., Zhang, G. Z., Xu, Y. M., and Sun, L. J. (2022). Exploration of inﬂuence factors on regional fractional vegetation cover based on a combination offactor regression and interaction–take the three-river headwaters region as an example. China Environ. Sci. 42 (8), 3903–3912. doi: 10.19674/j.cnki.issn1000-6923.20220329d011 Woebbecke, D., Meyer, G., Bargen, K., and Mortensen, D. (1995). Color indices for weed identiﬁcation under various soil, residue, and lighting conditions. T ASABE 38, 259–269. doi: 10.13031/2013.27838 Xu, X., Liu, L., Han, P., Gong, X., and Zhang, Q. (2022). Accuracy of vegetation indices in assessing different grades of grassland desertiﬁcation from UAV. Int. J. Env. Res. Pub. He. 19 (24), 16793. doi: 10.3390/ijerph192416793 Zhao, J., Yang, H. B., Lan, Y. B., Lu, L. Q., Jia, P., and Li, Z. M. (2019). Extraction method of summer corn vegetation coverage based on visible light image of unmanned aerial vehicle. Trans. Chin. Soc. Agric. Machinery 050 (005), 232–240. Xu, K. X., Su, Y. J., Liu, J., Hu, T. Y., Jin, S. C., Ma, Q., et al. (2020). Estimation of degraded grassland aboveground biomass using machine learning methods Zhou, T., Hu, Z. Q., Han, J. Z., and Zhang, H. (2021). Green vegetation extraction based on visible light image of UAV. China Environ. Sci. 41 (05), 2380–2390. 13 13 frontiersin.org Frontiers in Ecology and Evolution
https://openalex.org/W2172873824	https://hrcak.srce.hr/file/111570	English	null	Main Phases of Wood Formation in Chestnut (Castanea sativa) in Central Italy - Comparison of Seasons 2008 and 2009	Drvna industrija	2,011	cc-by	6,390	1 Authors are associate professor, master of science and Ph.D., respectively, at Tuscia University, Viterbo, Italy. 2Authors are young researcher – Ph.D. student and professor, respectively, at University of Ljubljana, Biotechnical Faculty, Slovenia. 3Author is assistant professor and se- nior research associate at Slovenian Forestry Institute, Ljubljana, Slovenia. y j j 1 Autori su redom, izvanredna profesorica, magistrica i doktor, Sveučilište Tuscia, Viterbo, Italija. 2Autori su, redom, znanstveni novak – dok- torand i redovna profesorica, Sveučilište u Ljubljani, Biotehnički fakultet, Slovenija. 3Autorica je docentica i viša znanstvena suradnica, Slovenski šumarski institut, Ljubljana, Slovenija. Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... 1 INTRODUCTION 1. UVOD wood for high-added-value products. They are consi- dered to occur as a consequence of growth stresses (ra- dial tensile stress) and structural weakness of portions of wood tissue (Fonti and Macchioni, 2003; Spina and Romagnoli, 2010). Sweet chestnut (Castanea sativa Mill.) is the only native species of the genus Castanea in Europe and its cultivation has a long tradition. Chestnut is valued for its wood, bark and fruit. COST action G4 “Multidiscipli- nary Chestnut Research” based on National Forest In- ventories reported in 1997 that 2.25 million hectares of forests in Europe were dominated by chestnut, with roughly 80% cultivated for wood and 20% for fruit pro- duction. Furthermore, three types of chestnut countries have been distinguished: (i) countries with a strong chestnut tradition (e.g. Italy, France, southern Switzer- land, Spain, Portugal and Greece), where the chestnut stands are cultivated with intensive and characteristic silvicultural systems (coppices and orchards); (ii) coun- tries with only a partially developed chestnut tradition due to the country’s particular geography (e.g. England) or history (e.g. Croatia, Turkey, Georgia); and (iii) coun- tries where the chestnut only sporadically occurs (e.g. Hungary, Bulgaria, Belgium) or has been recently intro- duced (e.g. Slovakia, Netherlands) (Conedera et al, 2004). Since wood quality depends on wood structure, which is deﬁ ned during the process of wood formation, a detailed knowledge of wood formation processes will improve our understanding of the relationship among wood structure and properties and the end-use of wood. We started wood formation studies in chestnut in Central Italy in 2008 and the ﬁ rst results on cambial activity, wood and phloem formation in ﬁ ve trees in 2008 have already been published (Čufar et al, 2011). Since considerable tree to tree and year to year varia- bility are expected, we evaluated the results of addi- tional ﬁ ve trees sampled in 2008 and continued with the experiment in 2009. The aim of the present study was to compare the seasonal dynamics of wood formation in ten trees in 2008 and 2009 and to discuss the effect of wood forma- tion dynamics on wood quality. Sweet chestnut is a very common and important tree species in Italy. It grows all over the peninsula and is characteristic of the phytoclimatic association Casta- netum, which grows in the altitudinal belt from 0 to 900 m a.s.l. in the north, and from 600 to 1200 m a.s.l. 1 INTRODUCTION 1. UVOD in the central and southern parts of Italy. The cultiva- tion and use of chestnut fruit and wood has a long tra- dition all over Central Italy and also in the Province of Viterbo (Romagnoli et al, 2005; Romagnoli, 2007). Glavne faze razvoja drva pitomog kestena (Castanea sativa) u središnjoj Italiji – usporedba sezone 2008 i 2009 Uspoređuje se dinamika stvaranja drva u te dvije godine i raspravlja o mogućem utjecaju na njego DRVNA INDUSTRIJA 62 (4) 269-275 (2011) 269 Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... ............. kvalitetu. Za tu su svrhu s deset stabala pitomog kestena u planinama Cimini, pokraj mjesta Viterbo u središnjoj Italiji, u tjednim intervalima skupljeni mikroizvrtci koji su sadržavali drvo, kambij i ﬂ oem. Početak stvaranja drva u 2008. godini dogodio se prije uzimanja prvih uzoraka 17. travnja 2008. Početak ligniﬁ kacije prvooblikovanih traheja primijećen je oko 23. travnja (dan u godini DOY 113,8 ± 5,3), a prve traheje kasnog drva primijećene su oko 5. lipnja (DOY 156,5 ± 7,7). Stvaranje kasnog drva nastavljeno je do 29. rujna 2008. (DOY 273,9 ± 10,5), kad su završne stanice novostvorenoga goda drva bile potpuno ligniﬁ cirane. U 2009. godini glavne su se faze stvaranja drva uglavnom pojavile ranije nego u 2008. godini. Širenje traheja ranog drva primijećeno je oko 10. travnja (DOY 99,7 ± 6,1), početak ligniﬁ kacije oko 22. travnja (DOY 111,9 ± 7,4), a prve traheje kasnog drva oko 28. svibnja 2009. (DOY 147,9 ± 4,7). Ligniﬁ kacija zadnjih proizvedenih stanica završena je do 26. rujna 2009. (DOY 273,9 ± 10,5). Prosječno trajanje stvaranja goda drva u 2008. godini iznosilo je 161 dan, a u 2009. godini 169 dana. Prosječne širine godova bile su 3296 ± 1514 µm u 2008. godini i 3166 ± 1073 µm u 2009. godini, a postotni udjel kasnog drva iznosio je 76 i 74% godova u 2008., odnosno u 2009. godini. Male razlike u vremenu početka pojedine faza stvaranja drva u dvjema promatranim godinama vjerojatno su posljedica malih varijacija klimatskih uvjeta između te dvije godine i čini se da nemaju velik utjecaj na širinu godova ni na postotak kasnog drva, a to su dva važna čimbenika koji utječu na kvalitetu drva prstenasto poroznih vrsta. Ključne riječi: Castanea sativa, pitomi kesten, središnja Italija, stvaranje drva, kvaliteta drva Glavne faze razvoja drva pitomog kestena (Castanea sativa) u središnjoj Italiji – usporedba sezone 2008 i 2009 Original scientiﬁ c paper • Izvorni znanstveni rad Received – prispjelo: 21. 7. 2011. Accepted – prihvaćeno: 22. 11. 2011. UDK: 630*811.1; 674.031.632.254.2 doi:10.5552/drind.2011.1124 ABSTRACT • We present wood formation in chestnut (Castanea sativa) during the growing seasons 2008 and 2009, compare its dynamics in the two years and discuss possible effects on wood quality. To this purpose, micro- cores containing wood, cambium and phloem were collected at weekly intervals from 10 chestnut trees growing at the Cimini mountains near Viterbo, Central Italy. In 2008, the onset of wood formation started before the ﬁ rst sampling on 17 April 2008. Onset of ligniﬁ cation of the ﬁ rst formed vessels was observed around 23 April (day of the year DOY 113.8 ± 5.3) and the ﬁ rst latewood vessels were observed around 5 June 2008 (DOY 156.5 ± 7.7). Latewood formation continued until 29 September 2008 (DOY 273.9 ± 10.5) when the terminal cells of the newly formed xylem ring were fully ligniﬁ ed. In 2009, the main phases of wood formation generally occurred earlier than in 2008. The expansion of earlywood vessels was observed around 10 April (DOY 99.7 ± 6.1), the onset of ligniﬁ cation around 22 April (DOY 111.9 ± 7.4) and the ﬁ rst latewood vessels around 28 May 2009 (DOY 147.9 ± 4.7). Ligniﬁ cation of the last formed cells was completed by 26 September 2009 (DOY 273.9 ± 10.5). The average duration of tree-ring formation was 161 days in 2008 and 169 days in 2009, the average ring widths were 3296 ± 1514 µm in 2008 and 3166 ± 1073 µm in 2009, and latewood percentages comprised 76% and 74% of the 2008 and 2009 tree-rings, respectively. The small differences in timing of wood formation phases in the two study years are probably due to small variations in climatic conditions between the two years and they did not seem to have a major impact on ring widths and latewood percentages, which are two important parameters affecting wood quality in ring porous wood species. Key words: Castanea sativa, sweet chestnut, Central Italy, wood formation, wood quality ETAK • Rad obrađuje stvaranje drva pitomog kestena (Castanea sativa) tijekom vegetacije u 2008. i 200 i. 2 MATERIALS AND METHODS 2. MATERIJALI I METODE 10,000 µm2 were considered to belong to latewood (Fonti et al, 2007). In the newly formed wood tissue, we could fol- low the development of the vessels, vasicentric trac- heids around them (referred to in brief as tracheids) and tracheids or ﬁ bres apart from the vessels (ﬁ bres) (Figs. 2, 3). We recorded the following phases of cell development: post-cambial growth (PC), secondary cell wall deposition and ligniﬁ cation (SW) and mature cells (MT) (Fig. 3). The PC cells contained thin, non- ligniﬁ ed cell walls that stained blue with astra blue We selected ten isolated dominant healthy chest- nut trees with approximate diameters of 17 cm, heights of 15 m, and ages of 30 years. The trees originate from stumps and ring shake defect was observed in some of them. Each tree represents a single shoot that had been left after cutting all other trees sprouting from the same stump, at the end of the rotation time in 2006. The sam- pling trees are now the standards of the site. Samples of tissues containing the bark, cambium and the last formed wood were collected by taking mi- cro-cores with Trephor (Rossi et al, 2006). The micro- cores (diameter 1.8 mm, length approx. 15 mm) were extracted from living trees, at the basal part of the stems, at weekly intervals from April until October 2008 and 2009. Figure 2 Cross-section of Castanea sativa tissues showing the last formed xylem ring divided into earlywood and latewood, cambial zone (CZ) and phloem. The cells of the wood are: earlywood vessels (EV), latewood vessels (LV), tracheids (TR) and ﬁ bres. The rays (R) are uniseriate. Slika 2. Poprečni presjek zadnjih proizvedenih tkiva Castanea sativa s ranim i kasnim drvom, kambijskom zonom (CZ) i ﬂ oemom. Stanice drva su traheje ranog drva (EV), traheje kasnog drva (LV), traheide (TR) i vlakanca. Traci (R) jednoredni su. Fibres vlakanca LV TR EV R Phloem ﬂ oem CZ Latewood kasno drvo Earlywood rano drvo Fibres vlakanca LV TR EV R Phloem ﬂ oem CZ Latewood kasno drvo Earlywood rano drvo Fibres vlakanca LV TR EV R Phloem ﬂ oem CZ Latewood kasno drvo Earlywood rano drvo Immediately after extraction from the tree, the samples were put in 70% ethanol for ﬁ xation and con- servation. 2 MATERIALS AND METHODS 2. MATERIJALI I METODE The study was carried out at a coppice chestnut (Castanea sativa Mill.) forest stand in the locality of the Cimini mountains, part of Comune di Soriano nel Cimi- no, Viterbo, Italy (approx. 42°17’N, 12°12’E, 850 m a. s.l.). The area is on volcanic soil, very close to the old beech forest described by Piovesan et al (2008). The cli- mate is Mediterranean. The amount of annual precipita- tion in nearby Soriano nel Cimino is 1180 mm (Servizio Idrograﬁ co, 1916-2000). The precipitation maximum is recorded from October until December and the driest period occurs in summer, although there is no signiﬁ cant evidence of drought according to the Bagnouls-Gaussen diagram (Piovesan et al, 2008). The mean annual tem- perature is 14 °C, with a maximum in August (up to 24.2 °C) and minimum in December (6.2 °C) (Servizio idrograﬁ co 1997-2001). The daily data (minimum and In this region, chestnut wood has been used in mo- dern and in many historical buildings (Romagnoli et al, 2004; Romagnoli et al, 2005). The species is currently cultivated for wood production using coppice sil vi- cultural management, in which new trees (shoots) grow from the stumps. The rotation time at least 14 years. Chestnut belongs to ring-porous hardwoods with a morphological structure similar to oak wood (Schwein- gruber, 1990; Nardi Berti, 2006). Ring shakes are the main wood defect, which reduces the use of chestnut DRVNA INDUSTRIJA 62 (4) 269-275 (2011) 270 Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... Figure 1 Daily climatic data, minimum, mean and maximum temperatures (lines), and precipitation (bars), for Soriano nel Cimino, Central Italy, in 2008 and 2009. Slika 1 Dnevni klimatski podatci u 2008. i 2009. za Soriano nel Cimino, središnja Italija: minimalne, prosječne i maksimalne temperature (krivulje) i oborine (trake) Temperature / temperatura, ˚C Precipitation / oborine, mm Figure 1 Daily climatic data, minimum, mean and maximum temperatures (lines), and precipitation (bars), for Soriano nel Cimino, Central Italy, in 2008 and 2009. Slika 1 Dnevni klimatski podatci u 2008. i 2009. za Soriano nel Cimino, središnja Italija: minimalne, prosječne i maksimalne temperature (krivulje) i oborine (trake) Cimino, Central Italy, in 2008 and 2009. Slika 1 Dnevni klimatski podatci u 2008. i 2009. za Soriano nel Cimino, središnja Italija: minimalne, prosječne i maksimalne temperature (krivulje) i oborine (trake) maximum temperature and precipitation) for 2008 and 2009 are presented in Fig. 1. 2 MATERIALS AND METHODS 2. MATERIJALI I METODE Stanice drva su traheje ranog drva (EV), traheje kasnog drva (LV), traheide (TR) i vlakanca. Traci (R) jednoredni su. Figure 2 Cross-section of Castanea sativa tissues showing the last formed xylem ring divided into earlywood and latewood, cambial zone (CZ) and phloem. The cells of the wood are: earlywood vessels (EV), latewood vessels (LV), tracheids (TR) and ﬁ bres. The rays (R) are uniseriate. Slika 2. Poprečni presjek zadnjih proizvedenih tkiva Castanea sativa s ranim i kasnim drvom, kambijskom zonom (CZ) i ﬂ oemom. Stanice drva su traheje ranog drva (EV), traheje kasnog drva (LV), traheide (TR) i vlakanca. Traci (R) jednoredni su. DRVNA INDUSTRIJA 62 (4) 269-275 (2011) 2 MATERIALS AND METHODS 2. MATERIJALI I METODE After the end of sampling, they were embed- ded in parafﬁ n using a Leica TP 1020-1 tissue proces- sor for dehydration in a graded series of ethanol (70%, 90%, 95% and 100%) and bio-clear (D-limonene) for parafﬁ n inﬁ ltration (Rossi et al, 2006). Cross-sections of 10 µm thickness were prepared on a Leica RM 2245 rotary microtome, using disposable Feather N35H blades. For better adhesion of the sections, slides were pre-treated with albumin. Sections were dried at 70 °C for half an hour and cleaned of residual parafﬁ n by im- mersing the slides in bio-clear and ethanol. Sections were ﬁ nally stained for light microscopy with a mix- ture of safranin and astra blue (40 mg safranin and 150 mg astrablue added to a solution of 100 ml demineral- ized water with 2 ml acetic acid) (van der Werf et al, 2007) and mounted on glass slides in Euparal. A Nikon Eclipse 800 light microscope (bright ﬁ eld and polarized light), Nikon digital sight DS-Fi1 video camera and the NIS elements BR 3.0 image anal- ysis system were used for observations and semi-auto- matic counting and measuring of cells and tissues at various stages of their development. We also measured the width of the current increment as well as early- and latewood widths (in µm). The measurements were al- ways done in each sample along three radial ﬁ les. The boundary between earlywood and latewood was de- ﬁ ned based on vessel dimensions; vessels smaller than Figure 2 Cross-section of Castanea sativa tissues showing the last formed xylem ring divided into earlywood and latewood, cambial zone (CZ) and phloem. The cells of the wood are: earlywood vessels (EV), latewood vessels (LV), tracheids (TR) and ﬁ bres. The rays (R) are uniseriate. Slika 2. Poprečni presjek zadnjih proizvedenih tkiva Castanea sativa s ranim i kasnim drvom, kambijskom zonom (CZ) i ﬂ oemom. Stanice drva su traheje ranog drva (EV), traheje kasnog drva (LV), traheide (TR) i vlakanca. Traci (R) jednoredni su. Figure 2 Cross-section of Castanea sativa tissues showing the last formed xylem ring divided into earlywood and latewood, cambial zone (CZ) and phloem. The cells of the wood are: earlywood vessels (EV), latewood vessels (LV), tracheids (TR) and ﬁ bres. The rays (R) are uniseriate. Slika 2. Poprečni presjek zadnjih proizvedenih tkiva Castanea sativa s ranim i kasnim drvom, kambijskom zonom (CZ) i ﬂ oemom. 3 RESULTS AND DISCUSSION 3. REZULTATI I DISKUSIJA Fonti et al. (2007) tried to identify the climatic signals contained in the earlywood vessel size of Cas- tanea sativa from Valle Mesolcina (46°14′N, 9°07′E, elevations 300, 600 and 900 m a.s.l.) in Switzerland and the physiological processes involved in the under- lying mechanisms. They reported that the ﬁ rst vessels appeared in late April to early May. They hypothesised that April temperatures are related to tree activation, whereby new hormone production fosters vessel ex- pansion. In 2008, the onset of cambial divisions and the ﬁ rst earlywood vessels in the phase of expansion oc- curred before our ﬁ rst sampling on 17 April (Čufar et al, 2011). In 2009, the ﬁ rst earlywood vessels in the phase of expansion were observed around 10 April (day of the year DOY 99.7 ± 6.1) (Fig. 4), while the onset of cambial cell production was recorded on 2 April 2009 (DOY 92.0 ± 6.4). Previous reports on wood formation in chestnut are extremely rare. In the 1940s, Ciampi (1951) sam- pled two chestnut trees at two sites in Tuscany in Italy: near San Giusto, at 500 m a.s.l. in the 1947 growing season, and near Vallombrosa at 1000 m a.s.l. in the 1949 growing season. It was observed that the onset of cambial activity and earlywood production differed be- tween the two localities. In San Giusto, earlywood ves- sels were already formed at the beginning of April and ligniﬁ cation of the initial vessels was concluded by mid-May. In Vallombrosa, the onset of cambial activity occurred much later; at the end of April, but ligniﬁ ca- tion was already evident at the beginning of May. Deposition of the secondary wall layers and lig- niﬁ cation of ﬁ rst formed vessels and surrounding ﬁ - bres/ﬁ bre tracheids started at almost the same time, around 23 April 2008 (DOY 113.8 ± 5.3) and around 22 April 2009 (DOY 111.9 ± 7.4) (Fig. 4). The onset of deposition was recognized due to birefringence of ves- sels and ﬁ bres observed under polarized light (Fig. 3b), whereas ligniﬁ cation was characterized by red staining of the cell walls observed in bright-ﬁ eld (Fig. 3a). DRVNA INDUSTRIJA 62 (4) 269-275 (2011) 271 Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... ....... mation started 13 days earlier than in oaks and ended around the beginning of May. stain (Fig. 3 a) and showed no birefringence under po- larized light (Fig. 3 b). In this phase, the cells enlarged in radial and tangential directions (vessels) or elongat- ed (tracheids, ﬁ bres). The beginning of the secondary wall deposition was detected under polarized light, since the cell walls showed birefringence (Fig. 3 b). The beginning of cell wall ligniﬁ cation could be ob- served under bright-ﬁ eld, when red stain safranin grad- ually replaced the blue staining (Fig. 3 a). It is generally known that the ﬁ rst earlywood ves- sels develop before bud break in ring-porous trees such as Castanea, Quercus, Fraxinus and Robinia, and therefore before the resumption of photosynthetic ac- tivity. Formation of the ﬁ rst earlywood vessels can oc- cur two to six weeks prior to bud break (Suzuki et al, 1996; Schmitt et al, 2000; Sass-Klaassen et al. 2011). The early phases of earlywood formation thus require the mobilization of reserves stored during the previous growing season (Barbaroux and Bréda, 2002). 3 RESULTS AND DISCUSSION 3. REZULTATI I DISKUSIJA As reported by several studies, both secondary wall formation and ligniﬁ cation start ﬁ rst in the vessels and in the cells that are in contact with the vessels (Mu- rakami et al, 1999; Terashima 2000; Grünwald et al, 2002; Marion et al, 2007; Prislan et al, 2009). Since Since other reports on wood formation in chest- nut are not available, we can compare our observations with those in other ring porous species, which have been conducted in various geographic areas. Gričar (2010) followed wood formation in Quercus petraea (sessile oak) in Ljubljana, Slovenia, at 323 m a.s.l. in 2007 and observed that the ﬁ rst earlywood vessels were created by mid-April. Horaček et al. (2003) pre- sented xylem formation in Quercus robur (pedunculate oak) in Czech Republic. They observed that the activ- ity of cambium started before the ﬁ rst sampling on 27 April 1998, when on average 6.66 radially enlarging cells were already observed. A study by Derr and Evert (1967) on young Robinia pseudoacacia (black locust) trees in Madison, USA, from October 1960 until Octo- ber 1962, reported that the ﬁ rst signs of cambial cell divisions were observed on 18 April 1961 and on 7 April 1962. The ﬁ rst expanding vessels were observed on 27 April 1962. Figure 3 Cells in different phases of formation observed under a light microscope: (a) bright ﬁ eld and (b) polarized light. Phases of cell formation: post-cambial growth (PC), secondary cell wall deposition and ligniﬁ cation (SW) and mature cells (MT) formed in the season. Slika 3. Stanice u različitim fazama razvoja promatrane svjetlosnim mikroskopom: (a) u svijetlom polju i (b) u polariziranoj svjetlosti. Faze razvoja stanica obuhvaćaju: postkambijski rast (PC), odlaganje sekundarnog sloja stijenke i ligniﬁ kacija (SW) te odrasle stanice (MT) proizvedene u sezoni. Figure 3 Cells in different phases of formation observed under a light microscope: (a) bright ﬁ eld and (b) polarized light. Phases of cell formation: post-cambial growth (PC), secondary cell wall deposition and ligniﬁ cation (SW) and mature cells (MT) formed in the season. Figure 3 Cells in different phases of formation observed under a light microscope: (a) bright ﬁ eld and (b) polarized light. Phases of cell formation: post-cambial growth (PC), secondary cell wall deposition and ligniﬁ cation (SW) and mature cells (MT) formed in the season. 3 RESULTS AND DISCUSSION 3. REZULTATI I DISKUSIJA Figure 4 Milestones of wood formation in 2008 and 2009 shown for days of the year (DOY) and calendar dates, with variation (± standard deviation). Slika 4. Prekretnice u stvaranju drva u 2008. i 2009. godini prikazane za dane u godini (DOY) i kalendarske datume, s varijacijom (± standardna devijacija) cambial activity in Robinia pseudoacacia ended in ear- ly September; however, xylem differentiation did not end until October. Termination of cambial activity, when no wood increment was noticed, occurred from the end of September to October in Quercus robur in Czech Republic (Horaček et al, 2003). ring-porous species mainly transport water through the wide early-wood vessels of the current growth ring (Suzuki et al, 1999), early establishment of water con- ducting pathways is particularly important (Sass, 1993). Earlywood formation was completed and the ﬁ rst newly formed late-wood vessels were observed around 5 June (DOY 156.5 ± 7.7) in 2008 and around 28 May (DOY 147.9 ± 4.7) in 2009 (Fig. 4). According to the presented data the xylem growth ring formation in the studied chestnuts lasted on avera- ge 160.7 ± 13.0 days in 2008 and slightly more, 169.4 ± 8.0 days, in 2009. The duration slightly varied from tree to tree and the differences were larger in 2008 (RSD = 10%) than in 2009 (RSD = 4%). Gričar (2010) observed a transition from early- to latewood in Quercus petraea slightly earlier; i.e., in the third week of May 2008. Derr and Evert (1967) obser- ved that the period of greatest cambial activity coinci- ded with earlywood formation in Robinia pseudoaca- cia. Furthermore, Schmitt et al. (2000), who investi gated wood production in Robinia pseudoacacia near Ham- burg, Germany reported that wood formation started around 3 May, and about 70% of wood was already formed by 5 July. The duration of earlywood formation was on average 43.9 ± 8.9 days in 2008 and 48.2 ± 6.4 days in 2009. The formation of latewood lasted on average 108.7 ± 18.8 days in 2008 and 110.7 ± 7.9 days in 2009 (Table 1). Early wood was thus formed in a period that lasted less than one third of the growing season. Xylem ring in 2008 was on average 3296 ± 1514 µm wide and slightly narrower in 2009, with 3166 ± 1073 µm, although the growth season was about one week longer in 2009 (Table 1). 3 RESULTS AND DISCUSSION 3. REZULTATI I DISKUSIJA Figure 3 Cells in different phases of formation observed under a light microscope: (a) bright ﬁ eld and (b) polarized light. Phases of cell formation: post-cambial growth (PC), secondary cell wall deposition and ligniﬁ cation (SW) and mature cells (MT) formed in the season. Slika 3. Stanice u različitim fazama razvoja promatrane svjetlosnim mikroskopom: (a) u svijetlom polju i (b) u polariziranoj svjetlosti. Faze razvoja stanica obuhvaćaju: postkambijski rast (PC), odlaganje sekundarnog sloja stijenke i ligniﬁ kacija (SW) te odrasle stanice (MT) proizvedene u sezoni. Sass-Klaassen et al. (2011) studied vessel forma- tion in Quercus robur and Fraxinus excelsior (pedun- culate oak and ash) in relation to leaf phenology in 2008 in the Netherlands (52°23’N, 5°37’E). Vessel for- mation in ash started well before bud swelling and much earlier than in oak. Earlywood vessel formation in oak on average already started at the end of March and ended at the beginning of May. In ash, vessel for- Slika 3. Stanice u različitim fazama razvoja promatrane svjetlosnim mikroskopom: (a) u svijetlom polju i (b) u polariziranoj svjetlosti. Faze razvoja stanica obuhvaćaju: postkambijski rast (PC), odlaganje sekundarnog sloja stijenke i ligniﬁ kacija (SW) te odrasle stanice (MT) proizvedene u sezoni. Slika 3. Stanice u različitim fazama razvoja promatrane svjetlosnim mikroskopom: (a) u svijetlom polju i (b) u polariziranoj svjetlosti. Faze razvoja stanica obuhvaćaju: postkambijski rast (PC), odlaganje sekundarnog sloja stijenke i ligniﬁ kacija (SW) te odrasle stanice (MT) proizvedene u sezoni. DRVNA INDUSTRIJA 62 (4) 269-275 (2011) 272 ............ Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... Mar Apr May Jun Jul Aug Sep Oct DOY 80 100 120 140 160 180 200 220 240 260 280 300 Year / godina End of lignification završetak lignifikacije First latewood vessels prva pora kasnog drva Onset of lignification začetak lignifikacije Earlywood vessel expansion rasprostranjenje pora ranog drva 2009 2008 Figure 4 Milestones of wood formation in 2008 and 2009 shown for days of the year (DOY) and calendar dates, with variation (± standard deviation). Slika 4. Prekretnice u stvaranju drva u 2008. i 2009. godini prikazane za dane u godini (DOY) i kalendarske datume, s varijacijom (± standardna devijacija) Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... . Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... 3 RESULTS AND DISCUSSION 3. REZULTATI I DISKUSIJA Earlywood width on average comprised 702 ± 430 µm in 2008 and 781 ± 209 µm in 2009 and its proportion was similar in both years, with 24 ±11% in 2008 and 26 ±5% in 2009. Con- sequently, the largest part of the xylem growth ring in both years consisted of latewood, the proportion of which was 76 ±12% in 2008 and 74 ±5% in 2009 (Ta- ble 1). The variation in earlywood proportion among Xylem growth ring formation in chestnut was completed when the last formed xylem cells were com- pletely ligniﬁ ed. In both years, this was observed at approximately the same time, i.e., around 29 Septem- ber 2008 (DOY 273.9 ± 10.5) and around 26 Septem- ber 2009 (DOY 269.1 ± 4.7) (Fig. 4). Ciampi (1951) observed that ligniﬁ cation of the terminal xylem cells in Castanea sativa concluded in October. Schmitt et al. (2000) noted the end of wood formation in Robinia pseudoacacia in the second week of September. Derr and Evert (1967) reported that Table 1 Main characteristics of wood formation in 2008 and 2009 Tablica 1. Glavna obilježja stvaranja drva u 2008. i 2009. Measured parameter / Mjereni parametar 2008 2009 Duration of wood formation, days / trajanje stvaranja drva, dani 160.7 ± 13.0 169.4 ± 8.0 Duration of earlywood formation, days / trajanje stvaranja ranog drva, dani 43.4 ± 9.3 48.2 ± 6.4 Duration of latewood formation, days / trajanje stvaranja kasnog drva, dani 117.2 ± 10.1 121.2 ± 5.0 Total tree-ring width, µm / širina cijeloga goda, µm 3295.6 ± 1513.6 3166.1 ± 1072.8 Proportion of latewood, % / udjel kasnog drva, % 75.7 ± 11.2 74.4 ± 4.7 Table 1 Main characteristics of wood formation in 2008 and 2009 Tablica 1. Glavna obilježja stvaranja drva u 2008. i 2009. Measured parameter / Mjereni parametar 2008 2009 Duration of wood formation, days / trajanje stvaranja drva, dani 160.7 ± 13.0 169.4 ± 8.0 Duration of earlywood formation, days / trajanje stvaranja ranog drva, dani 43.4 ± 9.3 48.2 ± 6.4 Duration of latewood formation, days / trajanje stvaranja kasnog drva, dani 117.2 ± 10.1 121.2 ± 5.0 Total tree-ring width, µm / širina cijeloga goda, µm 3295.6 ± 1513.6 3166.1 ± 1072.8 Proportion of latewood, % / udjel kasnog drva, % 75.7 ± 11.2 74.4 ± 4.7 Table 1 Main characteristics of wood formation in 2008 and 2009 Tablica 1. 3 RESULTS AND DISCUSSION 3. REZULTATI I DISKUSIJA Glavna obilježja stvaranja drva u 2008. i 2009. Table 1 Main characteristics of wood formation in 2008 and 2009 Table 1 Main characteristics of wood formation in 2008 and 200 Tablica 1. Glavna obilježja stvaranja drva u 2008. i 2009. Tablica 1. Glavna obilježja stvaranja drva u 2008. i 2009. Measured parameter / Mjereni parametar DRVNA INDUSTRIJA 62 (4) 269-275 (2011) ACKNOWLEDGEMENTS ZAHVALE The work was supported by PRAL 2003/53 of the Latium Region and by Comune del Soriano nel Cimino (Italy), LLP ERASMUS bilateral agreement between the University of Ljubljana and Tuscia Uni- versity, as well as programmes P4-0015 and P4-0107 and project L7-2393 funded by the Slovenian Research Agency (Ministry of Higher Education, Science and Technology). We thank Angela Bistoni, Manuel Agru- mi, Luka Krže and Matej Vovk for their help in the ﬁ eld and laboratory. The morphology of cells and the structure of the xylem growth ring are determined during wood forma- tion. All this crucially affects wood properties. Chest- nut is a typical representative of ring-porous wood spe- cies, with large earlywood vessels (mean diameters around 250 µm, range 150-300 µm), a more or less abrupt transition from early- to latewood, and small latewood vessels (mean diameters around 65 µm, range 36-125 µm) (Wagenführ, 1996). The morphological cell structure of chestnut is comparable to that of oak (e.g., Quercus petraea and Quercus robur). Chestnut and oak can be differentiated on the basis of the size of rays, which are exclusively uniseriate in chestnut and of two sizes, uniseriate and over 10 cells wide ones, in oaks (Schweingruber, 1990; Nardi Berti, 2006). The oven-dry density of chestnut wood is 530-590 kg/m3 (Wagenführ, 1996) and in ring porous wood species it mainly depends on the proportion of earlywood and latewood and increases with increasing ring width (Kollmann and Cote, 1968). 5 REFERENCES 5. LITERATURA 1. Barbaroux C.; Bréda, N., 2002. Contrasting distribution and seasonal dynamics of carbohydrate reserves in stem wood of adult ring-porous sessile oak and diffuse-porous beech trees. Tree Physiology 22: 1201-1210. 2. Ciampi, C., 1951: Evoluzione della cerchia in Castanea sativa Mill. Giornale Botanico Italiano 58 (2): 271-292, http://dx.doi.org/10.1080/11263505109431515 2. Ciampi, C., 1951: Evoluzione della cerchia in Castanea sativa Mill. Giornale Botanico Italiano 58 (2): 271-292, http://dx.doi.org/10.1080/11263505109431515 3. Conedera, M.; Manetti, M. C.; Guidici, F.; Amorini, E.. 2004. Distribution and economic potential of the Sweet chestnut (Castanea sativa Mill.) in Europe. Ecologia Mediterranea 30: 179-193. 4. Čufar, K.; Cherubini, M.; Gričar, J.; Prislan, P.; Spina, S.; Romagnoli, M., 2011: Xylem and phloem formation in chestnut (Castanea sativa Mill.) during the 2008 growing season. Dendrochronologia, 29: 127-134, http://dx.doi.org/10.1016/j.dendro.2011.01.006 The observed differences in the wood formation dynamics in chestnut in 2008 and 2009 did not seem to have a major impact on wood quality as characterized by tree-ring widths and latewood percentages. The climatic situation in the two years seemed to be similar (Fig. 1). However, to evaluate the effect of climate on wood formation and quality, we would need to explore in detail the climate-wood formation rela- tionships with speciﬁ c models, taking into account the climatic parameters that could affect each step of wood formation. A longer time series of wood formation would be necessary for this purpose. 5. Derr, W.F.; Evert, R. F., 1967: The cambium and seasonal development of the phloem in Robinia pseudoacacia. Amer. J. Bot. 54(2): 147-153, http://dx.doi.org/10.2307/2440792 6. Fonti, P.; Macchioni, N., 2003: Ring shake in chestnut: anatomical description, extent and frequency of failures. Ann. For. Sci. 60: 403-408, http://dx.doi.org/10.1051/forest:2003032 7. Fonti, P.; Solomonoff, N.; Garcia-Gonzalez, I., 2007: Earlywood vessels of Castanea sativa record temperature before their formation. New Phytol. 173, 562-570, http://dx.doi.org/10.1111/j.1469-8137.2006.01945.x DRVNA INDUSTRIJA 62 (4) 269-275 (2011) 273 Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... ....... the trees was much higher in 2008 (RSD = 46%) than in 2009 (RSD = 18%). on wood structure as characterized by tree-ring widths and earlywood/latewood percentages. We observed small differences in the timing of wood formation phases and duration of wood produc- tion between 2008 and 2009, and a comparable aver- age width of tree-rings formed in the two years. The observed similarity in ring width and earlywood/late- wood proportions are also expected to be reﬂ ected in a similar wood density. Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... 23. Schmitt, U.; Möller, R.; Eckstein, D., 2000: Seasonal wood formation dynamics of beech (Fagus sylvatica L.) and black locust (Robinia pseudoacacia L.) as deter- mined by the “pinning” technique. J. Appl. Bot. 74: 10- 16. 13. Murakami Y., Funada, R.; Sano, Y.; Ohtani, J., 1999: The differentiation of contact cells and isolation cells in the xylem ray parenchyma of Populus maximowiczii. Ann. Bot. 84: 429–435, http://dx.doi.org/10.1006/anbo.1999.0931 24. Schweingruber, F. H., 1990. Microscopic wood anatomy. Birmensdorf: Eidgenössische Anstalt für das Forstliche Versuchswesen. 14. Nardi Berti, R., 2006: La struttura anatomica del legno ed il riconoscimento dei legnami italiani di più corrente im- piego. 2. ed. CNR IVALSA. 25. Servizio idrograﬁ co 2009, URL: <http://www.idrograﬁ - co.roma.it/default.aspx> (date of accession 26 July 2011). 15. Piovesan, G.; Biondi, F.; Di Filippo, A.; Alessandrini, A.; Maugeri, M., 2008: Drought-driven growth reduction in old beech (Fagus sylvatica) forests of the central Apen- nines, Italy. Glob. Change Biol. 14 (6): 1265-1281, http://dx.doi.org/10.1111/j.1365-2486.2008.01570.x 26. Spina S.; Romagnoli M., 2010: Characterization of ring shake defect in chestnut wood in the Lazio Region (Ita- ly), Forestry 83: 315-327, http://dx.doi.org/10.1093/forestry/cpq014 16. Prislan, P.; Koch, G.; Čufar, K.; Gričar, J.; Schmitt, U., 2009: Topochemical investigations of cell walls in devel- oping xylem of beech (Fagus sylvatica L.). Holzforsc- hung, 63 (4): 482-490, http://dx.doi.org/10.1515/HF.2009.079 27. Suzuki, M.; Yoda, K.; Suzuki, H., 1996: Phenological comparison on the onset of vessel formation between ring-porous and diffuse-porous deciduous trees in a Japa- nese temperate forest. IAWA J. 17: 431-444. 17. Romagnoli, M.; Nocetti, M.; Sarlatto, M.; Evangelistella, L., 2004: Dendrochronological assessment of chestnut (Castanea sativa Mill.) for dating purposes in central It- aly. Dendrochronologia 21(3): 117-130. 28. Terashima, N., 2000: Formation and ultrastructure of li- gniﬁ ed plant cell walls. In: New Horizons in Wood Ana- tomy. Eds. Kim, Y.S. Chonnam National University Press, Kwangju. pp. 169-180. 18. Romagnoli, M.; Nocetti, M.; Sarlatto, M., 2005: Datazio- ne dendrocronologica di strutture lignee nei tetti in Italia centro-meridionale. In: Proceedings of the International Conference “Conservation of historic Wooden Struc- tures”. 22-27 Febbraio, Firenze: 19-24. 29. Wagenführ, R., 1996: Holzatlas. 4th edition. Leipzig: Fa- chbuchverlag. 30. Werf, van der G.W.; Sass-Klaassen, U.; Mohren, G.M.J., 2007: The impact of the 2003 summer drought on the intra-annual growth pattern of beech (Fagus sylvatica L.) and oak (Quercus robur L.) on a dry site in the Netherlan- ds. Dendrochronologia 25: 103-112. 19. Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... Romagnoli, M., 2007: Scienze del Legno nel territorio Tuscia tra arte archeologia e ambiente: casi di studio. In- formazioni, periodico dell’Ente Provincia di Viterbo – Ufﬁ cio Documentazione e Valorizzazione delle Risorse Territoriali, 36-46 20. Rossi, S.; Anfodillo, T.; Menardi, R., 2006: Trephor: a new tool for sampling microcores from tree stems, IAWA J. 27: 89-97. Prof. KATARINA ČUFAR, Ph.D. University of Ljubljana, Biotechnical Faculty, Department of Wood Science and Technology, Rožna dolina, Cesta VIII/34, SI-1000 Ljubljana, SLOVENIA e-mail: katarina.cufar@bf.uni-lj.si University of Ljubljana, Biotechnical Faculty, Department of Wood Science and Technology, Rožna dolina, Cesta VIII/34, SI-1000 Ljubljana, SLOVENIA e-mail: katarina.cufar@bf.uni-lj.si University of Ljubljana, Biotechnical Faculty, Department of Wood Science and Technology, Rožna dolina, Cesta VIII/34, SI-1000 Ljubljana, SLOVENIA e-mail: katarina.cufar@bf.uni-lj.si University of Ljubljana, Biotechnical Faculty, Department of Wood Science and Technology, Rožna dolina, Cesta VIII/34, SI-1000 Ljubljana, SLOVENIA e-mail: katarina.cufar@bf.uni-lj.si Department of Wood Science and Technology, 22. Sass-Klaassen, U.; Sabajo, C. R.; den Ouden, J., 2011: Vessel formation in relation to leaf phenology in pedun- culate oak and European ash. Dendrochronologia 29: 171-175, http://dx.doi.org/10.1016/j.dendro.2011.01.002 Corresponding address: 21. Sass, U., 1993. Die Gefäße der Buche als ökologische Variable – Bildanalytische Erfassung, dendroklimatolo- gische Prüfung, ökologische Bewertung. Doctoral disser- tation. Hamburg, Universität Hamburg. 4 CONCLUSIONS 4. ZAKLJUČCI 8. Gričar, J., 2010: Xylem and phloem formation in Sessile oak from Slovenia in 2007. Wood Research, 55 (4): 15- 22. The expansion of ﬁ rst formed earlywood vessels, the onset of cell wall development and ligniﬁ cation that ﬁ rst occurs in vessels and in tissues around them, the occurrence of the ﬁ rst latewood vessels and end of ligniﬁ cation of terminal xylem cells are crucial phases of wood formation in Castanea sativa. They helped us to link the dynamics of wood formation and its effect on wood structure. The variation in dynamics between the two years, 2008 and 2009, was generally smaller than the variation among the 10 study trees. Wood for- mation on average lasted about six months in both studied years and average tree ring widths were above 3 mm. Earlywood on average formed about one quarter and latewood about three quarters of xylem rings in both years. The recorded differences in timing of wood formation phases did not seem to have a major impact 9. Grünwald, C.; Ruel, K.; Schmitt, U., 2002: Differentia- tion of xylem cells in rolC transgenic aspen trees – a study of secondary wall development. Ann. For. Sci. 59: 679-685, http://dx.doi.org/10.1051/forest:2002056 10. Horáček, P.; Šlezingerová, J.; Gandelová, L., 2003. Anal- ysis of cambial activity and formation of wood in Quer- cus robur. Journal of Forestry Science. 2003. 49, 9: 412- 418. 11. Kollmann F.; Cote, W., 1968: Principles of Wood Science and Technology. Vol. I: Solid Wood. Berlin, Heidelberg, New York: Springer Verlag. 12. Marion, L.; Gričar, J., Oven, P., 2007: Wood formation in urban Norway maple trees studied by the micro-coring method. Dendrochronologia 25 (2): 97-102, http://dx.doi.org/10.1016/j.dendro.2007.05.001 DRVNA INDUSTRIJA 62 (4) 269-275 (2011) DRVNA INDUSTRIJA 62 (4) 269-275 (2011) 274 Romagnoli, Cherubini, Prislan, Gričar, Spina, Čufar: Main Phases of Wood... DRVNA INDUSTRIJA 62 (4) 269-275 (2011) 275
https://openalex.org/W1996306783	https://escholarship.org/content/qt58x4k1r2/qt58x4k1r2.pdf?t=qan99z	English	null	Identification of novel post-transcriptional features in olfactory receptor family mRNAs	Nucleic acids research	2,015	cc-by	10,711	UC San Diego UC San Diego Previously Published Works UC San Diego UC San Diego UC San Diego Previously Published Works Title Identification of novel post-transcriptional features in olfactory receptor family mRN Permalink https://escholarship.org/uc/item/58x4k1r2 Journal Nucleic Acids Research, 43(19) ISSN 0305-1048 Authors Shum, Eleen Y Espinoza, Josh L Ramaiah, Madhuvanthi et al. Publication Date 2015-10-30 DOI 10.1093/nar/gkv324 Peer reviewed UC San Diego UC San Diego Previously Published Works Title Identification of novel post-transcriptional features in olfactory receptor family mRN Permalink https://escholarship.org/uc/item/58x4k1r2 Journal Nucleic Acids Research, 43(19) ISSN 0305-1048 Authors Shum, Eleen Y Espinoza, Josh L Ramaiah, Madhuvanthi et al. Publication Date 2015-10-30 DOI 10.1093/nar/gkv324 Peer reviewed UC San Diego UC San Diego Previously Publis Title Identification of novel post-transcriptional fe Permalink https://escholarship.org/uc/item/58x4k1r2 Journal Nucleic Acids Research, 43(19) ISSN 0305-1048 Authors Shum, Eleen Y Espinoza, Josh L Ramaiah, Madhuvanthi et al. Publication Date 2015-10-30 DOI 10.1093/nar/gkv324 Peer reviewed ABSTRACT ceptors are thought to be derived from ancestral fish and ev- idence suggests that class-II receptors evolved from ances- tral amphibians (3). These two Olfr gene classes are respon- sible for generating receptors that detect different odorants; e.g. it has been shown that class-I OLFRs preferentially de- tect predator-related odorants (4). ceptors are thought to be derived from ancestral fish and ev- idence suggests that class-II receptors evolved from ances- tral amphibians (3). These two Olfr gene classes are respon- sible for generating receptors that detect different odorants; e.g. it has been shown that class-I OLFRs preferentially de- tect predator-related odorants (4). Olfactory receptor (Olfr) genes comprise the largest gene family in mice. Despite their importance in ol- faction, how most Olfr mRNAs are regulated remains unexplored. Using RNA-seq analysis coupled with analysis of pre-existing databases, we found that Olfr mRNAs have several atypical features suggest- ing that post-transcriptional regulation impacts their expression. First, Olfr mRNAs, as a group, have dra- matically higher average AU-content and lower pre- dicted secondary structure than do control mRNAs. Second, Olfr mRNAs have a higher density of AU- rich elements (AREs) in their 3′UTR and upstream open reading frames (uORFs) in their 5 UTR than do control mRNAs. Third, Olfr mRNAs have shorter 3′ UTR regions and with fewer predicted miRNA- binding sites. All of these novel properties correlated with higher Olfr expression. We also identiﬁed strik- ing differences in the post-transcriptional features of the mRNAs from the two major classes of Olfr genes, a ﬁnding consistent with their independent evolu- tionary origin. Together, our results suggest that the Olfr gene family has encountered unusual selective forces in neural cells that have driven them to acquire unique post-transcriptional regulatory features. In support of this possibility, we found that while Olfr mRNAs are degraded by a deadenylation-dependent mechanism, they are largely protected from this de- cay in neural lineage cells. p ( ) Olfr genes are regulated in a unique manner. Only a sin- gle Olfr gene allele from ∼1000 Olfr gene choices is selected to be expressed in a given OSN (5–8). The Olfr gene se- lected by each OSN is not only responsible for detecting odorants in the olfactory epithelium (OE), but it also di- rects the axons of OSNs that express the same Olfr gene to converge into the same glomerulus in the olfactory bulb (9,10). C⃝The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT By controlling both axon guidance and receptor ex- pression, this ‘1-receptor, 1-cell’ rule provides the founda- tion by which the olfactory system distinguishes different odorants (1,3,11). How precisely this 1-receptor, 1-cell rule is implemented at the molecular level remains enigmatic. In principal, it appears to be largely dictated by a selec- tive transcriptional mechanism in which one Olfr gene is transcriptionally activated and all other Olfr genes are tran- scriptionally repressed in a given OSN. Likely to be involved are transcription factors that regulate Olfr gene expression, including the LHX2 LIM/homeobox transcription factor and members of the OLF-1/EBF (O/E) helix-loop-helix (HLH) family (12–14). The regulation of Olfr gene choice may also be dictated by epigenetic signatures that correlate with transcriptional activity (15,16).i While considerable progress has been defining tran- scriptional mechanisms acting on Olfr genes, little is known about post-transcriptional mechanisms regulating Olfr mRNAs. This is a large gap in the field given that post-transcriptional regulation has the potential to be crit- ical for regulation of OLFR expression. For example, se- lective RNA decay mechanisms could contribute to the ‘1- receptor, 1-cell rule’ by degrading non-selected Olfr mR- NAs that are expressed from incompletely silenced Olfr genes. Post-transcriptional mechanisms also have the po- tential to control Olfr mRNA levels during OSN develop- ment, as well as in response to acute exposure to odorants. To whom correspondence should be addressed: Tel: +1 858 822 4819; Fax: +1 858 822 4701; Email: mfwilkinson@ucsd.edu †The authors contributed equally to the paper as first authors. Identiﬁcation of novel post-transcriptional features in olfactory receptor family mRNAs Eleen Y. Shum1,†, Josh L. Espinoza1,†, Madhuvanthi Ramaiah1 and Miles F. Wilkinson1,2, 1Department of Reproductive Medicine, School of Medicine, University of California, San Diego, La Jolla, CA 92093-0695, USA and 2Institute of Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA Received April 25, 2014; Revised March 27, 2015; Accepted March 30, 2015 Powered by the California Digital Library University of California eScholarship.org 9314–9326 Nucleic Acids Research, 2015, Vol. 43, No. 19 doi: 10.1093/nar/gkv324 Published online 23 April 2015 INTRODUCTION Olfactory receptors (OLFRs) are G protein-coupled recep- tors (GPCRs) essential for odor detection in olfactory sen- sory neurons (OSNs). These receptors are encoded by the largest gene family in mice, occupying ∼2% of the protein- coding genome (1–3). Olfr genes are divided into 2 classes, each of which has a different evolutionary origin: class-I re- Nucleic Acids Research, 2015, Vol. 43, No. 19 9315 Post-transcriptional regulation is typically directed to- ward the 5′ and 3′ untranslated regions (UTRs) of mRNAs, as they house a plethora of cis elements that impact mRNA stability and translation. For example, UTRs harbor se- quence motifs and secondary structures that recruit ribo- somes and RNA-binding proteins (RBPs) to govern rates of mRNA decay and translation (17–19). Also recruited to UTRs, particularly to 3′ UTRs, are microRNAs (miRNAs), which are short RNAs that elicit translational repression, mRNA destabilization, or both (20). None of these features have been investigated in Olfr mRNAs. encompassing Bowtie2, Tophat2 and Cufflinks programs (29,30) using the ‘novel gene discovery mode’ as outlined in the Tuxedo suite program manuals (30). Specifically us- ing the CuffCompare program, we extracted information such as the nearest Ensembl reference ID, exon start/end positions, chromosome, gene direction and gene symbol from each gene entry. Next, we fetched the CDS of all en- tries using the nearest reference IDs from the UCSC Ta- ble Browser from the NCBIm37/mm9 genome. Using the exon start/end positions for each reference ID entry, we extracted the exon sequences from the mm9 genome, and ligated them together in silico for each transcript. For each entry, the entire transcript sequence is subtracted from the known CDS sequence obtained above to identify the 5′ and 3′ UTRs. If a 30-nucleotide sequence was matched for the 5′ and 3′ peripheral regions of the nearest reference coding sequence with the corresponding in silico spliced transcript then the transcript was considered complete. For the com- plete transcripts, the 5′ and 3′ regions flanking the coding se- quence were considered 5′ and 3′ UTRs, respectively. Intron sequences were extracted by using the regions between the exon junctions for each transcript. The second set of Olfr transcripts were provided by the supplemental data sets pro- vided by Ibarra-Soria et al. (28). Only transcripts with both a detectable 5′ UTR and 3′UTR were considered in subse- quent analysis. GC-content, ARE and miRNA site search Once extracted, the lengths and GC-content were calculated for 5′/3′ UTRs, coding sequences and introns. To calculate global GC-content for Olfr, control and GPCR groups we created sets for every position, ranging from −50 to +50, flanking the splice-site of each intron. The GC-content was calculated for each of these positions to construct a general global GC analysis for each group of transcripts. To con- struct sequence logos, we used all sequences flanking splice- sites, ranging from −15 to +15, in UCB’s WebLogo appli- cation (http://weblogo.berkeley.edu/) (31). The 3′UTR se- quences generated during the annotation were used to scan for AREs. We scanned the 3′UTR sequences for the fol- lowing n-mers identified by AREsite (http://rna.tbi.univie. ac.at/cgi-bin/AREsite.cgi) (32): AUUUA and WWAUU- UAWW. W indicates either an adenine or uracil nucleotide. miRNA sites were discovered using TargetScan 6.0 (33– 36). In brief, 9 miRNA mature and whole sequences were scanned along all genes from the three gene groups to find putative miRNA binding sites. Compiling the Olfr mRNA transcriptome RNA-seq analysis was performed on two 8 week-old C57BL/6 female OE littermates, who were housed in ac- cordance with UCSD IACUC policies. In brief, dissected OE RNA was isolated using TriZOL (Life Technologies), followed by a secondary purification step using RNeasy columns (Qiagen). RNA-seq was performed at the UCSD Biogem Core. Total RNA was assessed for quality using an Agilent Bioanalyzer, and samples determined to have an RNA Integrity Number (RIN) of at least 8 or greater were used to generate RNA libraries using Illumina’s TruSeq RNA Sample Prep Kit following manufacturer specifica- tions, with the RNA fragmentation time adjusted to 5 min. 1 ug of total RNA was used per sample. RNA libraries were multiplexed and sequenced at 10 pM with 50 base- pair (bp) single end reads to a depth of approximately 40 million reads per sample on an Illumina HiSeq2000. The OE transcriptome was extracted using the Tuxedo suite, MATERIALS AND METHODS Compiling the Olfr mRNA transcriptome Compiling the Olfr mRNA transcriptome INTRODUCTION g f To address post-transcriptional regulatory mechanisms that regulate Olfr transcripts, it is critical to first define Olfr mRNA sequences. Zhang et al. and Shiao et al. detected the expression of ∼800–1200 Olfr mRNAs in the OE using a custom microarray and RNA-seq, respectively, but they did not define the 5′ and 3′ termini of these transcripts or iden- tifying alternative isoforms (21,22). Other studies have used transcription start-site mapping to identify the 5′ UTR and promoter regions of ∼200 Olfr mRNAs (23–26) and one study screened cDNA libraries to identify promoter and 5′ UTR sequences of ∼400 Olfr mRNAs (27). In our study, we employed RNA-seq analysis to analyze Olfr mRNAs with respect to their post-transcriptional features. Our analysis revealed that Olfr mRNAs tend to have several unique fea- tures, including a short 3′ UTR, high AU-content, and a high density of AREs and uORFs. After our manuscript de- scribing this work was submitted, another paper was pub- lished that characterized Olfr mRNAs using RNA-seq anal- ysis (28). While this Ibarra-Soria et al. paper did not focus on post-transcriptional features, it provided us an opportu- nity to examine such features using another Olfr data set. As described herein, the data sets from this paper corrobo- rated what we determined with our Olfr data sets. In sum- mary, we have uncovered unusual post-transcriptional fea- tures that are unique to the Olfr gene family, thereby shining light into how these genes are potentially regulated in OSNs. In support, we provide in vitro evidence that Olfr mRNAs are post-transcriptionally regulated in a neuronal-specific manner. GC-content, ARE and miRNA site search Secondary structure minimum free energy To select the minimum free energy of the 5′UTR sequences, we used University of Vienna’s RNAfold Web Server (http: //rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) (37) to calculate the folding energy. The 5′UTR sequences used were gener- ated from the RNA-seq reads, Ensembl reference IDs, and the annotation algorithms described above. In situ hybridization In situ hybridization was performed as previously described previously (38) using 10 m fresh frozen OE cryosections and LNA probes against miR-741, miR-9 and miR-128 (Ex- igen). Olfr mRNAs tend to have short 3′ UTRs To understand how Olfr mRNA characteristics potentially impact their expression, we analyzed Olfr mRNAs we de- fined by RNA-seq (Figure 2, Supplementary Figure S1A– B, Supplementary Table S3), as Olfr trancripts on publi- cally available databases such as Ensembl and RefSeq have inaccuracies. Unless otherwise noted, all data analysis be- low was performed in Olfr mRNAs we identified through our RNA-seq analysis. As a basis of comparison, we exam- ined two other gene groups: (i) Gpcr genes encoding 92 non- OLFR GPCR proteins defined by the International Union of Basic and Clinical Pharmacology Database [IUPHAR- DB] (43) that we found were significantly expressed in the OE, based on our RNA-seq analysis, and (ii) Ctrl genes, a randomly selected group of 587 non-Gpcr genes expressed in the OE. Programming language used Algorithms were generated with the Python programming language (version 2.7.6). Data analysis was performed using both R and GraphPad PRISM. This analysis revealed unique features of Olfr mRNAs as a group. One unusual feature of Olfr mRNAs is 3′ UTR length. The average 3′ UTR length of Olfr mRNAs in our data set (‘Olfr A’, 1094 nt) is dramatically shorter than that of the Ctrl (1895 nt) and Gpcr (1800 nt) group mRNAs (Fig- ure 2A; P < 0.0001). Olfr mRNAs defined by Ibarra-Soria et al. (28) (‘Olfr-B’) also had a shorter 3′ UTR length (1439 3′UTR cloning, cell culture and luciferase assay These reporter constructs were trans- fected by Lipofectamine 2000 reagent (Life Technologies) into P19 and Neuro2A cells. The P19 cells were cultured in 10% fetal calf serum (Stem Cell Technologies) and MEM media (Invitrogen); Neuro2A cells were cultured in 10% fetal calf serum (Stem Cell Technologies) and DMEM media (Invitrogen). Also transfected into these cells was the CCR4B expression vector (kindly provided by Jens Lykke-Andersen [UCSD]) and the Ccr4b siRNA and the ONTARGETplus siRNA control (ThermoScientific). The Firefly Renilla luciferase reporter was co-transfected as an internal control. Luciferase activity was assayed as previously described (38) using the Dual Luciferase Assay System (Promega). Cellular RNA was harvested using TriZOL (Invitrogen), followed by iScript Reverse Tran- scription Kit and SYBR Green qPCR Reagent (BioRad), all according to manufacturer’s protocol. The level of Ccr4b mRNA was measured using the following qPCR primers: UCSD8023: CGCGGAGGAGAATGAGACTA and UCSD8024: GTGCAGTGCTGTCAAGTGTG. The endogenous RNA control Rpl19 was used and was am- plified using the following qPCR primers: UCSD4667: CTGAAGGTCAAAGGGAATGTG and UCSD4668: GGACAGAGTCTTGATGATCTC. ( ) We observed that class-I Olfr genes tend to be expressed at strikingly lower level than most class-II Olfr genes (Fig- ure 1C–E). A likely explanation is that class-I Olfr genes are selected to be expressed less often than are class-II Olfr genes in individual OSNs, as class-I Olfr genes com- prise only ∼10% of all Olfr genes (40). In further support of this possibility, class-I OLFR-expressing OSNs are only found in the dorsal zone of the OE, whereas class-II OLFR- expressing OSNs are present in virtually all regions of the OE (41,42). A non-mutually exclusive explanation for the lower levels of class-I Olfr mRNAs in the OE is that class- I Olfr genes are transcribed at a lower rate per OSN than are class-II Olfr genes. Since class-I Olfr genes have a dis- tinct evolutionary origin to that of class-II Olfr genes (3), they may have distinct promoters that support lower levels of transcription than do class-II promoters. Finally, it is also possible that class-I Olfr mRNAs are intrinsically less stable than are class-II Olfr mRNAs. Identifying uORFs The 5′ UTR sequences identified through our annotation process (described above when compiling Olfr transcript above) were used to scan for upstream open reading frames that contained particular nucleotides from the Kozak con- sensus sequence critical for initiating translation (39). In particular, we only considered uORFs ≥30 nt long that had a purine at the −3 position or a guanine at the +1 posi- tion (relative to the A in the AUG initiation codon [+1]). To reduce the probability of identifying uORFs that can re- initiate translation (and thus escape NMD), one additional criteria is the uORF must not contain the main open read- ing frame. 3′UTR cloning, cell culture and luciferase assay The 3′ UTRs of Olfr212, Olfr1226 and Olfr1242 were amplified by PCR from adult OE cDNA and cloned 9316 Nucleic Acids Research, 2015, Vol. 43, No. 19 9316 downstream of the Firefly luciferase ORF (at the SpeI and HindIII restriction enzyme sites) in the pMIR-REPORT vector (Ambion). These reporter constructs were trans- fected by Lipofectamine 2000 reagent (Life Technologies) into P19 and Neuro2A cells. The P19 cells were cultured in 10% fetal calf serum (Stem Cell Technologies) and MEM media (Invitrogen); Neuro2A cells were cultured in 10% fetal calf serum (Stem Cell Technologies) and DMEM media (Invitrogen). Also transfected into these cells was the CCR4B expression vector (kindly provided by Jens Lykke-Andersen [UCSD]) and the Ccr4b siRNA and the ONTARGETplus siRNA control (ThermoScientific). The Firefly Renilla luciferase reporter was co-transfected as an internal control. Luciferase activity was assayed as previously described (38) using the Dual Luciferase Assay System (Promega). Cellular RNA was harvested using TriZOL (Invitrogen), followed by iScript Reverse Tran- scription Kit and SYBR Green qPCR Reagent (BioRad), all according to manufacturer’s protocol. The level of Ccr4b mRNA was measured using the following qPCR primers: UCSD8023: CGCGGAGGAGAATGAGACTA and UCSD8024: GTGCAGTGCTGTCAAGTGTG. The endogenous RNA control Rpl19 was used and was am- plified using the following qPCR primers: UCSD4667: CTGAAGGTCAAAGGGAATGTG and UCSD4668: GGACAGAGTCTTGATGATCTC. some of these Olfr genes to accurately determine their 5′ and 3′ termini (data not shown). To exclusively annotate Olfr mRNAs that encode functional proteins, we only ana- lyzed transcripts with RNA-seq reads matching the coding sequence (CDS) of known OLFR proteins, as defined in the Ensembl database. Using this criterion, we identified 805 full-length mRNAs encoded by 554 Olfr genes (Supplemen- tary Table S1). The alternative isoforms included those with different 5′ and 3′ UTR regions and those generated by al- ternative splicing (Supplementary Table S2). The Olfr tran- scripts we identified emanate from Olfr genes distributed on the various chromosomes known to have Olfr gene clusters (Figure 1B; 27), suggesting that our analysis was not biased for a particular sector of the genome. Analysis of the reads corresponding to individual Olfr genes showed they exhib- ited considerable heterogeneity in expression (Figure 1C and D), confirming findings from other studies on Olfr mR- NAs (27,28). downstream of the Firefly luciferase ORF (at the SpeI and HindIII restriction enzyme sites) in the pMIR-REPORT vector (Ambion). RESULTS AND DISCUSSION Genome-wide identification of class I and II Olfr transcripts Olfr transcripts were defined using RNA-seq data from adult mice OE as outlined in Figure 1A. We detected reads from 1101 Olfr genes, but there were insufficient reads from Genome-wide identification of class I and II Olfr transcripts Olfr transcripts were defined using RNA-seq data from adult mice OE as outlined in Figure 1A. We detected reads from 1101 Olfr genes, but there were insufficient reads from Nucleic Acids Research, 2015, Vol. 43, No. 19 9317 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X 0 50 100 150 200 250 Chromosome # # of Olfr Olfr Observed Olfr Known –4 –2 0 2 4 6 8 OLFRs from Chr1-ChrX log2(FPKM) Chr7 –10 –5 0 5 10 log2(FPKM) Class II Olfr Class I Olfr Olfr543-692 Olfr along Chr7 Olfr693-518 B C D E RNA-seq raw reads Tophat/Bowtie Cufflinks Olfr Transcript Start/Stop Sites In silico Translation for largest ORF, compare with known CDS Annotation of 5'UTR, CDS, 3'UTR sequences A I Olfr II Olfr –10 –5 0 5 10 log2(FPKM) ** Figure 1. RNA-seq analysis of Olfr mRNAs in the mouse adult OE. (A) Schematic of workflow used to assemble the Olfr transcriptome. (B) Full-length Olfr mRNA transcripts detected by our RNA-seq analysis versus all known Olfr genes in each mouse chromosome. (C) Bar graph of the expression level of individual Olfr genes plotted sequentially along all mouse chromosomes, expressed as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). (D) Bar graph displaying the expression level of individual Olfr genes along chromosome 7. (E) Boxplot of average expression level of class-I versus -II Olfr transcripts. P < 0.0001. RESULTS AND DISCUSSION RNA-seq raw reads Tophat/Bowtie Cufflinks Olfr Transcript Start/Stop Sites In silico Translation for largest ORF, compare with known CDS Annotation of 5'UTR, CDS, 3'UTR sequences A A Olfr Transcript Start/Stop Sites –4 –2 0 2 4 6 8 OLFRs from Chr1-ChrX log2(FPKM) Chr7 C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X 0 50 100 150 200 250 Chromosome # # of Olfr Olfr Observed Olfr Known B B C OLFRs from Chr1-ChrX D –10 –5 0 5 10 log2(FPKM) Class II Olfr Class I Olfr Olfr543-692 Olfr along Chr7 Olfr693-518 D E I Olfr II Olfr –10 –5 0 5 10 log2(FPKM) E Figure 1. RNA-seq analysis of Olfr mRNAs in the mouse adult OE. (A) Schematic of workflow used to assemble the Olfr transcriptome. (B) Full-length Olfr mRNA transcripts detected by our RNA-seq analysis versus all known Olfr genes in each mouse chromosome. (C) Bar graph of the expression level of individual Olfr genes plotted sequentially along all mouse chromosomes, expressed as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). (D) Bar graph displaying the expression level of individual Olfr genes along chromosome 7. (E) Boxplot of average expression level of class-I versus -II Olfr transcripts. P < 0.0001. nt; P < 0.0001) (Supplementary Figure S1C). Both class-I and -II Olfr mRNAs had shorter 3′ UTRs than control gene groups, but was most evident for the former (786 nt and 1121 nt average 3′ UTR length, respectively; P < 0.05; Fig- ure 2B and Supplementary Table S3). The longer average Olfr mRNA length determined by Ibarra-Soria et al. com- pared to that found in our study could be due to variabil- ity in sampling conditions or transcriptome assembly algo- rithm differences, or simply because different mice have dif- ferent repertoires of Olfr mRNAs (22). For example, Ibarra- Soria et al. used both males and female mice for their anal- ysis, while our analysis used only female mice (28). Given that males and females have a vastly different profile of Olfr expression, this experimental difference could explain some transcriptome annotation differences. nt; P < 0.0001) (Supplementary Figure S1C). RESULTS AND DISCUSSION Both class-I and -II Olfr mRNAs had shorter 3′ UTRs than control gene groups, but was most evident for the former (786 nt and 1121 nt average 3′ UTR length, respectively; P < 0.05; Fig- ure 2B and Supplementary Table S3). The longer average Olfr mRNA length determined by Ibarra-Soria et al. com- pared to that found in our study could be due to variabil- ity in sampling conditions or transcriptome assembly algo- rithm differences, or simply because different mice have dif- ferent repertoires of Olfr mRNAs (22). For example, Ibarra- Soria et al. used both males and female mice for their anal- ysis, while our analysis used only female mice (28). Given that males and females have a vastly different profile of Olfr expression, this experimental difference could explain some transcriptome annotation differences. pressure on Olfr genes to evade post-transcriptional regu- lation pathways that target the 3′ UTR. For example, previ- ous work has suggested that polyadenylation site selection shifts during development and cancer progression to differ- entially expose recognition sites for RNA-binding proteins (RBPs) or miRNAs for regulatory purposes (44–49). Both RBPs and miRNAs dictate translation efficiency and RNA decay rates, and thus mechanisms that control their ability to be recruited to an mRNA are critical for the fate of an mRNA (50). Below, we consider how the unique character- istics of Olfr 3′ UTRs, including their short length, might shape the post-transcriptional fate of Olfr transcripts. We also found that there is considerably heterogeneity in the length in Olfr mRNAs encoded by different Olfr genes (Supplementary Figure S1D). This is largely attributable to the heterogeneity of 3′ UTR length, since the coding region (CDS) length of Olfr mRNAs are nearly invariant (Sup- plementary Figure S1E) and the 5′ UTR region does not p The finding that Olfr 3′ UTRs tend to be shorter than control 3′ UTRs (Figure 2A, Supplementary Figure S1C) raised the possibility that there has been strong selection 9318 Nucleic Acids Research, 2015, Vol. 43, No. 19 I II 0 500 1000 Olfr Class 5'UTR Length Ctrl Gpcr Olfr-A 0 200 400 600 800 1000 5'UTR Length n.s. Ctrl Gpcr Olfr-A 0 1000 2000 3000 4000 5000 3'UTR Length **** I II 0 1000 2000 3000 4000 5000 Olfr Class 3'UTR Length * A B C D Figure 2. Most Olfr transcripts have short 3′ UTRs. (A, B) Boxplots of 3′ UTR length. RESULTS AND DISCUSSION (C, D) Boxplots of 5′ UTR length. P < 0.05, P < 0.01, *P < 0.0001; n.s. not statistically significant. Ctrl Gpcr Olfr-A 0 1000 2000 3000 4000 5000 3'UTR Length ** A I II 0 1000 2000 3000 4000 5000 Olfr Class 3'UTR Length * B tion (44,47,52). In the case of Olfr mRNAs, we hypothesized that they were selected to harbor short 3′ UTRs to escape repression mediated by miRNAs. B A p y To test this possibility, we used the miRNA target- prediction program, TargetScan, to screen for miRNA- binding seed sequences in the Olfr mRNAs we defined as well as the data set from Ibarra-Soria et al. In particular, we used this in silico screen to examine the following miRNAs: miR-200a/miR-141a, the only miRNA known to have a role in olfactory neurogenesis (53), and eight well-studied neu- rally expressed miRNAs: miR-7, -9, -124, -128, -132, -134, -138 and let-7 (38,54–61). We found that at least 2 of these miRNAs––miR-9 and -128––are expressed in the OSN layer of the OE (Figure 3A). We functionally tested one of the Olfr 3′ UTRs (Olfr1226) with a predicted miR-9 target site. In support of the accuracy of the TargetScan algorithm, we found that the Olfr1226 3′ UTR conferred repression in re- sponse to exogenously expressed miR-9 (Figure 3B). Ctrl Gpcr Olfr-A 0 200 400 600 800 1000 5'UTR Length n.s. C 1 C I II 0 500 1000 Olfr Class 5'UTR Length ** D D Analysis of predicted target sites revealed less predicted binding sites for 8 out of the 9 miRNAs in Olfr tran- scripts compared to the Gpcr and Ctrl transcript groups (Figure 3C–D, Supplementary Table S4). As another means to analyze miRNA-regulatory potential, we examined the percentage of mRNAs in a given group predicted to be tar- geted by the nine neurally expressed miRNAs. We found that a significantly lower percentage of Olfr mRNAs had predicted target sites for these nine miRNAs than did the other mRNA groups (P < 0.0001; Figure 3C). Olfr Class Figure 2. Most Olfr transcripts have short 3′ UTRs. (A, B) Boxplots of 3′ UTR length. (C, D) Boxplots of 5′ UTR length. P < 0.05, P < 0.01, **P < 0.0001; n.s. not statistically significant. contribute much to the variation in Olfr mRNAs (Supple- mentary Figure S1F). Olfr mRNAs are AU-rich Previous studies have demonstrated that the average AU- content of mammalian mRNAs is ∼45% (18). In contrast, we found that most Olfr mRNAs have an AU-content of greater than 50%. This characteristic was evident through- out the length of Olfr mRNAs, including both UTRs and the CDS, and even the splice junction region (Figure 4A– B, Supplementary Figure S2A–C). Particularly striking was the Olfr 5′ UTR region, which had ∼60% average AU- content versus the ∼40% average AU-content of the 5′ UTR region in the Ctrl and Gpcr mRNAs (Figure 4B, P < 0.0001, Supplementary Table S5). Of note, both class-I and –II Olfr mRNAs tended to be AU-rich (Supplementary Fig- RESULTS AND DISCUSSION Another contributing factor to Olfr mRNA length heterogeneity is the difference in the average length of class-I and-II Olfr mRNAs; we found that class-I mRNAs are modestly, but significantly shorter than class-II mRNAs (Supplementary Figure S1G). g p ( ; g ) We considered the possibility that the only reason that Olfr mRNAs have fewer miRNA-target sites than control mRNAs is because their 3′ UTRs are shorter. If this was the case, then the density of miRNA-target sites should be similar between the three groups. Instead, we found that Olfr mRNAs had significantly lower density of predicted binding sites for the nine miRNAs than the control groups (Figure 3E). Thus, the low number of predicted miRNA- target sites in Olfr mRNAs is not only because they tend to have short 3′ UTRs. Together, these data are consistent with a model in which Olfr genes evolved to reduce miRNA- mediated repression by both shortening their 3′ UTRs and reducing the number of miRNA-target sites by mutation. During the course of this analysis, we noted that a relatively large percentage of Gpcr genes (>15%) contain predicted miR-7 and -128 binding sites (Figure 3C), suggesting that miR-7, -200a, -9 and -128, in particular, may be broad reg- ulators of Gpcr mRNAs. ( pp y g ) In contrast to the 3′ UTR, the 5′ UTR was not signifi- cantly different in median length in Olfr mRNAs (223 nt in Olfr-A, 241 nt in Olfr-B) as compared to either Ctrl (212 nt) or Gpcr (242 nt) mRNAs (Figure 2C). However, we found that Olfr 5′ UTRs were quite heterogeneous in length, in large part because class-I Olfr 5′ UTRs (129 nt) are, on av- erage, half the length of Class-II Olfr 5′ UTRs (240 nt, Fig- ure 2D; P < 0.01, Supplementary Table S3). Thus, Class- I Olfr 5′ UTRs tend to be much shorter than the average length of 5′ UTRs in mRNAs in general. We suggest that this likely reflects the different evolutionary origins and/or unique selection pressures exerted on class-I Olfr genes (3). 5′ UTR regions are known to be major sites for regulation of translation efficiency and RNA decay rate (19) and thus class-I Olfrs may have been selected to have short 5′ UTR regions over evolutionary time for regulatory purposes. Olfr mRNAs have few predicted binding sites for OE- expressed miRNAs (B) Dual luciferase analysis of the effect of the Olfr1226 3′ UTR in response to ectopic expression of miR-9 in Neuro2A cells. (C) The percentage of mRNAs from the indicated gene groups with predicted miRNA-binding site defined using the TargetScan algorithm. (D) Boxplot representation of the average percentage of miRNA-binding sites determined across all 9 miRNAs, determined from the data in panel C. (E) Average frequency of miRNA sites per kb of 3′UTR in each gene group. P < 0.05; ***P < 0.0001. mRNAs with low GC-content and secondary structure are thought to be more efficiently translated than those with a high GC-content (19). This is supported by genome-wide studies observing a significant correlation between 5′ UTR secondary structure folding free energy and protein abun- dance (62). While the molecular mechanism for this is not known, a likely possibility is that less structured 5′ UTRs permit more efficient scanning of the 40S ribosomal subunit as it traverses from the 5′ cap to the start AUG codon of the main ORF (63). This raises the possibility that Olfr genes have been selected to have low GC-content over evolution- ary time so that they can translate high levels of Olfr pro- teins. Because translation can protect mRNAs from decay (64), this also predicts that Olfr mRNAs harboring 5′ UTRs with low GC-content would be more stable than those with- out. In agreement with this prediction, we found a negative correlation between Olfr expression level and the 5′ UTR GC-content of Olfr mRNAs (Figure 4D). In contrast, we observed a statistically significant positive correlation be- ure S2D–F), suggesting that this quality was independently selected for during the generation of both classes of Olfr genes. g The discovery that Olfr mRNAs have a high AU-content raised the possibility that this feature is important for Olfr post-transcriptional regulation. One possibility is that this reduces secondary structure that would otherwise reduce translation efficiency or lead to steric hindrance of inter- action with RNA-binding proteins (19). To assess whether Olfr mRNAs have reduced secondary structure relative to control mRNAs, we utilized the RNAFold algorithm (37), which calculates minimum free energy (mfe), a measure of the difficulty to linearize a given sequence. Since 5′ UTR length contributes to mfe calculations, we selected 50 5′ UTRs from each mRNA group that have similar 5′ UTR length. Olfr mRNAs have few predicted binding sites for OE- expressed miRNAs We first considered the possibility that Olfr mRNAs have short 3′ UTRs to avoid repression by miRNAs. miRNAs are short (18-22 nucleotides) RNAs that bind by comple- mentarity with their mRNA targets and recruit RBPs that lead to mRNA stabilization and/or translational repres- sion (51). miRNAs typically bind to the 3′ UTR region of their target mRNAs and thus there has been consider- able interest in identifying mechanisms that dictate which 3′ UTR regions are accessible to miRNA-mediated regula- Nucleic Acids Research, 2015, Vol. 43, No. 19 9319 Ctrl miR-9 0.0 0.5 1.0 1.5 RLU Olfr1226 B D A C Ctrl Gpcr Olfr-A Olfr-B 0.0 0.5 1.0 1.5 2.0 2.5 miR sites per kb ** miR-741 miR-9 miR-128 E Ctrl Gpcr Olfr-A Olfr-B 0.0 0.1 0.2 0.3 0.4 0.5 % of Genes All Predicted Targets ** miR-7 miR-9 miR-124 miR-128 miR-132 miR-134 miR-138 miR-200a let-7 0.0 0.1 0.2 0.3 0.4 0.5 % of Genes Ctrl Gpcr Olfr-B All Predicted Targets Olfr-A 3. Olfr mRNAs tend to have few predicted target sites for neurally expressed miRNAs. (A) In situ hybridization of neurally expressed miRNAs in E. (B) Dual luciferase analysis of the effect of the Olfr1226 3′ UTR in response to ectopic expression of miR-9 in Neuro2A cells. (C) The percentage RNAs from the indicated gene groups with predicted miRNA-binding site defined using the TargetScan algorithm. (D) Boxplot representation of the ge percentage of miRNA-binding sites determined across all 9 miRNAs, determined from the data in panel C. (E) Average frequency of miRNA sites b of 3′UTR in each gene group. P < 0.05; **P < 0.0001. A miR-741 A miR-741 miR-9 miR-128 A miR-9 Ctrl miR-9 0.0 0.5 1.0 1.5 RLU Olfr1226 B C miR-7 miR-9 miR-124 miR-128 miR-132 miR-134 miR-138 miR-200a let-7 0.0 0.1 0.2 0.3 0.4 0.5 % of Genes Ctrl Gpcr Olfr-B All Predicted Targets Olfr-A B C Ctrl Gpcr Olfr-A Olfr-B 0.0 0.5 1.0 1.5 2.0 2.5 miR sites per kb ** E D Ctrl Gpcr Olfr-A Olfr-B 0.0 0.1 0.2 0.3 0.4 0.5 % of Genes All Predicted Targets ** D E Figure 3. Olfr mRNAs tend to have few predicted target sites for neurally expressed miRNAs. (A) In situ hybridization of neurally expressed miRNAs in the OE. Olfr 3′ UTRs confer stability Our finding that Olfr mRNAs tend to be AU-rich (Fig- ure 4 and Supplementary Figure S2) raised the possibil- ity that they are enriched for AU-rich binding elements (AREs). AREs are typically permutations of the core se- quence AUUUA (or, more rarely, short stretches of U) that serve as cis elements to promote the degradation of mR- NAs through a deadenylation-dependent mechanism called ARE-mediated RNA decay (AMD) (65,66).i We examined whether the unique 3′ UTR characteristics of Olfr mRNAs confer stable expression in neural cells. To test this possibility, we cloned the 3′ UTRs of two Olfr genes––Olfr1226 and Olfr1242––downstream of the fire- fly luciferase ORF in the pMIR-REPORT reporter vector (Figure 5D). We transfected these constructs into a neu- ronal cell line, Neuro2A, together with an endogenous con- trol containing a Renilla plasmid reporter and measured the stability of the encoded transcripts. We found that both the Olfr1226 and Olfr1242 3′ UTRs confer an extremely long RNA half-life in Neuro2A cells, as measured by treatment with the transcriptional inhibitor, Actinomycin D (>10 h; Figure 5E). This suggests that these 3′ UTRs confer stabil- ity to Olfr mRNA in neuronal cells. AREs have been computationally defined in mammalian transcriptomes and deposited in several databases, includ- ing ARED and AREsite (32,67). However, these databases do not contain most Olfr mRNAs, including those anno- tated recently (28). Our annotation of Olfr genes expressed in the mouse OE together with another data set provided an opportunity to fill this gap. We analyzed the average ARE density in Olfr mRNAs and found it is twice as high as in the Gpcr and Ctrl mRNA groups (Figure 5A, Supplementary Table S6). We found this to also be the case when we ana- lyzed the recently defined Olfr mRNA data set from Ibarra- Soria et al. (28) (‘Olfr-B’ in Figure 5A). Given that Olfr mR- NAs have much shorter average 3′ UTR length than these other groups (see below), this is consistent with the possi- bility that there has been strong selection pressure for en- richment of AREs to maintain regulation by ARE-binding proteins. The net result is that AREs are as prevalent in Olfr mRNAs as in the other gene groups (Figure 5B). y f A key rate-limiting step of mRNA decay is deadeny- lation (65). Olfr mRNAs have few predicted binding sites for OE- expressed miRNAs This analysis revealed that Olfr mRNAs have a strik- ingly higher average mfe compared to Gpcr and Ctrl tran- scripts (Figure 4C). 9320 Nucleic Acids Research, 2015, Vol. 43, No. 19 9320 fr mRNAs are AU-rich. (A) GC-content along exon-intron junctions (top) and intron-exon junctions (bottom). (B) Boxplot of% R. (C) Boxplot of minimal free energy (mfe) in the 5′ UTR. (D) Scatterplot and linear regression analysis of expression level ve * P < 0.05; P < 0.01; ** P < 0.0001; n.s., not statistically significant. Figure 4. Olfr mRNAs are AU-rich. (A) GC-content along exon-intron junctions (top) and intron-exon junctions (bottom). (B) Boxplot of% of AU-content in the 5′ UTR. (C) Boxplot of minimal free energy (mfe) in the 5′ UTR. (D) Scatterplot and linear regression analysis of expression level versus 5′ UTR% GC-content. * P < 0.05; P < 0.01; ** P < 0.0001; n.s., not statistically significant. Figure 4. Olfr mRNAs are AU-rich. (A) GC-content along exon-intron junctions (top) and intron-exon junctions (bottom). (B) Boxplot of% of AU-content in the 5′ UTR. (C) Boxplot of minimal free energy (mfe) in the 5′ UTR. (D) Scatterplot and linear regression analysis of expression level versus 5′ UTR% GC-content. * P < 0.05; P < 0.01; ** P < 0.0001; n.s., not statistically significant. Nucleic Acids Research, 2015, Vol. 43, No. 19 9321 Nucleic Acids Research, 2015, Vol. 43, No. 19 9321 Nucleic Acids Research, 2015, Vol. 43, No. 19 9321 ARE density raises the possibility that Olfr mRNAs are a new class of transcripts that are positively regulated by ARE-binding proteins. While AREs are normally associ- ated with decay, they can also promote mRNA stabiliza- tion. Some ARE-binding proteins serve as mRNAs stabi- lizers, including members of the embryonic lethal abnormal vision (ELAV) family member (68). At least one member of this family, HuC, is expressed in the developing OE and other neuronal tissues (69). Another family member, HuD, is neuron-specific in its expression and is responsible for regulating Gap43 mRNA, which encodes a neuron-specific protein critical for neurogenesis (70,71). This raised the pos- sibility that AREs promote the expression of transcripts in a neuronal setting. tween the GC-content of Gpcr mRNAs and their expres- sion. Olfr mRNAs have few predicted binding sites for OE- expressed miRNAs While we do not know the underlying mechanism for this, one possibility is higher GC-content alters the spec- trum of ribonucleoprotein complexes that form on the 5′ UTR of Gpcr mRNAs in a manner that favors the stability of these mRNAs. No correlation was observed for the Ctrl mRNAs, possibly because they represent a broad group of genes with confounding variables. While there are several explanations for these findings, we suggest that the most parsimonious interpretation is that Olfr mRNAs evolved to have unstructured 5′ UTRs as a means to increase their translation efficiency, which in turn increased the stability of their mRNAs. Olfr 3′ UTRs confer stability The difference in Olfr 3′ UTR sensitivity to CCR4B depletion in neuronal g ) Our finding that Olfr 3′ UTRs are both enriched in AREs and expressed in a pattern that positively correlates with 9322 Nucleic Acids Research, 2015, Vol. 43, No. 19 gure 5. Olfr mRNAs are ARE-rich. (A) ARE pentamer density measured in silico. (B) Density distribution plot of the number of AUUUA ARE pen- mers, assessed as in panel (A). (C) Scatterplots and linear regression analysis of expression level versus AUUUA pentamer density, assessed as in panel (D) Schematic of luciferase and renilla reporters used. (E,F) Two Olfr 3′ UTRs influence mRNA steady-state level and mRNA half-life in response to CR4B depletion (using a siRNA targeting CCR4B [siCcr4b]) and CCR4B overexpression (using a CCR4B expression vector). Panel E is measurement relative mRNA levels (RQ) and panel (F) is measurement of relative luciferase values (RLU). P < 0.05; n.s. not significant. Figure 5. Olfr mRNAs are ARE-rich. (A) ARE pentamer density measured in silico. (B) Density distribution plot of the number of AUUUA ARE pen- tamers, assessed as in panel (A). (C) Scatterplots and linear regression analysis of expression level versus AUUUA pentamer density, assessed as in panel A. (D) Schematic of luciferase and renilla reporters used. (E,F) Two Olfr 3′ UTRs influence mRNA steady-state level and mRNA half-life in response to CCR4B depletion (using a siRNA targeting CCR4B [siCcr4b]) and CCR4B overexpression (using a CCR4B expression vector). Panel E is measurement of relative mRNA levels (RQ) and panel (F) is measurement of relative luciferase values (RLU). P < 0.05; n.s. not significant. versus non-neuronal cells suggests that the Olfr 3′ UTRs have evolved to permit stable mRNA expression specifically in neural cells. acts as a biological switch to control biological events, including developmental pathways (38,75–80). Thus, we investigated whether Olfr mRNAs might be targeted for decay by NMD. The best-characterized context that triggers NMD is the presence of an exon-exon junction (intron) at least 55 nt downstream of the stop codon defining the end of the main ORF (81). Most Olfr mRNAs do ‘not’ have this architec- ture (Supplementary Figure S4). Nevertheless, we identi- fied a small number of Olfr mRNAs (15 of 805) defined by RNA-seq analysis that do possess an ‘NMD-inducing’ ar- chitecture; i.e. an exon-exon junction >55 nt downstream of the stop codon (Supplementary Figure S4). Olfr 3′ UTRs confer stability To examine whether Olfr 3′ UTRs confer deadenylation-dependent mRNA downregulation, we mea- sured the effect of depleting or overexpressing the core deadenylase, CCR4B, on reporter mRNAs harboring the two Olfr 3′ UTRs described above. Forced overexpression of CCR4B (Supplementary Figure S3C) downregulated Olfr 3′ UTR-dependent reporter expression and destabi- lized the reporter mRNA transcript in Neuro2A cells (Fig- ure 5E–F), suggesting that, indeed, Olfr 3′ UTRs are sensi- tive to deadenylation-dependent decay. In contrast, siRNA- mediated knockdown of CCR4B (Supplementary Figure S3C) did not significantly affect Olfr 3′ UTR-dependent re- porter expression (Figure 5F), consistent with our finding that Olfr 3′ UTRs are normally stable in neural cells (Fig- ure 5E).i To assess the possible impact of AREs on Olfr expression, we asked whether there is an association between ARE den- sity and expression level. We found a strong positive corre- lation between AUUUA pentamer density and expression level, as assessed by RNA-seq reads (Figure 5C). Linear re- gression analysis demonstrated that this was highly statisti- cally significant (P < 0.0001). In contrast, there was a neg- ative correlation between ARE density and expression for the Ctrl group and no significant correlation for the Gpcr group (Figure 5C). To extend this analysis, we evaluated the density of another functional ARE motif: the WWAU- UUAWW nonamer (32). As with AUUUA pentamer den- sity, WWAUUUAWW density was higher in Olfr mRNAs than in the other groups (Supplementary Figure S3A). The frequency of these nonomers per transcript positively cor- related with Olfr mRNA expression level (Supplementary Figure S3B).i To assess the specificity of this response, we tested whether Olfr 3′UTR responds to deadenylation-dependent decay in non-neuronal cells. We used P19 embryonal carci- noma cells for this experiment, which are stem-like cells that transfect efficiently (72). As we observed in Neuro2A cells, overexpression of CCR4B from an expression vector in P19 cells (Supplementary Figure S3D) elicited reduced reporter expression from the two Olfr 3′ UTR reporters (Supplemen- tary Figure S3E). In striking contrast to the results obtained with Neuro2A cells, knocking down CCR4B in P19 cells (Supplementary Figure S3D) increased the expression the two Olfr 3′UTR reporters and stabilized its mRNA half- life (Supplementary Figure S3E and F). Olfr 3′ UTRs confer stability Since virtually all mRNAs with this feature are targeted for decay by NMD (73,74), it is likely that this small subset of Olfr transcripts FUNDING National Institutes of Health (NIH) [GM111838 to M.F.W.]. Funding for open access charge: National Insti- tutes of Health [GM111838 to M.F.W.].l Conflict of interest statement. None declared. Conflict of interest statement. None declared. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. Supplementary Data are available at NAR Online. SUMMARY A comprehensive landscape of mRNA features present in Olfr mRNAs revealed several unusual mRNA features in both the 5′ and 3′ UTR of Olfr mRNAs that we posit were selected over evolutionary time to direct the post- transcriptional regulation of Olfr genes. We provide evi- dence that Olfr mRNAs are extremely stable, which we posit results from one or more of these post-transcriptional fea- tures. Mechanisms potentially impacted by the unique fea- tures in Olfr mRNAs include ARE-mediated RNA decay, miRNA-mediated regulation and NMD. p g y g y Additionally, it has been shown that upstream open read- ing frames (uORFs) can trigger NMD, perhaps by virtue of the ability of the stop codon in the uORF to trigger pre- mature translation termination and thereby recruit mRNA decay enzymes (19,74). We found that majority of the 805 Olfr mRNAs expressed in the OE (67%) have uORFs (Fig- ure 6A, Supplementary Table S7). This is a significantly higher proportion than in the Gpcr and Ctrl groups. Only 38% of the Ctrl mRNA group have uORFs, a value consis- tent with the average value recently reported for all human mRNAs (84). In addition, Olfr mRNAs from both our data set and Ibarra-Soria et al. have a significantly higher average number of uORFs per transcript than Ctrl and Gpcr group (Figure 6B) despite having a similar average 5′ UTR length as the Ctrl and Gpcr groups (Figure 2C). ACKNOWLEDGEMENT p g p ( g ) Even though uORFs can trigger NMD, few uORF- containing mRNAs are likely to be subject to NMD reg- ulation (85). This follows from the fact that while ∼40% of human and rodent mRNAs have uORFs (84,86,87) (Fig- ure 6A), at most 5% of mammalian mRNAs are targeted for decay by NMD, based on microarray and RNA-seq anal- yses of cells depleted of NMD factors (85). Indeed, sev- eral mRNAs have been defined that escape uORF-induced NMD (88,89). This predicts that a majority of uORFs found in Olfr mRNAs are not bona fide targets of NMD. Consistent with this prediction, we found that Olfr mRNA harboring uORFs were statistically more highly expressed, when analyzed as a group, than those without (Figure 6C). We also observed a statistically significant positive corre- lation between the number of uORFs per transcript and Olfr mRNA level (Figure 6D). Finally, we also observed a positive correlation between average uORF length and Olfr mRNA level (Figure 6E). As evidence for specificity, we did not observe a correlation between any of these pa- rameters and the expression of the Gpcr or Ctrl gene groups (Figure 6C–E). Together, these results suggest that uORFs have an overall positive effect on Olfr mRNA expression. It will be interesting in the future to determine the under- lying mechanism. One means by which uORF-containing mRNAs may escape uORF-induced NMD is by allowing ribosomes that terminate translation in uORFs to reiniti- ate downstream. In S. cerevisiae, the 5′ UTR environment surrounding uORFs has been shown to influence this. For example, it has been shown that the AU-rich region down- stream of the uORF in gcn4 mRNA allows for efficient ri- bosome reinitiation, thereby avoiding triggering NMD (90). Given that we found that Olfr 5′ UTRs tend to be AU-rich (Figure 4), this may provide a general mechanism by which they escape uORF-induced NMD. Given that translation is generally regarded as providing a protective role from mRNA degradation (64), one explanation is that the AU- rich environment of Olfr uORFs promotes stabilization of Olfr mRNAs through their ability to promote re-initiation Eleen Y. Shum is a Howard Hughes Medical Institute In- ternational Student Fellow. Josh L. Espinoza is a Bridges intern at the California Institute of Regenerative Medicine. We thank the entire Wilkinson Laboratory for their help and support on this manuscript. Olfr mRNAs are uORF-rich Another well-studied deadenylation-dependent RNA degradation mechanism is Nonsense-mediated RNA decay (NMD). This pathway that was originally identified by virtue of its ability to degrade aberrant mRNAs containing premature stop codons as a result of mutations or biosyn- thetic (e.g. RNA splicing) errors (73,74). More recently, it was discovered that NMD also targets normal mRNAs that have an in-frame stop codon in a context that is recognized as premature (73,75,76). Increasing evidence indicates that the ability of NMD to degrade a subset of normal mRNAs Nucleic Acids Research, 2015, Vol. 43, No. 19 9323 , , , r mRNAs are uORF-rich. (A) The percentage of transcripts from the three groups with the indicated number of uORFs. (B) The average RFs. (C) Box plot comparing the expression level of transcripts harboring or lacking uORFs from the three groups. (D) Scatterplot and on analysis of expression level versus uORFs density. (E) Scatterplot and linear regression analysis of expression level versus average uORF < 0.0001; n.s., not statistically significant. Figure 6. Olfr mRNAs are uORF-rich. (A) The percentage of transcripts from the three groups with the indicated number of uORFs. (B) The average number of uORFs. (C) Box plot comparing the expression level of transcripts harboring or lacking uORFs from the three groups. (D) Scatterplot and linear regression analysis of expression level versus uORFs density. (E) Scatterplot and linear regression analysis of expression level versus average uORF length. **** P < 0.0001; n.s., not statistically significant. Nucleic Acids Research, 2015, Vol. 43, No. 19 9324 is subject to rapid decay by NMD, perhaps to provide de- velopmental regulation. of translation. We note, however, that our results do not rule out that a subset of Olfr mRNAs are targeted for decay through their uORFs by NMD. Another NMD-inducing feature is the presence of a long 3′ UTR (82,83). While no specific 3′ UTR length has been shown to trigger NMD, our finding that Olfr mRNAs tend to have considerably shorter 3′ UTRs than do control mR- NAs (Figure 2A) suggests that it is unlikely that Olfr tran- scripts are generally degraded by this mechanism. REFERENCES 1. DeMaria,S. and Ngai,J. (2010) The cell biology of smell. J. Cell Biol., 191, 443–452. 2. Hoover,K.C. (2013) Evolution of olfactory receptors. Methods Mol. Biol., 1003, 241–249. 3. Firestein,S. (2001) How the olfactory system makes sense of scents. Nature, 413, 211–218. 4. Kobayakawa,K., Kobayakawa,R., Matsumoto,H., Oka,Y., Imai,T., Ikawa,M., Okabe,M., Ikeda,T., Itohara,S., Kikusui,T. et al. (2007) Innate versus learned odour processing in the mouse olfactory bulb. Nature, 450, 503–508. 5. Ressler,K.J., Sullivan,S.L. and Buck,L.B. (1993) A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell, 73, 597–609. 6. Vassar,R., Ngai,J. and Axel,R. (1993) Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell, 74, 309–318. 7. Chess,A., Simon,I., Cedar,H. and Axel,R. (1994) Allelic inactivation regulates olfactory receptor gene expression. Cell, 78, 823–834. 8. Malnic,B., Hirono,J., Sato,T. and Buck,L.B. (1999) Combinatorial receptor codes for odors. Cell, 96, 713–723. 9. Barnea,G., O’Donnell,S., Mancia,F., Sun,X., Nemes,A., Mendelsohn,M. and Axel,R. (2004) Odorant receptors on axon termini in the brain. Science, 304, 1468. 10. Shykind,B.M., Rohani,S.C., O’Donnell,S., Nemes,A. 10. Shykind,B.M., Rohani,S.C., O’Donnell,S., Nemes,A., Mendelsohn,M., Sun,Y., Axel,R. and Barnea,G. (2004) Gene switching and the stability of odorant receptor gene choice. Cell, 117, 801–815. Nucleic Acids Research, 2015, Vol. 43, No. 19 9325 11. Buck,L. and Axel,R. (1991) A novel multigene family may encode odorant receptors: A molecular basis for odor recognition. Cell, 65, 175–187. 33. Lewis,B.P., Burge,C.B. and Bartel,D.P. (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 120, 15–20. 34. Friedman,R.C., Farh,K.K.-H., Burge,C.B. and Bartel,D.P. (2009) Most mammalian mRNAs are conserved targets of microRNAs. Genome Res., 19, 92–105. 12. Wang,S.S., Lewcock,J.W., Feinstein,P., Mombaerts,P. and Reed,R.R. (2004) Genetic disruptions of O/E2 and O/E3 genes reveal involvement in olfactory receptor neuron projection. Development, 131, 1377–1388. 35. Grimson,A., Farh,K.K.-H., Johnston,W.K., Garrett-Engele,P., Lim,L.P. and Bartel,D.P. (2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol. Cell, 27, 91–105 13. Wang,S.S., Tsai,R.Y.L. and Reed,R.R. (1997) The characterization of the Olf-1/EBF-like HLH transcription factor family: implications in olfactory gene regulation and neuronal development. J. Neurosci., 17, 4149–4158. 36. Garcia,D.M., Baek,D., Shin,C., Bell,G.W., Grimson,A. and Bartel,D.P. (2011) Weak seed-pairing stability and high target-site abundance decrease the proficiency of lsy-6 and other microRNAs. Nat. Struct. Mol. Biol., 18, 1139–1146. 14. McClintock,T.S. (2010) Achieving singularity in mammalian odorant receptor gene choice. Chem. Senses, 35, 447–457. 37. Hofacker,I.L. (2003) Vienna RNA secondary structure server. Nucleic Acids Res., 31, 3429–3431. 15. REFERENCES Magklara,A., Yen,A., Colquitt,B.M., Clowney,E.J., Allen,W., Markenscoff-Papadimitriou,E., Evans,Z.A., Kheradpour,P., Mountoufaris,G., Carey,C. et al. (2011) An epigenetic signature for monoallelic olfactory receptor expression. Cell, 145, 555–570. 38. Bruno,I.G., Karam,R., Huang,L., Bhardwaj,A., Lou,C.H., Shum,E.Y., Song,H.-W.W., Corbett,M.A., Gifford,W.D., Gecz,J. et al. (2011) Identification of a microRNA that activates gene expression by repressing nonsense-mediated RNA decay. Mol. Cell, 42, 500–510.il 16. Lyons,D.B., Allen,W.E., Goh,T., Tsai,L., Barnea,G. and Lomvardas,S. (2013) An epigenetic trap stabilizes singular olfactory receptor expression. Cell, 154, 325–336. 39. Kozak,M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell, 44, 283–292. 17. Bolognani,F. and Perrone-Bizzozero,N.I. (2008) RNA-protein interactions and control of mRNA stability in neurons. J. Neurosci. Res., 86, 481–489. 40. Niimura,Y. and Nei,M. (2007) Extensive gains and losses of olfactory receptor genes in mammalian evolution. PLoS One, 2, e708. 18. Pesole,G., Mignone,F., Gissi,C., Grillo,G., Licciulli,F. and Liuni,S. (2001) Structural and functional features of eukaryotic mRNA untranslated regions. Gene, 276, 73–81. 41. Miyamichi,K., Serizawa,S., Kimura,H.M. and Sakano,H. (2005) Continuous and overlapping expression domains of odorant receptor genes in the olfactory epithelium determine the dorsal/ventral positioning of glomeruli in the olfactory bulb. J. Neurosci., 25, 3586–3592. g 19. Barrett,L.W., Fletcher,S. and Wilton,S.D. (2012) Regulation of eukaryotic gene expression by the untranslated gene regions and other non-coding elements. Cell. Mol. Life Sci., 69, 3613–3634. 42. Tsuboi,A., Miyazaki,T., Imai,T. and Sakano,H. (2006) Olfactory sensory neurons expressing class I odorant receptors converge their axons on an antero-dorsal domain of the olfactory bulb in the mouse. Eur. J. Neurosci., 23, 1436–1444. g f 20. Krol,J., Loedige,I. and Filipowicz,W. (2010) The widespread regulation of microRNA biogenesis, function and decay. Nat. Rev. Genet., 11, 597–610. 21. Zhang,X., Rogers,M., Tian,H., Zhang,X., Zou,D.-J., Liu,J., Ma,M., Shepherd,G.M. and Firestein,S.J. (2004) High-throughput microarray detection of olfactory receptor gene expression in the mouse. Proc. Natl. Acad. Sci. U.S.A., 101, 14168–14173. 43. Sharman,J.L., Benson,H.E., Pawson,A.J., Lukito,V., Mpamhanga,C.P., Bombail,V., Davenport,A.P., Peters,J.A., Spedding,M. and Harmar,A.J. (2013) IUPHAR-DB: updated database content and new features. Nucleic Acids Res., 41, D1083–D1088. 22. Shiao,M.-S., Chang,A.Y.-F., Liao,B.-Y., Ching,Y.-H., Lu,M.-Y.J., Chen,S.M. and Li,W.-H. (2012) Transcriptomes of mouse olfactory epithelium reveal sexual differences in odorant detection. Genome Biol. Evol., 4, 703–712. 44. Spies,N., Burge,C.B. and Bartel,D.P. (2013) 3′UTR-isoform choice has limited influence on the stability and translational efficiency of most mRNAs in mouse fibroblasts. Genome Res., 23, 2078–2090. 23. Sosinsky,A., Glusman,G. and Lancet,D. (2000) The genomic structure of human olfactory receptor genes. Genomics, 70, 49–61. 45. REFERENCES Mayr,C. and Bartel,D.P. (2009) Widespread shortening of 3′UTRs by alternative cleavage and polyadenylation activates oncogenes in cancer cells. Cell, 138, 673–684. 24. Lane,R.P., Cutforth,T., Young,J., Athanasiou,M., Friedman,C., Rowen,L., Evans,G., Axel,R., Hood,L. and Trask,B.J. (2001) Genomic analysis of orthologous mouse and human olfactory receptor loci. Proc. Natl. Acad. Sci. U.S.A., 98, 7390–7395.i 46. Sandberg,R., Neilson,J.R., Sarma,A., Sharp,P.A. and Burge,C.B. (2008) Proliferating cells express mRNAs with shortened 3′ untranslated regions and fewer microRNA target sites. Science, 320, 1643–1647. 25. Michaloski,J.S., Galante,P.A.F. and Malnic,B. (2006) Identification of potential regulatory motifs in odorant receptor genes by analysis of promoter sequences. Genome Res., 16, 1091–1098. 47. Stark,A., Brennecke,J., Bushati,N., Russell,R.B. and Cohen,S.M. (2005) Animal MicroRNAs confer robustness to gene expression and have a significant impact on 3′UTR evolution. Cell, 123, 1133–1146. ′ 26. Clowney,E.J., Magklara,A., Colquitt,B.M., Pathak,N., Lane,R.P. and Lomvardas,S. (2011) High-throughput mapping of the promoters of the mouse olfactory receptor genes reveals a new type of mammalian promoter and provides insight into olfactory receptor gene regulation. Genome Res., 21, 1249–1259. 48. Hogg,J.R. and Goff,S.P. (2010) Upf1 senses 3′UTR length to potentiate mRNA decay. Cell, 143, 379–389. 49. Ji,Z., Lee,J.Y., Pan,Z., Jiang,B. and Tian,B. (2009) Progressive lengthening of 3′ untranslated regions of mRNAs by alternative polyadenylation during mouse embryonic development. Proc. Natl. Acad. Sci. U.S.A., 106, 7028–7033. 27. Young,J.M., Shykind,B.M., Lane,R.P., Tonnes-Priddy,L., Ross,J.A., Walker,M., Williams,E.M. and Trask,B.J. (2003) Odorant receptor expressed sequence tags demonstrate olfactory expression of over 400 genes, extensive alternate splicing and unequal expression levels. Genome Biol., 4, R71. 50. Szostak,E. and Gebauer,F. (2013) Translational control by 3′-UTR-binding proteins. Brief. Funct. Genomics, 12, 58–65. 28. Ibarra-Soria,X., Levitin,M.O., Saraiva,L.R. and Logan,D.W. (2014) The olfactory transcriptomes of mice. PLoS Genet., 10, e1004593. g p f 51. Bushati,N. and Cohen,S.M. (2007) microRNA functions. Annu. Rev. Cell Dev. Biol., 23, 175–205. 52. Bhattacharyya,S.N., Habermacher,R., Martine,U., Closs,E.I. and Filipowicz,W. (2006) Relief of microRNA-mediated translational repression in human cells subjected to stress. Cell, 125, 1111–1124. 29. Trapnell,C., Pachter,L. and Salzberg,S.L. (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics, 25, 1105–1111. 30. Trapnell,C., Roberts,A., Goff,L., Pertea,G., Kim,D., Kelley,D.R., Pimentel,H., Salzberg,S.L., Rinn,J.L. and Pachter,L. (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc., 7, 562–578. 53. Choi,P.S., Zakhary,L., Choi,W.-Y., Caron,S., Alvarez-Saavedra,E., Miska,E.A., McManus,M., Harfe,B., Giraldez,A.J., Horvitz,H.R. et al. (2008) Members of the miRNA-200 family regulate olfactory neurogenesis. Neuron, 57, 41–55. 31. Crooks,G.E., Hon,G., Chandonia,J.-M. and Brenner,S.E. (2004) WebLogo: a sequence logo generator. REFERENCES Genome Res., 14, 1188–1190. 54. Chen,H., Shalom-Feuerstein,R., Riley,J., Zhang,S.-D., Tucci,P., Agostini,M., Aberdam,D., Knight,R.A., Genchi,G., Nicotera,P. et al. (2010) miR-7 and miR-214 are specifically expressed during neuroblastoma differentiation, cortical development and embryonic stem cells differentiation, and control neurite outgrowth in vitro. Biochem. Biophys. Res. Commun., 394, 921–927. 32. Gruber,A.R., Fallmann,J., Kratochvill,F., Kovarik,P. and Hofacker,I.L. (2011) AREsite: a database for the comprehensive investigation of AU-rich elements. Nucleic Acids Res., 39, D66–D69. 9326 Nucleic Acids Research, 2015, Vol. 43, No. 19 72. McBurney,M.W. (1993) P19 embryonal carcinoma cells. Int. J. Dev. Biol., 37, 135–140. 55. Sempere,L.F., Freemantle,S., Pitha-Rowe,I., Moss,E., Dmitrovsky,E. and Ambros,V. (2004) Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation. Genome Biol., 5, R13. 73. Popp,M.W.-L. and Maquat,L.E. (2013) Organizing principles of mammalian nonsense-mediated mRNA decay. Annu. Rev. Genet., 47, 139–165. 74. Chang,Y.-F., Imam,J.S. and Wilkinson,M.F. (2007) The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem., 76, 51–74. 56. Zhao,C., Sun,G., Li,S. and Shi,Y. (2009) A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination. Nat. Struct. Mol. Biol., 16, 365–371. 57. Makeyev,E. V, Zhang,J., Carrasco,M.A. and Maniatis,T. (2007) The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol. Cell, 27, 435–448. 75. Huang,L. and Wilkinson,M.F. (2012) Regulation of nonsense-mediated mRNA decay. Wiley Interdiscip. Rev. RNA, 3, 807–828. 76. Karam,R. and Wilkinson,M. (2012) A conserved microRNA/NMD regulatory circuit controls gene expression. RNA Biol., 9, 22–26. 58. Edbauer,D., Neilson,J.R., Foster,K.A., Wang,C.-F., Seeburg,D.P., Batterton,M.N., Tada,T., Dolan,B.M., Sharp,P.A. and Sheng,M. (2010) Regulation of synaptic structure and function by FMRP-associated microRNAs miR-125b and miR-132. Neuron, 65, 373–384. 77. Lareau,L.F., Inada,M., Green,R.E., Wengrod,J.C. and Brenner,S.E. (2007) Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements. Nature, 446, 926–929. 78. Gong,C., Kim,Y.K., Woeller,C.F., Tang,Y. and Maquat,L.E. (2009) SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs. Genes Dev., 23, 54–66. 59. Lagos,D., Pollara,G., Henderson,S., Gratrix,F., Fabani,M., Milne,R.S.B., Gotch,F. and Boshoff,C. (2010) miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator. Nat. Cell Biol., 12, 513–519. 79. Colak,D., Ji,S.-J., Porse,B.T. and Jaffrey,S.R. (2013) Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay. Cell, 153, 1252–1265. 60. Siegel,G., Obernosterer,G., Fiore,R., Oehmen,M., Bicker,S., Christensen,M., Khudayberdiev,S., Leuschner,P.F., Busch,C.J.L., Kane,C. et al. (2009) A functional screen implicates microRNA-138-dependent regulation of the depalmitoylation enzyme APT1 in dendritic spine morphogenesis. REFERENCES Nat. Cell Biol., 11, 705–716. 80. Lou,C.H., Shao,A., Shum,E.Y., Espinoza,J.L., Huang,L., Karam,R. and Wilkinson,M.F. (2014) Posttranscriptional control of the stem cell and neurogenic programs by the nonsense-mediated RNA decay pathway. Cell Rep., 6, 748–764. 61. Wulczyn,F.G., Smirnova,L., Rybak,A., Brandt,C., Kwidzinski,E., Ninnemann,O., Strehle,M., Seiler,A., Schumacher,S. and Nitsch,R. (2007) Post-transcriptional regulation of the let-7 microRNA during neural cell specification. FASEB J., 21, 415–426. 81. Nagy,E. and Maquat,L.E. (1998) A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. Trends Biochem. Sci., 23, 198–199. 62. Ringn´er,M. and Krogh,M. (2005) Folding free energies of 5′-UTRs impact post-transcriptional regulation on a genomic scale in yeast. PLoS Comput. Biol., 1, e72. 82. Nicholson,P., Yepiskoposyan,H., Metze,S., Zamudio Orozco,R., Kleinschmidt,N., M¨uhlemann,O. and Muhlemann,O. (2010) Nonsense-mediated mRNA decay in human cells: mechanistic insights, functions beyond quality control and the double-life of NMD factors. Cell. Mol. life Sci., 67, 677–700. 63. Kozak,M. (1989) The scanning model for translation: an update. J. Cell Biol., 108, 229–241. 64. Roy,B. and Jacobson,A. (2013) The intimate relationships of mRNA decay and translation. Trends Genet., 29, 691–699. 83. Rebbapragada,I. and Lykke-Andersen,J. (2009) Execution of nonsense-mediated mRNA decay: what defines a substrate? Curr. Opin. Cell Biol., 21, 394–402. 65. Chen,C.-Y.A. and Shyu,A.-B. (1995) AU-rich elements: characterization and importance in mRNA degradation. Trends Biochem. Sci., 20, 465–470. 84. Calvo,S.E., Pagliarini,D.J. and Mootha,V.K. (2009) Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. Proc. Natl. Acad. Sci. U.S.A., 106, 7507–7512. 66. Barreau,C., Paillard,L. and Osborne,H.B. (2005) AU-rich elements and associated factors: are there unifying principles? Nucleic Acids Res., 33, 7138–7150. 85. Hurt,J.A., Robertson,A.D. and Burge,C.B. (2013) Global analyses of UPF1 binding and function reveal expanded scope of nonsense-mediated mRNA decay. Genome Res., 23, 1636–1650. 67. Bakheet,T., Frevel,M., Williams,B.R., Greer,W. and Khabar,K.S. (2001) ARED: human AU-rich element-containing mRNA database reveals an unexpectedly diverse functional repertoire of encoded proteins. Nucleic Acids Res., 29, 246–254. 86. Iacono,M., Mignone,F. and Pesole,G. (2005) uAUG and uORFs in human and rodent 5′untranslated mRNAs. Gene, 349, 97–105. 68. Ford,L.P., Watson,J., Keene,J.D. and Wilusz,J. (1999) ELAV proteins stabilize deadenylated intermediates in a novel in vitro mRNA deadenylation/degradation system. Genes Dev., 13, 188–201. 87. Matsui,M., Yachie,N., Okada,Y., Saito,R. and Tomita,M. (2007) Bioinformatic analysis of post-transcriptional regulation by uORF in human and mouse. FEBS Lett., 581, 4184–4188. 69. Okano,H.J. and Darnell,R.B. (1997) A Hierarchy of Hu RNA Binding Proteins in Developing and Adult Neurons. J. Neurosci., 17, 3024–3037. 88. REFERENCES Stockklausner,C., Breit,S., Neu-Yilik,G., Echner,N., Hentze,M.W., Kulozik,A.E. and Gehring,N.H. (2006) The uORF-containing thrombopoietin mRNA escapes nonsense-mediated decay (NMD). Nucleic Acids Res., 34, 2355–2363. 70. Anderson,K.D., Morin,M.A., Beckel-Mitchener,A., Mobarak,C.D., Neve,R.L., Furneaux,H.M., Burry,R. and Perrone-Bizzozero,N.I. (2002) Overexpression of HuD, but not of its truncated form HuD I+II, promotes GAP-43 gene expression and neurite outgrowth in PC12 cells in the absence of nerve growth factor. J. Neurochem., 75, 1103–1114. 89. Silva,A.L., Pereira,F.J.C., Morgado,A., Kong,J., Martins,R., Faustino,P., Liebhaber,S.A. and Rom˜ao,L. (2006) The canonical UPF1-dependent nonsense-mediated mRNA decay is inhibited in transcripts carrying a short open reading frame independent of sequence context. RNA, 12, 2160–2170. sequence context. RNA, 12, 2160–2170. 71. Mobarak,C.D., Anderson,K.D., Morin,M., Beckel-Mitchener,A., Rogers,S.L., Furneaux,H., King,P. and Perrone-Bizzozero,N.I. (2000) The RNA-binding protein HuD is required for GAP-43 mRNA stability, GAP-43 gene expression, and PKC-dependent neurite outgrowth in PC12 cells. Mol. Biol. Cell, 11, 3191–3203. 90. Hinnebusch,A.G. (1997) Translational regulation of yeast GCN4. A window on factors that control initiator-trna binding to the ribosome. J. Biol. Chem., 272, 21661–21664.
https://openalex.org/W2782793186	http://www.scielo.br/pdf/abmvz/v69n6/1678-4162-abmvz-69-06-01551.pdf	Portuguese	null	Sonda uretral flexível como método alternativo para aferição invasiva da pressão intracraniana em trauma cranioencefálico induzido em coelhos	Arquivo Brasileiro de Medicina Veterinária e Zootecnia/Arquivo brasileiro de medicina veterinária e zootecnia	2,017	cc-by	4,953	RESUMO O objetivo deste estudo foi utilizar a sonda uretral flexível como método alternativo para aferição da pressão intracraniana em coelhos com trauma cranioencefálico induzido pelo cateter de Fogarty 4 Fr (balão epidural) e comparar os dados obtidos com o método convencional de cateter de ventriculostomia. Foram utilizados 12 coelhos, machos, adultos, distribuídos aleatoriamente em dois grupos, denominados de G1: mensuração da PIC com cateter de ventriculostomia (n=6) e G2: mensuração com sonda uretral (n=6). Foram realizadas duas craniotomias na região parietal direita e esquerda para a implantação do cateter de ventriculostomia ou sonda uretral flexível e o balão epidural, respectivamente. A PAM, a PPC, a FC, a FR e a TR foram mensurados antes e após a craniotomia. A PIC foi avaliada após a craniotomia e a cada 10 minutos depois do preenchimento do balonete com 0,3mL de NaCl 0,9%, durante 40 minutos, e com 0,6mL, pelo mesmo período de tempo, totalizando 80 minutos. A PIC aumentou em ambos os grupos, sendo menores os valores registrados com a sonda uretral flexível. Foi possível reproduzir o aumento da PIC com o modelo experimental de TCE utilizando o cateter de Fogarty 4 Fr na região epidural e, embora haja a necessidade de outros estudos, a sonda uretral flexível demonstra ser um método alternativo de mensuração da PIC em coelhos com trauma cranioencefálico. Palavras-chave: cateter de ventriculostomia, autorregulação, neurologia, cirurgia [Flexible urethral catheter as alternative method to invasive measurement of intracranial pressure in induced head trauma in rabbits] [Flexible urethral catheter as alternative method to invasive measurement of intracranial pressure in induced head trauma in rabbits] G. Aiello1, A.O. Andrades1, A. Ripplinger1, A.V. Soares2, D. Polidoro1, M.A.B. Vaz1, A.C.Colvero3, R.P. Santos1, R. Conceição3, R.O. Chaves1, A. Mazzanti2* G. Aiello1, A.O. Andrades1, A. Ripplinger1, A.V. Soares2, D. Polidoro1, M.A.B. Vaz1, A.C.Colvero3, R.P. Santos1, R. Conceição3, R.O. Chaves1, A. Mazzanti2* 1Aluno de pós-graduação – Universidade Federal de Santa Maria – UFSM – Santa Maria, RS 2Universidade Federal de Santa Maria – UFSM – Santa Maria, RS 3 3Aluno de graduação – Universidade Federal de Santa Maria – UFSM – Santa Maria, RS 3Aluno de graduação – Universidade Federal de Santa Maria – UFSM – Santa Maria, RS 3Aluno de graduação – Universidade Federal de Santa Maria – UFSM – Santa Maria, RS http://dx.doi.org/10.1590/1678-4162-9184 http://dx.doi.org/10.1590/1678-4162-9184 Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 Recebido em 19 de maio de 2016 Aceito em 19 de outubro de 2016 Autor para correspondência (corresponding author) E-mail: alexamazza@yahoo.com.br INTRODUÇÃO O traumatismo cranioencefálico (TCE) está associado à alta taxa de mortalidade e morbidade em humanos e pequenos animais (Dewey e Fletcher, 2016). Pode ser dividido em eventos primários, que ocorrem no momento da injúria, e secundários, provocados pelos processos bioquímicos, inflamatórios e metabólicos (Lorenz et al., 2011). Foram utilizados 12 coelhos, machos, da raça Nova Zelândia, entre 3,3-4,0kg de peso vivo, provenientes do Biotério Central da Instituição. Os animais permaneceram alojados em gaiolas individuais, com água e ração comercial ad libitum, por um período de 10 dias antes do início do experimento. Este trabalho foi aprovado pelo Comitê de Ética em Pesquisa da Instituição, sob o número 045/2014. O traumatismo cranioencefálico (TCE) está associado à alta taxa de mortalidade e morbidade em humanos e pequenos animais (Dewey e Fletcher, 2016). Pode ser dividido em eventos primários, que ocorrem no momento da injúria, e secundários, provocados pelos processos bioquímicos, inflamatórios e metabólicos (Lorenz et al., 2011). A mensuração da pressão intracraniana (PIC) é empregada para o diagnóstico da hipertensão intracraniana e também para o valor prognóstico, especialmente nos pacientes com graves sinais clínicos devido ao TCE. Ainda, pode ser utilizada como parâmetro na avaliação das medidas terapêuticas empregadas para reduzir a PIC, sendo imprescindível para determinar os valores da pressão de perfusão cerebral (PPC) (Carlotti et al., 1998). Os coelhos foram distribuídos aleatoriamente em dois grupos experimentais, com seis indivíduos cada: grupo 1 – G1: avaliação contínua da PIC pelo método do cateter de ventriculostomia (Kit Kompacto, Ventura, Brasil) e grupo 2 – G2: avaliação contínua da PIC pelo método intraventicular por sonda uretral flexível (Mark Med, Brasil). Ambos os dispositivos foram acoplados à um monitor multiparamétrico. Para realização da craniotomia e acesso ao ventrículo lateral do encéfalo, foi realizada a tricotomia da face externa das orelhas direita e esquerda e do crânio (ossos frontal e parietal). Como medicação pré-anestésica (MPA), foi administrado midazolam (1mg.kg-1, intramuscular) associado à morfina (2mg.kg-1, intramuscular), 10 minutos antes de acessar a veia marginal da orelha esquerda. Em seguida, os coelhos foram submetidos à anestesia geral, por meio da vaporização de isoflurano na máscara facial, intubados com máscara laríngea e mantidos em anestesia geral inalatória com o mesmo agente anestésico, em vaporizador calibrado na concentração de 1,5 CAM (concentração alveolar mínima). Para a mensuração da PIC, existem métodos invasivos e não invasivos (Bratton et al., 2007). ABSTRACT The aim of this study was to evaluate the use of flexible urethral catheter as an alternative method for measuring intracranial pressure in rabbits with head trauma induced by 4 F Fogarty catheter (epidural balloon) and compare the data obtained with the conventional method of ventriculostomy catheter. In this study, New Zealand rabbits were randomly distributed into two groups, G1: measuring the ICP with ventriculostomy catheter (n=6) and G2: measuring the ICP with urethral catheter (n=6). Two craniotomies were performed in the right and left parietal region for the implantation of a ventriculostomy catheter and/or flexible urethral catheter and epidural 4 Fr Fogarty arterial embolectomy catheter, respectively. MAP, CPP, HR, RF and RT values were measured before and after of the craniotomy. The ICP value was measured after craniotomy, every five minutes during 40 minutes after the balloon was inflated with 0.3 ml with NaCl and further 40 minutes after the balloon was inflated with 0.6 ml. The ICP value increased in both groups; however, the ICP values were lower in the flexible urethral catheter. The flexible urethral catheter can be used as an alternative method to measure ICP values in rabbits with head injury. Keywords: ventriculostomy catheter, autoregulation, neurology, surgery Recebido em 19 de maio de 2016 Aceito em 19 de outubro de 2016 Autor para correspondência (corresponding author) E-mail: alexamazza@yahoo.com.br Aiello et al. INTRODUÇÃO O uso de cateter intraventricular é considerado o “padrão ouro” para a monitorização invasiva (Maniker et al., 2006) por permitir a aferição precisa da PIC (Smith, 2008). Existem vários equipamentos utilizados para monitorização intraventricular, entre eles, pode ser citado o aparelho por fibra óptica, considerado de alto custo, e o uso de cateter intraventricular acoplado a um monitor multiparamétrico (Roux, 2013), que, além do menor custo, tem a possibilidade terapêutica de drenagem liquórica (Giugno et al., 2003). A monitorização da PIC é rotineiramente utilizada em pacientes humanos (Dewey e Fletcher, 2016), porém poucos são os hospitais e as clínicas veterinárias que mensuram a PIC em pacientes com trauma cranioencefálico. Vários são os motivos, entre eles, o custo dos métodos empregados (Packer et al., 2011), sendo necessária a busca por métodos alternativos de fácil aplicação e de baixo custo. A artéria auricular direita foi acessada com cateter 22G acoplado a um transdutor de pressão e este sistema a um monitor multiparamétrico para mensuração da pressão arterial média (PAM). A temperatura corporal foi monitorada utilizando-se uma probe retal e mantida entre 38 e 39°C durante todo o procedimento cirúrgico. Todos os animais foram posicionados em decúbito esternal. Antes da intervenção cirúrgica, foram mensuradas a pressão arterial média (PAM), a frequência cardíaca (FC), a frequência respiratória (FR) e a temperatura retal (TR), três vezes, com intervalo de cinco minutos (denominados de C0, C5 e C10). Foi colhido Diante disso, o objetivo deste estudo foi utilizar a sonda uretral flexível como método alternativo para a aferição da pressão intracraniana em coelhos com trauma cranioencefálico induzido por cateter Fogarty 4 Fr (balão epidural) e comparar os dados obtidos com o método convencional de cateter de ventriculostomia. 1552 Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 Sonda uretral flexível... uretral flexível número seis (G2), em uma profundidade de 6mm, compatível com a entrada no interior do ventrículo lateral direito. A confirmação do posicionamento do dispositivo dentro do ventrículo ocorreu ao se visualizar o líquido cerebroespinhal (LCE) fluindo pelo cateter ou pela sonda uretral (Fig. 1B). Para evitar extravasamento de LCE e fixar o cateter ou a sonda uretral, o orifício da craniotomia foi selado com resina acrílica autopolimerizável. Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 INTRODUÇÃO Para a realização do modelo experimental de TCE por balão epidural, o cateter de Fogarty 4 Fr (ED - alas Lifesciences LLC Irvine, EUA) foi introduzido no interior do orifício da região parietal esquerda (Fig. 1C), posicionado no espaço epidural (entre o osso do crânio e a dura- máter), e foi realizado o selamento hermético com resina acrílica autopolimerizável (Fig. 1D). Na sequência, o balão do cateter de Fogarty 4 Fr foi preenchido com 0,3mL de solução salina isotônica (NaCl a 0,9%), durante 40 minutos, para mensuração dos valores da pressão intracraniana (PIC), da PAM, da FC, da FR, do TPC, do CO2 e da TR a cada cinco minutos. A PPC foi calculada por meio da fórmula: PPC=PAM-PIC (Dewey e Fletcher, 2016). máxima observada antes do óbito foi 86mmHg no G1 e 78mmHg no G2, o que correspondeu sete vezes o valor da PIC normal em coelhos (5 a 13mmHg) (Çagavi et al., 2005). Nesses animais, foi realizada a craniotomia descompressiva e verificado deslocamento do cerebelo através do forame magno, caracterizando herniação foraminal. Para Freeman e Platt (2011), esse tipo de herniação é frequentemente fatal, pois causa parada respiratória por compressão dos centros respiratórios na medula oblonga. As três principais complicações associadas à inserção de cateteres intraventriculares são: hemorragia intracraniana, infecção e obstrução (Li et al., 2010). Binz et al. (2009) observaram complicações hemorrágicas associadas a cateteres intraventriculares em 5,7% dos pacientes avaliados, porém, estas foram clinicamente significativas em menos de 1%. O hematoma subdural é outra possível complicação (Lang et al., 2012). No presente estudo, não foram observadas complicações associadas à inserção (hemorragia, hematoma e obstrução) em ambos os grupos. No estudo de Çagavi et al. (2005), que também mensurou a PIC intraventricular em coelhos, não foi observada a presença de hematomas. Decorridos 40 minutos, o balão foi preenchido com mais 0,3mL, totalizando 0,6mL de NaCl 0,9%, que permaneceu inflado pelo mesmo período de tempo, finalizando 80 minutos de mensuração, ou seja, 40 minutos com balão epidural preenchido com 0,3mL e 40 minutos com 0,6mL de NaCl 0,9%. Ao término das avaliações, os animais foram submetidos à eutanásia por meio de sobredose anestésica com isoflurano, seguida da administração de cloreto de potássio (KCl a 10%) até a interrupção dos batimentos cardíacos. O cateter/sonda uretral flexível e o cateter de Fogarty 4 Fr foram removidos, e os locais de inserção inspecionados em busca de hematomas. INTRODUÇÃO Com o cateter de ventriculostomia (G1) ou a sonda uretral flexível (G2) inseridos, e antes de introduzir o balão epidural, foram mensurados novamente a PAM, a FC, a FR, o TPC e a TR, três vezes, com intervalo de cinco minutos (denominados de Cr0, Cr5 e Cr10). Em seguida, as aferições foram anotadas, juntamente com os valores da PIC basal, em todos os coelhos dos dois grupos. 0,3mL de sangue arterial para hemogasometria, com início antes da craniotomia e a cada 15 minutos até o término, totalizando oito avaliações. ç Para a realização do procedimento cirúrgico, foi realizada a antissepsia com álcool-iodo-álcool e uma incisão longitudinal da pele, do subcutâneo, e do periósteo, sobre a linha média do crânio de, aproximadamente, 5,0cm. Utilizando-se um elevador de periósteo, iniciou-se o deslocamento dele na região parietal esquerda e direita. Com um motor de suspensão elétrico de baixa rotação acoplado a uma broca sulcada de 3,0mm de diâmetro, dois orifícios no crânio foram criados sobre o osso parietal, situados a 2,5mm em posição lateral direita e esquerda da sutura sagital e 5mm caudal à sutura coronária (Fig. 1A). Através do orifício direito, foi introduzido o cateter de ventriculostomia (G1) ou a sonda Figura 1. Implantação da sonda uretral flexível intraventricular para aferir a PIC e confecção do modelo experimental de TCE em coelhos. A) Mensurações na região parietal direita para a realização da craniotomia; B) LCE no interior da sonda uretral flexível, confirmando a localização da sonda uretral no ventrículo lateral direito. C) Sonda uretral flexível inserida na craniotomia direita e o cateter de Fogarty 4 FR na esquerda. D) Oclusão das craniotomias e fixação dos cateteres com resina acrílica autopolimerizável. Figura 1. Implantação da sonda uretral flexível intraventricular para aferir a PIC e confecção do modelo experimental de TCE em coelhos. A) Mensurações na região parietal direita para a realização da craniotomia; B) LCE no interior da sonda uretral flexível, confirmando a localização da sonda uretral no ventrículo lateral direito. C) Sonda uretral flexível inserida na craniotomia direita e o cateter de Fogarty 4 FR na esquerda. D) Oclusão das craniotomias e fixação dos cateteres com resina acrílica autopolimerizável. 1553 Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 Aiello et al. INTRODUÇÃO Os valores de PaCO2 considerados normais para coelhos variam entre 20 e 46mmHg (Barzago et al., 1992). Neste estudo, todos os animais apresentaram hipercapnia em todos os tempos avaliados, inclusive na avaliação da PaCO2 basal (Fig. 2B). A hipercapnia observada na mensuração basal, antes de se realizar a craniotomia e o modelo experimental de TCE, pode estar relacionada ao tempo entre a indução anestésica utilizando a máscara facial e a introdução da máscara laríngea, à ausência de ventilação mecânica e aos fármacos administrados. A morfina é um µ agonista, que produz analgesia, euforia e uma importante depressão respiratória em coelhos (Donnelly e Warren, 2014). Vários estudos mostram que os benzodiapínicos, como o midazolam, causam média a moderada hipercapnia em coelhos e humanos. (Forster et al., 1980; Chang et al., 2009). Porém, no estudo de Schroeder e Smith (2011), os quais avaliaram a taxa respiratória e realizaram as análises de gases em coelhos medicados com opioides (buprenorfina, butorfanol), midazolam e a associação do midazolam aos opioides isoladamente, não foi Foi realizada análise estatística pelo modelo de regressão linear clássico, aplicando-se o teste t de Student para verificar a significância dos coeficientes (P<0,05) para comparar os métodos de aferição da PIC. RESULTADOS E DISCUSSÃO Neste estudo, o modelo experimental de TCE por redução de complacência, utilizando-se um cateter de Fogarty 4 Fr na região epidural, foi de fácil execução, de baixo custo e permitiu elevar a pressão intracraniana. Esse mesmo método foi realizado com êxito por Douzinas et al. (1999) e Çagavi et al. (2005), utilizando-se cateter de Fogarty, e por Abe et al. (1984); Mizumoto et al. (2005), utilizando sonda de Foley. Dois coelhos foram excluídos das análises dos dados deste estudo (G1 e G2), pois foram a óbito antes do término das mensurações. A PIC 1554 Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 Sonda uretral flexível... balonete com 0,6mL de solução salina) no grupo que mensurou a PIC com cateter de ventriculostomia. evidenciada hipercapnia. Neste estudo, não houve diferença estatística entre os tempos avaliados e entre os grupos. Mesmo não havendo diferença estatística entre os tempos, houve uma tendência de aumento crescente dos valores da PaCO2, após se iniciar o modelo experimental de TCE. O valor médio do pH de sangue arterial em coelhos não anestesiados varia entre 7,2 - 7,5 (Donnelly e Warren, 2014). No estudo de Benato et al. (2013), o valor médio estimado do pH em coelhos anestesiados foi de 7,33 (±0,07), sendo utilizado fentanil, midazolam e isoflurano. Neste estudo, o valor estimado do pH não apresentou diferença entre os grupos. O pH manteve-se dentro do intervalo de normalidade antes e após a craniotomia e até 15 minutos após preencher o balonete com 0,3mL de solução salina. Porém, após esse período de avaliação, foi evidenciada acidose em ambos os grupos até a finalização das mensurações. A PaO2 demonstra o balanço entre a quantidade de oxigênio fornecida pela ventilação e o quanto é transferido dos alvéolos para os capilares (Amaral et al., 1992). O valor estimando da PaO2 em coelhos respirando ar ambiente é 90mmHg (Barzago et al., 1992). Neste estudo, todos os coelhos apresentaram valores superiores ao preconizado como normal para a espécie. Não houve diferença estatística entre os grupos e entre os tempos, exceto na última avaliação (35minutos de avaliação, após preencher o Figura 2. Mensurações da PAM dos grupos cateter de ventriculostomia e sonda uretral (A), do PaCO2 (B), da PPC (C) e da PIC (D) em coelhos submetidos ao modelo experimental de TCE, utilizando-se o cateter de Fogarty 4 Fr. RESULTADOS E DISCUSSÃO Nota-se que a PIC registrada com a sonda uretral flexível foi menor e acompanhou as variações da pressão registradas em todos os momentos pelo cateter de ventriculostomia. Figura 2. Mensurações da PAM dos grupos cateter de ventriculostomia e sonda uretral (A), do PaCO2 (B), da PPC (C) e da PIC (D) em coelhos submetidos ao modelo experimental de TCE, utilizando-se o cateter de Fogarty 4 Fr. Nota-se que a PIC registrada com a sonda uretral flexível foi menor e acompanhou as variações da pressão registradas em todos os momentos pelo cateter de ventriculostomia. Os valores do bicarbonato (HCO3) estavam dentro do intervalo de normalidade (22,37 a 35,08) nos dois grupos avaliados, e não houve diferença significativa entre os tempos. Portanto, o tipo de acidose observado nos coelhos deste estudo, após os 15minutos de avaliação do preenchimento do balonete com 0,3mL de solução salina, foi a respiratória, pois foi 1555 Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 Aiello et al. balonete com 0,6mL, a PAM aumentou nos dois grupos e os valores do G1 continuaram superiores ao G2. Após cinco minutos de avaliação, a PAM voltou a diminuir até que os valores do G1 e G2 se igualaram. É interessante observar que, mesmo com o aumento da PIC no momento do preenchimento do balonete com 0,3mL, a PAM aumentou somente com cinco minutos de compressão, persistindo a elevação até o 25º minuto (Fig. 2A). A provável explicação da não elevação da PAM nos primeiros cinco minutos se deve à doutrina de Monro-Kellie, cujo deslocamento do LCE no sentido caudal em direção ao canal central da medula espinhal, bem como a redução na produção, pode auxiliar na manutenção da pressão intracraniana nos momentos iniciais de aumento da PIC (Freeman e Platt, 2011). observada hipercapnia nos coelhos de ambos os grupos e normalidade nos valores do bicarbonato. A autorregulação da PIC é a ocorrência de mecanismos para manter constantes os componentes da cavidade craniana (parênquima encefálico, LCE e sangue) e controlar o FSC quando a PAM estiver entre 50-150mmHg (Dewey e Fletcher, 2016). Outro mecanismo para a autorregulação da PIC é a química (Carlotti et al., 1998), que se baseia na capacidade de resposta direta da vasculatura cerebral quando houver variação na pressão parcial de dióxido de carbono no sangue arterial (PaCO2). Sonda uretral flexível... As médias estimadas dos valores basais da PIC, ou seja, imediatamente após a craniotomia e decorridos cinco e 10 minutos antes do preenchimento do balão epidural, apresentaram diferença significativa (P<0,01) entre os grupos (G1: 13,93mmHg e em G2: 5,93mmHg), porém não houve variação entre os tempos em ambos os grupos (Fig. 2D). No estudo de Çagavi et al. (2005), também foi utilizado o cateter intraventricular em coelhos e foi obtido o valor médio da PIC basal intraventricular de 12,7mmHg. PIC e nas variações dos valores obtidos entre eles. Assim, recomendam-se novas pesquisas para comprovação dessa hipótese acima mencionada. Mesmo havendo diferença nos valores da PIC entre as duas formas de mensurações, cabe salientar que a variação dos valores ocorreu em ambos os métodos, obtendo-se, com isso, uma relação linear aparente. Assim, foi possível determinar, com o auxílio do teste de regressão, uma diferença da PIC aferida com o cateter de ventriculostomia em relação à sonda uretral flexível, obtendo-se a fórmula: PIC cateter ventriculostomia= 11,43297+0,83634xPICsonda, cuja precisão é em torno de 54,87%. Outra fórmula obtida com precisão de 86,13%, nos testes estatísticos foi PIC=0,001241+ 0,01661.sonda² + FR.(-9,023+0,01741.FR) + FC.(-4,882 + 0,002305.FC + 0,01115.sonda + 0,00002452.FR) + oximetria.(-1,102 -0,02606.Sonda + 0,1666.horas + 0,06586.FR + 0,03754.FC - 0,01395.CO2) + CO2.(0,003611.CO2 - 0,009116.sonda + 0,02059.horas + 0,01034.FR + 0,002975.FC) + horas.(-7,155 - 0,1777.horas + 0,05662.FR - 0,01978.FC), porém com mais limitações em se aplicar por depender de outras variáveis. Ao preencher o balonete do cateter de Fogarty 4 Fr com 0,3mL de solução salina, a PIC em ambos os grupos aumentou significativamente comparada aos valores da PIC basal (Fig. 2D). O valor médio estimado da PIC nos 40 minutos de avaliação foi de 36,71mmHg no G1 e de 27,77mmHg no G2. Em todo o tempo de avaliação, os valores estimados da PIC mensurados pelo cateter foram significativamente (P<0,01) superiores aos da sonda uretral flexível. Quando o balonete foi preenchido com 0,6 mL de NaCl, os valores da PIC foram significativamente maiores do que os valores da PIC basal e quando comparados ao preenchimento com 0,3mL (P<0,05) (Fig. 2D). O valor médio da PIC nos 40 minutos finais de avaliação foi de 39,68mmHg no G1 e de 36,68mmHg no G2 e foi estatisticamente superior (P<0,05) aos valores da sonda uretral flexível até 15 minutos de avaliação, não diferindo a partir dos 20 minutos. Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 RESULTADOS E DISCUSSÃO Os níveis elevados de PaCO2 causam vasodilatação cerebral, enquanto a diminuição dos níveis de PaCO2 causa vasoconstrição cerebral (Dewey e Fletcher, 2016). Neste estudo, provavelmente ocorreram mecanismos de autorregulação (pressão e química), pois a PAM variou dentro dos valores limites para a sua realização (41,4 e 73mmHg), bem como os valores da PaCO2 (Fig. 2B), que permaneceram altos durante o preenchimento do balonete, com 0,3 e 0,6mL, ao serem comparados com o tempo antes da craniotomia, condição para a ativação da autorregulação química. A pressão de perfusão cerebral (PPC) em coelhos varia de 85 a 117mmHg (Çagavi et al., 2005). Neste estudo, foi verificado um valor médio de 29,84mmHg no G1 e de 33,69mmHg no G2 quando o balonete foi preenchido com 0,3mL e 15,15mmHg no G1 e 11,49mmHg no G2 com 0,6mL de NaCl 0,9% (Fig. 2C). Esse resultado demonstrou que o aumento da pressão intracraniana no decorrer do tempo provocou a redução da PPC, provavelmente pela diminuição do FSC (Fig. 3). Tal fato indica que, mesmo com os prováveis mecanismos autorregulatórios da PIC presentes, a pressão de perfusão cerebral se manteve baixa e não evitou uma possível isquemia do tecido encefálico (Çagavi et al., 2005). Quanto à PAM, houve diferença estatística entre os tempos e entre os métodos de mensuração nos tempos (P<0,01). Após o preenchimento do balonete com 0,3mL de NaCl, a PAM aumentou em ambos os grupos, porém os valores em G1 foram maiores que em G2. Após preencher o Figura 3. Relação da pressão de perfusão cerebral (PPC) e da pressão intracraniana (PIC) nos grupos cateter de ventriculostomia (A) e sonda uretral flexível (B), em coelhos submetidos ao modelo experimental de TCE, utilizando-se o cateter de Fogarty 4 Fr. Nota-se a diminuição da PPC, conforme o aumento da PIC no grupo com cateter de ventriculostomia após o início do preenchimento do balão com 0,3mL de NaCl 0,9%. Figura 3. Relação da pressão de perfusão cerebral (PPC) e da pressão intracraniana (PIC) nos grupos cateter de ventriculostomia (A) e sonda uretral flexível (B), em coelhos submetidos ao modelo experimental de TCE, utilizando-se o cateter de Fogarty 4 Fr. Nota-se a diminuição da PPC, conforme o aumento da PIC no grupo com cateter de ventriculostomia após o início do preenchimento do balão com 0,3mL de NaCl 0,9%. Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 1556 Sonda uretral flexível... CONCLUSÃO CHANG, C.; UCHIYAMA, A.M.A.L.; MAHIMO, T.; FUJINO, Y. A comparison of the effects of respiratory carbon dioxide response, arterial blood pressure, and heart rate of dexmedetomidine, propofol, and midazolam in sevoflurane-anesthetized rabbits. Anesth. Analg., v.109, p.84-89, 2009. Conclui-se que a sonda uretral flexível é de fácil aplicação, de baixo custo e pode ser considerada um material alternativo para a mensuração da PIC intraventricular em coelhos. Porém, os valores mensurados são inferiores aos encontrados pelo cateter de ventriculostomia, necessitando da aplicação de uma fórmula de correção, caso se queira obter os valores exatos da PIC. CZOSNYKA, M.; SMIELEWSKI, P.; TIMOFEEV, I. et al. Intracranial pressure: more than a number. Neurosurg. Focus, v.22, p.E10, 2007. DEWEY, C.W.; FLETCHER, D.J. Head-trauma management. In: DEWEY, C.W.; COSTA, R.C. (Eds.). Practical guide to canine and feline neurology. Ames: JohnWiley & Sons, 2016. p.237-248. Sonda uretral flexível... Com os resultados obtidos neste estudo, há possibilidade da utilização da sonda uretral flexível em coelhos, como alternativa ao cateter de ventriculostomia disponível comercialmente, cujo custo é extremamente elevado (R$1300,00) quando comparado ao método empregando a sonda uretral flexível (R$0,89). Porém, mesmo não fornecendo os valores exatos da PIC em comparação com o cateter de ventriculostomia e, portanto, havendo a necessidade de aplicação de uma fórmula de correção, a relevância deste estudo foi observar que a sonda uretral flexível foi capaz de mensurar as oscilações da PIC durante a monitoração do paciente. Esta etapa é considerada essencial na determinação da evolução do quadro clínico e na resposta terapêutica empregada. Esse achado corrobora os de Czosnyka et al. (2007), ao mencionarem que o valor numérico da PIC não é o dado mais importante obtido durante a monitorização, mas sim a dinâmica da PIC e as formas das ondas, pois, com estes, podem-se obter informações importantes sobre a homeostase cerebral. Tanto o cateter de ventriculostomia como a sonda uretral flexível são materiais cilíndricos com diâmetros internos similares e com dois orifícios drenantes que se situavam dentro do ventrículo lateral do encéfalo dos coelhos para aferição da PIC. O princípio de mensuração da PIC pelo método intraventricular ocorre pelo deslocamento da coluna líquida e do grau de deformação da parede do cateter, que contribui para o deslocamento do LCE e resulta na leitura de um valor que é registrado pelo monitor multiparamétrico (Srinivasan et al., 2014). Acredita-se que a maneira de aferição da PIC com a utilização da sonda uretral flexível seja a mesma do cateter de ventriculostomia. Por outro lado, a diferença no comprimento entre os cateteres pode ter influenciado na mensuração da 1557 Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 Aiello et al. Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 REERÊNCIAS ABE, T.; BLACK, P.M.; FOLEY, L. Changes in parenchymal and ventricular pressure with experimental epidural compression. Surg. Neurol., v.22, p.477-480, 1984. DONNELLY, T.M.; WARREN, K.S. Textbook of rabbit medicine. London: Elsevier, 2014. p.187-248. AMARAL, J.L.G.; FERREIRA, A.C.P.; FEREZ, D.; GERETTO, P. Monitorização da respiração: oximetria e capnografia. Rev. Bras. Anestesiol., v.42, p.51-58, 1992. DOUZINAS, E.E.; KOSTOPOULOS, V.; KYPRIADES, E. et al. Brain eigen frequency shifting as a sensitive index of cerebral compliance in an experimental model of epidural hematoma in the rabbit: preliminary study. Crit. Care. Med., v.27, p.978-984, 1999. BARZAGO, M.M.; BORTOLOTTI, A.; OMARINI, D. et al. Monitoring of blood gas parameters and acid-base balance of pregnant and non-pregnant rabbits (Oryctolaguscuniculus) in routine experimental conditions. Lab. Anim., v.26, p.73-79, 1992. FORSTER, A.; GARDAZ, J.P.; SUTER, P.M.; GEMPERLE, M. Respiratory depressin by midazolam and diazepam. Anesthesiology, v.53, p.494-497, 1980. BENATO, L.; CHESNEL, M.; EATWELL, K.; MEREDITH, A. Arterial blood gas parameters in pet rabbits anaesthetized using a combination of fentanyl-fluanisonemidazolam-isofluorane. J. Small Anim. Pract., v.54, p.343-346, 2013. FREEMAN, A.C.; PLATT, S.R. Head trauma. In: PLATT, S.R.; GAROSI, L.S. (Eds.). Small animal neurological emergencies. London: Manson, 2011. p.323-382. BINZ, D.D.; TOUSSAINT, L.G.; FRIEDMAN, J.A. Hemorrhagic complications of ventriculostomy placement: a meta-analysis. Neurocrit. Care, v.10, p.253-256, 2009. GIUGNO, K.M.; MAIA, T.R.; KUNRATH, C.L.; BIZZI, J.J. Tratamento da hipertensão intracraniana. J. Pediat., v.79, p.287-96, 2003. LANG, S.S.; KOFKE, W.A.; STIEFEL, M.F. Monitoring and intraoperative management of elevated intracranial pressure and decompressive craniectomy. Anesthesiol. Clin., v.30, 289-310, 2012. BRATTON, S.L.; BULLOCK, M.R.; CARNEY, N. et al. Guidelines for the management of severe traumatic brain injury. J. Neurotrauma, v.24, p.S37-S44, 2007. ÇAGAVI, F.; KALAVCI, M.; OZER, Y. et al. Dispersion of cerebral temperature, cerebral perfusion and intracranial pressure in rabbits placed with epidural balloons. Brain Res. Bull., v.64, p.481-485, 2005. LI, L.M.; TIMOFEEV, I.; CZOSNYKA, M.; HUTCHINSON, P.J. Review article: the surgical approach to the management of increased intracranial pressure after traumatic brain injury. Anesth. Analg., v.111, p.736-748, 2010. CARLOTTI JR, C.G.; COLLI, B.O.; DIAS, L.A.A. Hipertensão intracraniana. Rev. Med., v.31, p.552-562, 1998. 1558 Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 Sonda uretral flexível... ROUX, P.L. Physiological Monitoring of the severe traumatic brain injury patient in the intensive care unit. Cur. Neurol. Neurosci. Rep., v.13, p.1-16, 2013. LORENZ, M.D.; COATES, J.R.; KENT, M. Stupor or coma. In: LORENZ, M.D.; DACVIM, D.V.M.; COATES, J. Handbook of veterinary neurology. 5.ed. [Amsterdã]: Elsevier, 2011. p.346-383. Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 REERÊNCIAS SCHROEDER, C.A.; SMITH, L.J. Respiratory rates and arterial blood gas tensions in healthy rabbits given buprenorphine, butorphanol, midazolam, or their combinations. J. Am. Assoc. Lab. Anim. Sci., v.50, p.205-2011, 2011. MANIKER, A.H.; VAYNMAN, A.Y.; KARIMI, R.J. et al. Hemorrhagic complications of external ventricular drainage. Neurosurgery, v.59, p.419- 424, 2006. SMITH, M. Monitoring intracranial pressure in traumatic brain injury. Anesth. Analg., v.106, p.240-248, 2008. MIZUMOTO, N.; TANGO, H.K.; PAGNOCCA, M.L. Efeitos da hipertensão arterial induzida sobre a complacência e pressão de perfusão encefálica em hipertensão intracraniana experimental: comparação entre lesão encefálica criogênica e balão subdural. Rev. Bras. Anestesiol., v.55, p.289-298, 2005. SRINIVASAN, V.M.; O´NEILL, B.R.; JHO, D. et al. The history of external ventricular drainage. Neurosurgery, v.120, p.228-236, 2014. PACKER, R.A.; SIMMONS, J.P.; DAVIS, N.M.; CONSTABLE, P.D. Evaluation of an acute focal epidural mass model to characterize the intracranial pressure-volume relationship in healthy Beagles. Am. J. Vet. Res., v.72, p.103-8, 2011. 1559 Arq. Bras. Med. Vet. Zootec., v.69, n.6, p.1551-1559, 2017 1559
https://openalex.org/W4231158504	https://europepmc.org/articles/pmc6245833?pdf=render	English	null	Correction: Cataract surgery and age-related cognitive decline: A 13-year follow-up of the English Longitudinal Study of Ageing	PloS one	2,018	cc-by	260	CORRECTION Asri Maharani, Piers Dawes, James Nazroo, Gindo Tampubolon, Neil Pendleton, on behalf of the SENSE-Cog WP1 group The following information is missing from the Funding section: PD is supported by the NIHR Manchester Biomedical Research Centre. OPEN ACCESS Citation: Maharani A, Dawes P, Nazroo J, Tampubolon G, Pendleton N, on behalf of the SENSE-Cog WP1 group (2018) Correction: Cataract surgery and age-related cognitive decline: A 13-year follow-up of the English Longitudinal Study of Ageing. PLoS ONE 13(11): e0208045. https://doi.org/10.1371/journal.pone.0208045 1. Maharani A, Dawes P, Nazroo J, Tampubolon G, Pendleton N, on behalf of the SENSE-Cog WP1 group (2018) Cataract surgery and age-related cognitive decline: A 13-year follow-up of the English Longitudinal Study of Ageing. PLoS ONE 13(10): e0204833. https://doi.org/10.1371/journal.pone. 0204833 PMID: 30307960 Reference 1. Maharani A, Dawes P, Nazroo J, Tampubolon G, Pendleton N, on behalf of the SENSE-Cog WP1 group (2018) Cataract surgery and age-related cognitive decline: A 13-year follow-up of the English Longitudinal Study of Ageing. PLoS ONE 13(10): e0204833. https://doi.org/10.1371/journal.pone. 0204833 PMID: 30307960 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Correction: Cataract surgery and age-related cognitive decline: A 13-year follow-up of the English Longitudinal Study of Ageing Asri Maharani, Piers Dawes, James Nazroo, Gindo Tampubolon, Neil Pendleton, on behalf of the SENSE-Cog WP1 group Published: November 20, 2018 Copyright: © 2018 Maharani 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. 1 / 1 PLOS ONE \| https://doi.org/10.1371/journal.pone.0208045 November 20, 2018
https://openalex.org/W3134202461	https://www.medrxiv.org/content/medrxiv/early/2021/03/11/2021.03.08.21253172.full.pdf	English	null	Comparing Interventions Aimed at Improving Influenza Vaccination Coverage Among Vulnerable Populations: A Systematic Review and Meta-Analysis Protocol	medRxiv (Cold Spring Harbor Laboratory)	2,021	cc-by	5,721	. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint reprint reports new research that has not been certified by peer review and should not be used to guide clinical practice Comparing Interventions Aimed at Improving Influenza Vaccination Coverage Among Vulnerable Populations: A Systematic Review and Meta-Analysis Protocol CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint Background Influenza is a major contributor to global disease burden. Vaccination recommendations specifically target populations at increased risk of serious influenza sequelae. The aim of this study is to conduct a systematic review and meta-analysis to evaluate the effectiveness of different quality improvement interventions to increase vaccination rates in high-risk populations. Comparing Interventions Aimed at Improving Influenza Vaccination Coverage Among Vulnerable Populations: A Systematic Review and Meta-Analysis Protocol Pei Chen Wu BSc1,2 Lyn McPherson GradDipHlthSc2,3 Stephen B Lambert PhD4,5 Peter Wnukowski-Mtonga MClinEpi4,5 Nicholas G Lennox PhD2 Robert S Ware PhD2,3 1. School of Public Health and Health Systems. University of Waterloo, ON N2L3G1, Canada. 2. Queensland Centre for Intellectual and Developmental Disability, MRI-UQ, The University of Queensland, South Brisbane QLD 4101, Australia. 3. Menzies Health Institute Queensland, Griffith University, Nathan QLD 4111, Australia. 4. UQ Centre for Child Health Research, The University of Queensland, Herston QLD 4006, Australia. 5. National Centre for Immunisation Research & Surveillance, The Children’s Hospital at Westmead, Westmead, NSW 2145, Australia. Corresponding Author: Robert Ware Postal Address: Menzies Health Insitute Queensland, Griffith University, 170 Kessels Road, Nathan QLD 4111, Australia Email Address: R.Ware@griffith.edu,au Telephone Number: +61 7 334 64836 2. Queensland Centre for Intellectual and Developmental Disability, MRI-UQ, The University of Queensland, South Brisbane QLD 4101, Australia. 2. Queensland Centre for Intellectual and Developmental Disability, MRI-UQ, The University of Queensland, South Brisbane QLD 4101, Australia. 3. Menzies Health Institute Queensland, Griffith University, Nathan QLD 4111, Australia. 3. Menzies Health Institute Queensland, Griffith University, Nathan QLD 4111, Australia. 3. Menzies Health Institute Queensland, Griffith University, Nathan QLD 4111, Australia. 4. UQ Centre for Child Health Research, The University of Queensland, Herston QLD 4006, Australia. 4. UQ Centre for Child Health Research, The University of Queensland, Herston QLD 4006, Australia. 4. UQ Centre for Child Health Research, The University of Queensland, Herston QLD 4006, Australia. 5. National Centre for Immunisation Research & Surveillance, The Children’s Hospital at Westmead, Westmead, NSW 2145, Australia. 5. National Centre for Immunisation Research & Surveillance, The Children’s Hospital at Westmead, Westmead, NSW 2145, Australia. 5. National Centre for Immunisation Research & Surveillance, The Children’s Hospital at Westmead, Westmead, NSW 2145, Australia. Corresponding Author: Robert Ware Postal Address: Menzies Health Insitute Queensland, Griffith University, 170 Kessels Road, Nathan QLD 4111, Australia Email Address: R.Ware@griffith.edu,au Telephone Number: +61 7 334 64836 NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . BACKGROUND Influenza is a vaccine-preventable acute respiratory infection directly responsible for three to five million cases of severe illness annually, resulting in 250,000 to 500,000 deaths.1 The financial burden of influenza and its sequelae are substantial: estimates from the United States are of annual direct losses of US$10.4 billion as a direct consequence of influenza, with an additional US$16.3 billion lost in sick days and productivity.2 Influenza disproportionately affects infants, young children, adults aged 65 years or older, pregnant women, immunocompromised individuals, First Nations peoples, and those suffering from chronic disease; all of whom are at risk of experiencing increased morbidity and mortality due to influenza.3,4 Current health care guidelines in most developed nations recommend annual vaccination for the aforementioned high-risk groups. In order to maximize the benefits these populations derive from immunization, the US Department of Health and Human Services has launched the Healthy People 2020 initiative in order to improve preventive health care.5 The initiative aims to increase influenza vaccination rates for various high-risk groups to targets ranging from 70 to 90 percent. Vaccination coverage rates for vulnerable populations have been rising substantially over the past two decades in most developed countries. In Australia, between 1 March and 19 April 2020, over 2.1 million influenza vaccinations were administered and recorded in the Australian Immunisation Register compared to 624,000 at the same time in 2019 and 235,000 in 20186, as government, GPs and other vaccine providers encouraged people to get vaccinated to avoid the combination of COVID-19 and influenza which could be life threatening, but inconsistent and even decreasing trends in coverage have been recently reported in the U.S,7 Europe8 and Canada,9 indicating that coverage rates still fall short of the goals proposed by the Healthy People 2020 initiative. In developed nations, the majority of influenza vaccinations take place in community settings. community-based primary care centers remain the most effective way to promote health to the public, and various outreach initiatives have been trialled in the community with the aim to increase influenza vaccination coverage in vulnerable populations. Ompad et al.10 previously reviewed over 50 studies examining interventions directed towards high-risk populations, and found the majority of interventions did not result in vaccination rates that met the Healthy People 2010 goals. Moreover, the study did not perform statistical analyses to evaluate the effectiveness of each intervention, and did not focus on community-settings. Methods Randomized and nonrandomized studies with concurrent control groups will be identified. Interventions to increase vaccination rates will be categorized by strategy type. Overall intervention effects will be calculated using random effects models. Study quality will be assessed using a modified Cochrane Risk of Bias tool. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint BACKGROUND A previous systematic review and meta-analysis by Lau et al.11 found that various quality improvement . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. ch was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: v preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint interventions were effective in increasing influenza and pneumococcal vaccination coverage in elderly adults, but did not examine intervention effects on other vulnerable populations, and did not focus exclusively on primary care interventions. The aim of this study is to systematically evaluate, compare, and analyze the effectiveness of different intervention strategies for improving influenza vaccination coverage, with the goal of identifying the most effective strategies to improve vaccination coverage among vulnerable populations in the community. This review protocol will follow the guidelines for the Preferred Reporting Items for Systematic Reviews and Metanalysis (PRISMA)12 and is reported here using the Guidance notes for registering a systematic review protocol with PROSPERO provided by the Centre for Reviews and Dissemination.13 1. Review title Comparing Interventions Aimed at Improving Influenza Vaccination Coverage Among Vulnerable Populations: A Systematic Review and Meta-Analysis 2. Original language title As above 3. Anticipated or actual start date 15 March 2021 4. Anticipated completion date 30 September 2021 2. Original language title As above 3. Anticipated or actual start date 15 March 2021 10. Organisational affiliation Menzies Health Institute Queensland, Griffith University 9. Named contact phone number +61 7 3735 9117 9. Named contact phone number +61 7 3735 9117 11. Review team members and their organisation affiliations Pei Chen Wu; Faculty of Medicine, The University of Queensland; Queensland Centre for Intellectual and Developmental Disability, The University of Queensland Lyn McPherson; Menzies Health Institute Queensland, Griffith University; Queensland Centre for Intellectual and Developmental Disability, The University of Queensland Stephen B Lambert, Faculty of Medicine, The University of Queensland Peter Wnukowski-Mtonga; National Centre for Immunisation Research & Surveillance, The Children’s Hospital at Westmead Nicholas G Lennox; Queensland Centre for Intellectual and Developmental Disability, MRI- UQ, The University of Queensland Robert S. Ware; Menzies Health Institute Queensland, Griffith University; Queensland Centre for Intellectual and Developmental Disability, The University of Queensland 5. Stage of review at time of this submission This review topic was first explored in December 2016/January 2017 and then again in December 2017/January 2018 as part of a University of Queensland Summer Scholarship Program. During this time the search strategy was developed and the publications appearing prior to December 2017 were identified, the study characteristics were recorded, and data was . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint extracted. After this Protocol is submitted the search will be re-run and eligible publications will be identified for inclusion in meta-analyses. extracted. After this Protocol is submitted the search will be re-run and eligible publications will be identified for inclusion in meta-analyses. 8 .Named contact address Centre for Applied Health Economics, Griffith University, 170 Kessels Road, Nathan QLD 4111, AUSTRALIA 17. URL to search strategy The final search strategies as used in the first version of this systematic review are detailed in Appendix I. Appendix I. 12. Funding sources This work was supported by a University of Queensland Summer Scholarship (PW). . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint 16. Searches We will identify relevant studies for synthesis using four databases (Cochrane Library via John Wiley & Sons Inc., MEDLINE via EBSCOHost, EMBASE via Elsevier, and CINAHL via EBSCOHost). Search strategies for each database were prepared by PW in consultation with other team members and a university library Information specialist. Preliminary searches were conducted using the various suggested terms to pilot the study selection process. After screening search results against eligibility criteria, modifications were made as considered necessary. 15. Review question Which vaccination strategies are the most effective for improving influenza vaccination coverage among vulnerable populations in the community? 13. Conflicts of interest All authors declare they have no known conflicts of interest. 19. Participants/population Studies will be included if they compared the effectiveness of a quality improvement intervention against a contemporaneous comparison group in a community setting. For inclusion, studies will need to report vaccination rates, be published in an English language peer-reviewed journal and involve a high-risk population living in the community. A population is defined as being high risk if it was included in the guidelines provided by the . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: edRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint relevant health authorities in the USA, Australia, Canada, or the United Kingdom (See Appendix 2). 20. Interventions/exposures Strategies will be categorized based on the classification system proposed by Shojania et al.14 and subsequently adapted by Lau et al.11. Categories will be audit and feedback, case management, clinician education, clinician reminders, community engagement, continuous quality improvement, delivery site change, financial incentives for clinicians, financial incentives for patients, patient outreach, changes in care team personnel (team change), and changes to patient visit routine (visit structure change). Where appropriate, quality improvement strategies with a sufficient number of comparisons will be organized into sub- categories, and meta-analyses will be conducted within each sub-category. 21. Comparator/control Comparator group will be either usual care, non-vaccination intervention, or, when intervention group received additional strategy, whatever strategy the control group received. 22. Types of study to be included Randomised Controlled Trials (RCTs) and quasi RCTs, 23. Context Studies need to be conducted among groups living in the community. Studies need to be conducted among groups living in the community. 24. Main outcomes 24. Main outcomes Vaccination rate Vaccination rate 26. Data extraction (selection and coding) The bibliographic software, EndNote, will be employed to organize, store, and manage all the citations. References retrieved from the four databases will be imported into Endnote. After removal of duplicate references, reviewers (PW and RW) will select studies for inclusion in the meta-analyses, first by screening of the titles and abstracts, and secondly, using full texts. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint We will attempt to obtain any references not available on-line or locally with the assistance of the University Library. Any differences in the selection of studies for inclusion will be resolved by discussion between reviewers and the other members of the research team if necessary. PW will extract data from each selected study. 27. Risk of bias (quality) assessment Study quality will be measured using a modified version of the Cochrane Risk of Bias (ROB) tool,15 a 6-item questionnaire assessing the appropriateness of random sequence generation, allocation concealment, blinding of participants, personnel, and outcome assessors, data completeness, and other sources of biases. For quality assessment we summed individual scores. 28. Strategy for data synthesis All studies reporting sufficient data to derive odds ratios and standard errors will be included in meta-analyses. Comparisons will be eligible to be included in the meta-analysis if the control group received usual care, or if the control intervention was focused towards non-vaccination behaviour, or if the intervention group received an additional strategy as well as whatever strategy was applied to both the intervention and control groups. When a given intervention can be classified into multiple quality improvement strategies, the number of subjects in the intervention group will divided between the strategies to avoid double-counting participants. Results from individual studies will be synthesized using random-effects models. Results will be reported overall, and by quality improvement strategy. Heterogeneity will be measured using the I2 statistic. The number needed to target for vaccination will be calculated. Sensitivity analyses will be conducted to investigate any association between study quality (assessed by ROB scores) and reported effect sizes using meta-regression. Publication bias will be tested by generating funnel plots and applying Harbord’s test.16 Analyses will be performed using Stata v14.0 (StataCorp LP, College Station, TX, USA). 30. Type and method of review Type of review - Systematic review and meta-analysis Health area of review – Public Health Type of review - Systematic review and meta-analysis Type of review - Systematic review and meta-analysis Health area of review – Public Health . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint 31. Language English 32. Country Australia 33. Other registration details Nil 38. Current review status 39. Any additional information Nil See below See below 35. Dissemination plans p It is intended to publish the results of this study in a peer-reviewed journal. Any amendments to this proposal will be documented and communicated in the final published manuscript. 36. Keywords Influenza, Vaccination, Immunization, Vulnerable Populations, Risk Factors . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint REFERENCES 1. Troeger C, Forouzanfar M, Rao PC, Khalil I, Brown A, Swartz S, et al. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower 1. Troeger C, Forouzanfar M, Rao PC, Khalil I, Brown A, Swartz S, et al. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint respiratory tract infections in 195 countries: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Infect Dis. 2017;17(11):1133-1161. respiratory tract infections in 195 countries: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Infect Dis. 2017;17(11):1133-1161. 2. Molinari NAM, Ortega-Sanchez IR, Messonnier ML, Thompson WW, Wortley PM, Weintraub E, et al. The annual impact of seasonal influenza in the US: measuring disease burden and costs. Vaccine. 2007;25(27):5086-5096. 3. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. J Amer Med Assoc. 2003;289(2):179-186. 3. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N, Anderson LJ, et al. Mortality associated with influenza and respiratory syncytial virus in the United States. J Amer Med Assoc. 2003;289(2):179-186. 4. Harper SA, Fukuda K, Uketi TM, Cox NJ, Bridges CB. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep. 2005;54(8):1-41. 4. Harper SA, Fukuda K, Uketi TM, Cox NJ, Bridges CB. Prevention and control of influenza: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep. 2005;54(8):1-41. 5. Office of Disease Prevention and Health Promotion. Immunization and Infectious Diseases. Healthy People 2020. Rockville, MD: ODPHP. https://www.healthypeople.gov/2020/topics-objectives/topic/immunization-and- infectious-diseases/objectives. Accessed Oct 20, 2020. 5. Office of Disease Prevention and Health Promotion. Immunization and Infectious Diseases. Healthy People 2020. Rockville, MD: ODPHP. https://www.healthypeople.gov/2020/topics-objectives/topic/immunization-and- infectious-diseases/objectives. Accessed Oct 20, 2020. 6. REFERENCES Hunt G. Record 16.5 million flu vaccines to protect Australians (media release). Canberra, Australia: Department of Health; 19 April 2020. https://www.health.gov.au/ministers/the-hon-greg-hunt-mp/media/record-165- million-flu-vaccines-to-protect- australians#:~:text=Since%201%20March%202020%20over,2019%20and%20235%2 C000%20in%202018. Accessed Oct 20, 2020. australians#:~:text=Since%201%20March%202020%20over,2019%20and%20235%2 C000%20in%202018. Accessed Oct 20, 2020. 7. Centers for Disease Control and Prevention. Flu Vaccination Trends. Atlanta,GA: CDC. https://www.cdc.gov/flu/fluvaxview/trends.htm. Accessed Oct 20, 2020. 7. Centers for Disease Control and Prevention. Flu Vaccination Trends. Atlanta,GA: CDC. https://www.cdc.gov/flu/fluvaxview/trends.htm. Accessed Oct 20, 2020. 8. European Centre for Disease Prevention and Control. Seasonal Influenza Vaccination in Europe: Vaccination Recommendations and Coverage Rates in the EU Member States for Eight Influenza Seasons, 2007-2008 to 2014-2015. Stockholm, Sweden: ECDC; 2017. https://ecdc.europa.eu/sites/portal/files/documents/influenza- vaccination-2007–2008-to-2014–2015.pdf. Accessed Oct 20, 2020. 9. Buchan SA, Kwong JC. Trends in influenza vaccine coverage and vaccine hesitancy in Canada, 2006/07 to 2013/14: results from cross-sectional survey data. Can Med Assoc J Open. 2016;4(3):E455-462. 10. Ompad DC, Galea S, Vlahov D. Distribution of influenza vaccine to high-risk groups. Epidemiol Rev. 2006;29(1):54-70. . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint 11. Lau D, Hu J, Majumdar SR, Storie DA, Rees SE, Johnson JA. Interventions to improve influenza and pneumococcal vaccination rates among community-dwelling adults: a systematic review and meta-analysis. Ann Fam Med. 2012;10(6):538-546. 12. Moher D, Shamseer L, Clarke M, Ghersi D, Liberati A, Petticrew M, el al. Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Systematic reviews. 2015 Dec 1;4(1):1. 13. Centre for Reviews and Dissemination. Guidance notes for registering a systematic review protocol with PROSPERO. York, UK: University of York; 2016. https://www.crd.york.ac.uk/prospero/documents/Registering%20a%20review%20on %20PROSPERO.pdf. Accessed Oct 20, 2020 14. Shojania KG, Ranji SR, McDonald KM, Grimshaw JM, Sundaram V, Rushakoff RJ, et al. Effects of quality improvement strategies for type 2 diabetes on glycemic control: a meta-regression analysis. J Amer Med Assoc. 2006;296(4):427-440. 15. Higgins JP, Altman DG, Gøtzsche PC, Jüni P, Moher D, Oxman AD, et al. REFERENCES The Cochrane Collaboration’s tool for assessing risk of bias in randomized trials. BMJ Brit Med J. 2011;343:d5928. 16. Harbord RM, Egger M, Sterne JA. A modified test for small-study effects in meta- analyses of controlled trials with binary endpoints. Stat Med. 2006;25(20):3443-345 . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint Appendix I: Database Search Strategies Appendix I: Database Search Strategies MedLine: Search performed on November 18th, 2016; returning 1090 articles. # Query 1 (MH "Influenza Vaccines") 2 MH vaccines 3 MH immunization 4 vaccin* 5 "immuni" 6 "inoculat" 7 "prevent" 8 1 OR 2 OR 3 OR 4 OR 5 OR 6 OR 7 9 (MH "Influenza, Human") 10 (MH "Influenza B virus") OR (MH "Influenza A virus") OR (MH "Influenza C virus") 11 (MH "Influenzavirus A") OR (MH "Influenzavirus B") OR (MH "Influenzavirus C" ) 12 AB influenza OR TI influenza 13 AB flu OR TI flu 14 9 OR 10 OR 11 OR 12 OR 13 15 AB primary care OR TI primary care 16 AB General practi OR TI General practi* 17 AB Primary health* OR TI Primary health* 18 AB Community mental health* OR TI Community mental health* 19 AB Family practice OR TI Family practice 20 AB Family medicine OR TI Family medicine 21 AB Family physician* OR TI Family physician* 22 TI Home care OR AB Home care 23 AB Home based OR TI Home based 24 AB Home health* OR TI Home health* 25 AB Community health* OR TI Community health* 26 AB Community nurs* OR TI Community nurs* 27 AB health visit* OR TI health visit* 28 AB Community pharmac* OR TI Community pharmac* 29 AB Preventive care OR TI Preventive care 30 AB Prevention program* OR TI Prevention program* 31 AB Preventive OR TI Preventive 32 15 OR 16 OR 17 OR 18 OR 19 OR 20 OR 21 OR 22 OR 23 OR 24 OR 25 OR 26 OR 27 OR 28 OR 29 OR 30 OR 31 33 PT randomized controlled trial 34 "random" . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. MedLine: ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint 35 "control" 36 "intervention" 37 "evaluat" 38 "compar" 39 "impact" 40 33 OR 34 OR 35 OR 36 OR 37 OR 38 OR 39 41 "rates" 42 "rate" 43 "distribution" 44 "coverage" 45 "status" 46 "delivery" 47 "program" 48 "intervention" 49 "promotion" 50 "initiative" 51 "strategy" 52 "uptake" 53 41 OR 42 OR 43 OR 44 OR 45 OR 46 OR 47 OR 48 OR 49 OR 50 OR 51 OR 52 54 MH humans 55 8 AND 14 AND 32 AND 40 AND 53 AND 54 Limiters - English Language; Age Related: Infant, Newborn: birth-1 month, Infant: 1-23 months, All Infant: birth-23 months, Child, Preschool: 2-5 years, Young Adult: 19-24 years, Adult: 19-44 years, Middle Aged: 45-64 years, Middle Aged + Aged: 45 + years, Aged: 65+ years, Aged, 80 and over, All Adult: 19+ years Update: Search performed on Nov 27th, 2017; returning 1,189 results. Filter for papers published between 2016-2017 yielded an additional 109 results. Update: Search performed on Nov 27th, 2017; returning 1,189 results. Filter for papers published between 2016-2017 yielded an additional 109 results. Embase: ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint care':ab,ti OR 'home based':ab,ti OR 'home health':ab,ti OR 'health visit':ab,ti OR 'health workers' OR 'community nurs':ab,ti OR 'community pharmac':ab,ti OR 'community health':ab,ti OR 'community health services'/exp OR 'preventive health services'/exp OR 'preventive care':ab,ti OR 'prevention program':ab,ti 5 randomized:it AND controlled:it AND trial:it OR random OR control* OR intervention* OR compar* OR impact OR evaluat* 6 'influenza'/exp OR 'influenza b virus'/exp OR 'influenzavirus a'/exp OR 'influenzavirus c'/exp OR influenza:ab,ti OR flu:ab,ti 7 [english]/lim AND [humans]/lim 8 #1 AND #2 AND #3 AND #4 AND #5 AND #6 AND #7 9 [adult]/lim OR [aged]/lim OR [infant]/lim OR [middle aged]/lim OR [newborn]/lim OR [preschool]/lim OR [very elderly]/lim OR [young adult]/lim 10 #8 AND #9 care':ab,ti OR 'home based':ab,ti OR 'home health':ab,ti OR 'health visit':ab,ti OR 'health workers' OR 'community nurs':ab,ti OR 'community pharmac':ab,ti OR 'community health':ab,ti OR 'community health services'/exp OR 'preventive health services'/exp OR 'preventive care':ab,ti OR 'prevention program':ab,ti 5 randomized:it AND controlled:it AND trial:it OR random* OR control* OR intervention* OR compar* OR impact OR evaluat* 6 'influenza'/exp OR 'influenza b virus'/exp OR 'influenzavirus a'/exp OR 'influenzavirus c'/exp OR influenza:ab,ti OR flu:ab,ti 7 [english]/lim AND [humans]/lim 8 #1 AND #2 AND #3 AND #4 AND #5 AND #6 AND #7 9 [adult]/lim OR [aged]/lim OR [infant]/lim OR [middle aged]/lim OR [newborn]/lim OR [preschool]/lim OR [very elderly]/lim OR [young adult]/lim 10 #8 AND #9 Update: Search performed on Nov 27th 2017; returning 1 215 results Filter for papers randomized:it AND controlled:it AND trial:it OR random* OR control* OR intervention* OR compar* OR impact OR evaluat* Update: Search performed on Nov 27th, 2017; returning 1,215 results. Filter for papers published between 2016-2017 yielded an additional 136 results. Search performed on November 18th, 2016; returning 1283 articles. # Query 1 (immuniz* or immunis* or inoculat* or vaccin):ti,ab,kw and (influenza):ti,ab,kw (Word variations have been searched) 2 rate or rates or uptake or delivery or distribution or coverage or status:ti,ab,kw or "quality improvement" or qi or "quality management" or "case management" or "patient care" or interdisciplinary:ti,ab,kw or registr or audit or education or reminder* or "self care" or "self management" or "medical record" or program* or intervention:ti,ab,kw or "primary care":ti,ab,kw (Word variations have been searched) 3 #1 and #2 Update: Search performed on Nov 27th, 2017; returning 1,453 results. Filter for papers published between 2016-2017 yielded an additional 216 results. Update: Search performed on Nov 27th, 2017; returning 1,453 results. Filter for papers published between 2016-2017 yielded an additional 216 results. Update: Search performed on Nov 27th, 2017; returning 1,453 results. Filter for papers published between 2016-2017 yielded an additional 216 results. Embase: Search performed on November 18th, 2016; returning 1149 articles. # Query 1 ('influenza' OR influenza OR flu) AND 'influenza' OR influenza OR flu 2 'influenza vaccines'/exp OR 'vaccine'/exp OR 'immunization'/exp OR vaccin OR immuni* OR inoculat* OR prevent* 3 rates OR rate OR uptake OR determinant* OR tool* OR distribution OR coverage OR status OR delivery OR program* OR interven* OR promot* OR initiat* OR strateg* 4 'primary care'/exp OR 'primary health care'/exp OR 'primary health':ab,ti OR 'general practise' OR 'general practi':ab,ti OR 'family practice':ab,ti OR 'family physician' OR 'family medicine':ab,ti OR 'community mental health':ab,ti OR 'community mental health services':ab,ti OR 'home care services'/exp OR 'home . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. CINAHL: CINAHL: Search performed on November 18th, 2016; returning 140 articles. # Query 1 MH immunization 2 TI vaccin OR AB vaccin* 3 TI immunis* OR AB immunis* 4 TI immuniz* OR AB immuniz* 5 TI inoculat* OR AB inoculat* Search performed on November 18th, 2016; returning 140 articles. # Query 1 MH immunization 2 TI vaccin* OR AB vaccin* 3 TI immunis* OR AB immunis* 4 TI immuniz* OR AB immuniz* 5 TI inoculat* OR AB inoculat* Search performed on November 18th, 2016; returning 140 articles. # Query 1 MH immunization 2 TI vaccin* OR AB vaccin* 3 TI immunis* OR AB immunis* 4 TI immuniz* OR AB immuniz* 5 TI inoculat* OR AB inoculat* Search performed on November 18th, 2016; returning 140 articles. Search performed on November 18th, 2016; returning 140 articles. # Query 1 MH immunization 2 TI vaccin* OR AB vaccin* 3 TI immunis* OR AB immunis* 4 TI immuniz* OR AB immuniz* 5 TI inoculat* OR AB inoculat* . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted March 11, 2021. ; https://doi.org/10.1101/2021.03.08.21253172 doi: medRxiv preprint 6 MH Influenza 7 TI influenza OR AB influenza 8 MH Influenza Vaccine 9 S1 OR S2 OR S3 OR S4 OR S5 10 S6 OR S7 11 S9 AND S10 12 S8 OR S11 13 S8 OR S11 Limiters - English Language; Exclude MEDLINE records; Human; Age Groups: Infant, Newborn: birth-1 month, Infant: 1-23 months, Child, Preschool: 2-5 years, Adult: 19-44 years, Middle Aged: 45-64 years, Aged: 65+ years, Aged, 80 and over, All Infant, All Adult Update: Search performed on Nov 27th, 2017; returning 209 results. Filter for papers published between 2016-2017 yielded an additional 92 results. Update: Search performed on Nov 27th, 2017; returning 209 results. Filter for papers published between 2016-2017 yielded an additional 92 results. Update: Search performed on Nov 27th, 2017; returning 209 results. Update: Search performed on Nov 27th, 2017; returning 209 results. Filter for papers published between 2016-2017 yielded an additional 92 results. CINAHL: Filter for papers published between 2016-2017 yielded an additional 92 results.
https://openalex.org/W4308354474	https://bmcwomenshealth.biomedcentral.com/counter/pdf/10.1186/s12905-022-02024-1	English	null	Effect of vitamin D supplementation on the severity of stress urinary incontinence in premenopausal women with vitamin D insufficiency: a randomized controlled clinical trial	BMC women's health	2,022	cc-by	6,574	Shahraki et al. BMC Women’s Health (2022) 22:431 https://doi.org/10.1186/s12905-022-02024-1 Shahraki et al. BMC Women’s Health (2022) 22:431 https://doi.org/10.1186/s12905-022-02024-1 Abstract Background: Urinary incontinence, especially stress urinary incontinence (SUI), is one of the problems experienced by premenopausal women. Given the role of vitamin D in enhancing muscle strength and function, this study explored the effect of vitamin D3 supplementation on SUI in premenopausal women. Methods: A randomized controlled trial was performed with 60 premenopausal women referring to Kerman gynecological clinic in 2020 and 2021. Eligible women received a 5000-unit vitamin D supplement or placebo weekly for 3 months. The International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form (ICIQ-SF) was utilized to assess SUI severity before and after the intervention. The t-test, Chi-square test, and repeated measures ANOVA were carried out in SPSS software (version 22) to analyze the data. P-values smaller than 0.05 were considered significant. Results: Before the intervention, there was no significant difference between the intervention and control groups in SUI severity (P = 0.652) and the impact of SUI severity on premenopausal women’s lives (P = 0.804). In contrast, after 8-12 weeks of vitamin D supplementation, these scores decreased significantly in the intervention group relative to the control group (P < 0.001). In addition, after vitamin D supplementation, the number of SUI and urinary leakage symptoms decreased in the intervention group (P < 0.001). Conclusion: Vitamin D supplementation improves SUI in premenopausal women. Trial registration: This trial was registered with the Iranian Registry of Clinical Trials; https://fa.irct.ir/trial/53474 (IRCT20190724044318N2) on 11/02/2021. Keywords: Vitamin D3, Stress urinary incontinence, Premenopausal Keywords: Vitamin D3, Stress urinary incontinence, Premenopausal Sedigheh Khodabandeh Shahraki1, Seyedeh Fatemeh Emadi2, Mahla Salarfard3, Zahra Chenari2, Faezeh Tadayyonfar4 and Maryam Alikamali5* Sedigheh Khodabandeh Shahraki1, Seyedeh Fatemeh Emadi2, Mahla Salarfard3, Zahra Chenari2, Faezeh Tadayyonfar4 and Maryam Alikamali5* © 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. Introduction Prior to menopause, a woman goes through perimen- opause or premenopause, a period characterized by irregular bleeding and some endocrine disorder symp- toms. Aging is associated with a gradual loss of mus- cle strength [1]. Alongside this, weakened pelvic floor Correspondence: M_kamali1984@yahoo.com 5 Student Research Committee, Kerman University of Medical Sciences, Kerman, Iran Kerman, Iran Full list of author information is available at the end of the article Effect of vitamin D supplementation on the severity of stress urinary incontinence in premenopausal women with vitamin D insufficiency: a randomized controlled clinical trial Sedigheh Khodabandeh Shahraki1, Seyedeh Fatemeh Emadi2, Mahla Salarfard3, Zahra Chenari2, Faezeh Tadayyonfar4 and Maryam Alikamali5 Methods muscles are linked with urinary incontinence, the most frequently reported symptom among pelvic floor disor- ders [2]. Stress urinary incontinence (SUI) is the most common type of urinary incontinence, with a preva- lence of 50% [3]; its prevalence among Iranian women aged 40 to 50 is approximately 38.4% [4]. SUI occurs during times of elevated intraabdominal pressure (e.g., sneezing, coughing, and exercise) [5]. Since leva- tor muscles play an important role in maintaining the urethra’s closure by supporting pelvic organs, strength- ening the pelvic floor muscles through exercise and training is recommended as an effective treatment for stress incontinence [6]. muscles are linked with urinary incontinence, the most frequently reported symptom among pelvic floor disor- ders [2]. Stress urinary incontinence (SUI) is the most common type of urinary incontinence, with a preva- lence of 50% [3]; its prevalence among Iranian women aged 40 to 50 is approximately 38.4% [4]. SUI occurs during times of elevated intraabdominal pressure (e.g., sneezing, coughing, and exercise) [5]. Since leva- tor muscles play an important role in maintaining the urethra’s closure by supporting pelvic organs, strength- ening the pelvic floor muscles through exercise and training is recommended as an effective treatment for stress incontinence [6]. Research setting, design, and participants This study is designed as a parallel group randomized clinical trial. The protocol of the trial was approved by 82the Ethics Committee of Kerman University of Medical Sciences (ethics code: KMU.REC.1399.555) and registered on the Iranian Registry of Clinical Trials (identifier: IRCT20190724044318N2). i From March 2020 to September 2021, the study enrolled premenopausal women with SUI who were referred to a gynecological clinic affiliated with Kerman University of Medical Sciences. Prior to sampling, all candidates were assured of the study’s objectives and procedures and were requested to sign informed consent forms as a sign of willingness to participate in the study. Moreover, they were assured they could withdraw from the study anytime. It is plausible from a biological standpoint that the epidemic of vitamin D deficiency may have significant clinical effects on the pelvic floor muscles [7]. Accord- ing to in vivo studies, the vitamin D receptor is located in the bladder neck, and consists of the urothelium and the inner length of the circle, as well as the middle and smooth muscle layers of the outer length of the bladder wall [8]. Randomizationh The random assignment sequence was generated using a computer program (http://www.random.org) with a 1:1 allocation ratio (blocks 4 and 6). Methods The presence of vitamin D receptors on both the smooth and skeletal muscles of the bladder, as well as 1-alpha hydroxylase in prostate cells, indicates that vitamin D can aid in both stress and emergency uri- nary incontinence, which are the most typical types of urinary incontinence in adults [8–10]. It is, therefore, conceivable that vitamin D receptors are dispersed throughout the bladder wall [7]. © 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. Shahraki et al. BMC Women’s Health (2022) 22:431 Shahraki et al. BMC Women’s Health (2022) 22:431 Page 2 of 8 Inclusion and exclusion criteria Literate women aged 40 to 49 years who had serum vitamin D levels below 30 ng/ml(18) and confirmed SUIs were included in the study [8]. Exclusion crite- ria included being reluctant to continue participation, using hormonal medications, and undergoing urogen- ital-related surgery. Participants were not included if they had any disorder that interfered with vitamin D absorption, such as inflammatory bowel disease, intes- tinal bypass surgery, or chronic liver or kidney disease [7], and any neurological disease affecting the urinary system or bowel movements, such as multiple sclerosis, degenerative muscle disease, a history of stroke with spinal cord injury, and chronic or high-grade diabetes [7, 8, 17]. Moreover, all chronic or current infectious diarrhea cases were excluded [7]. Epidemiological data regarding the connection between vitamin D status and urinary incontinence are contradictory [11]. According to some previous observational cohort studies, a higher concentration of 25-hydroxyvitamin D has been linked to a lower preva- lence and incidence of urinary incontinence [12–15]. Other studies have not found a significant correlation [8–10, 16]. Elin et al. found in a 2011–2014 study exam- ining the effect of vitamin D supplementation on uri- nary incontinence in older women that the widespread use of moderate vitamin D supplementation did not reduce urinary incontinence [11]. On the other hand, recent studies have found prevalent vitamin D defi- ciency in women with various types of urinary incon- tinence, particularly SUI. Indeed, using vitamin D in women to improve urinary incontinence symptoms has produced conflicting results. Midwives can prescribe vitamin D given the low levels of vitamin D in premen- opausal women, the higher prevalence of SUI in older women, as well as vitamin D’s safety, economic value, and ease of consumption. Accordingly, we conducted a study to determine the effect of vitamin D supplemen- tation, along with Kegel exercises, on SUI severity in premenopausal women with insufficient vitamin D. Sample size estimation As per GPower 3.1 computations, a minimum sam- ple size of n = 60 was required, given an effect size of d = 0.8 (based on a pilot study on urinary incontinence severity variable), β = 0.80, α level = 0.05, and 10% attrition. Blindingh The one who selected the research subjects, the data collector, the participants, and the data analyst were unaware of the groupings in this experiment (dou- ble-blind study). Identical, opaque, consecutively Shahraki et al. BMC Women’s Health (2022) 22:431 Page 3 of 8 were tested by the LIAISON method in the DIaSorin kits using a Chemiluminescence device. The standard labora- tory value was 30–100 ng/ml. were tested by the LIAISON method in the DIaSorin kits using a Chemiluminescence device. The standard labora- tory value was 30–100 ng/ml. numbered bottles containing codes A (vitamin D cap- sule) and code B (placebo capsule) were used to conceal the allocation and ensure blinding. The bottles were distributed to the participants in the order in which they entered the study. Someone uninvolved in sam- pling and evaluating the results prepared the bottles and determined the allocation sequence. Statistical methods T-test and Chi-square tests were utilized to compare the demographic variables of the intervention and con- trol groups using the SPSS 22 statistical software. The paired t-test was used to compare the severity of urinary incontinence and the quality of life of women before and after the intervention in each group. Moreover, the repeated measures ANOVA was employed to compare post-treatment severity of urinary incontinence and life quality between the two groups over time. In addition, the chi-square test was utilized to compare the qualita- tive variables of urinary incontinence and urinary leakage between the two groups. The significance level of P < 0.05 was considered. Intervention program and patient educationi p g p In the first session, the researcher educated both the pla- cebo and experimental group members on how to per- form the Kegel exercises. The individual lies in a supine position and contracts the pelvic floor muscles for 10 sec- onds while keeping the legs slightly apart and completely relaxed. The muscles are then released and relaxed for another 10 s. The performer can use her hands to check that the abdominal muscles are relaxed and not tense. The movement is performed 15–20 times and is imme- diately followed by a posture where the legs are slightly bent/reflexed and tilted toward the abdomen. Alter- natively, the exercise can be performed with the knees slightly apart and the arms resting on a table in a set of 15–20 repetitions at rest. The sets can be increased to 25 repetitions when the individual perceives stronger contractions. Alternately, the exercise can be performed with palms and knees on the ground, muscles succes- sively contracted and relaxed for 10 seconds, continuing this sequence for 15–20 repetitions [8]. The Kegel exer- cise brochure was also distributed to the participants. A weekly telephone check-in ensured that medication was used and Kegel exercises were performed. As the intervention concluded, a urologist re-evaluated the two groups. Characteristics of the patients We had no attrition in this study due to the good follow- up. Figure 1 presents the patient flowchart. Two hundred patients were initially screened, 120 were deemed ineligi- ble for the research, and 20 refused to participate. Sixty patients were randomized equally into vitamin D or pla- cebo groups (Fig. 1). Of 60 participants, members of the intervention (n = 30) and control (n = 30) groups com- pleted the questionnaires (Table 1). Main outcome measuresh The mean SUI severities were similar in the groups at baseline (P = 0.65) and 4 weeks after the intervention (P = 0.66). Nonetheless, at 8 and 12 weeks after the inter- vention, the vitamin D group’s SUI intensity was signifi- cantly lower than that of the placebo group (P < 0.0001) (Table 2). SUI was assessed by completing the standard ICIQ- SF (Incontinence Questionnaire - Urinary Incontinence Short) questionnaire at four points: baseline, month 1, month 2, and month 3. This questionnaire includes six questions that examine the SUI condition over the past 4 weeks. Items 1 and 2 pertain to demographic variables, while item 3 concerns urinary incontinence frequency. Item 4 measures leakage volume, while item 5 evaluates SUI’s impact on quality of life. Before the intervention, SUI severity (P = 0.652) and SUI’s impact on quality of life (P = 0.804) did not dif- fer significantly between the study groups (Table 2). In addition, despite a reduction in SUI severity (P = 0.666) and an improvement in SUI’s impact on life (P = 0.442) in the intervention group, no significant difference was observed between the intervention group and the control group 4 weeks after the intervention. Eight and 12 weeks after the intervention, there was a significant differ- ence between the two groups regarding a decline in SUI severity and improvement in life quality in the interven- tion group compared with the control group. More spe- cifically, a significant difference was observed four, eight, and twelve weeks after the intervention compared to before the intervention (Table 3). In contrast, the mean changes over time were insignificant in the control group. The scores from items 3, 4, and 5 represent the actual score. Item 6 is an unscored question that specifies the date and type of leakage. ICIQ-SF’s total score may range from 0 to 21 (1–5 mild, 6–12 moderate, 13–18 severe, and 19–21 very severe). Haj Ebrahimi et al. (2012) eval- uated the questionnaire’s validity and reliability in the Iranian context. Cronbach’s alpha was 0.75, while the Pearson correlation was 0.93 [16]. Before the participants began to complete the ques- tionnaire, blood samples were taken to measure vitamin D levels. Serum concentrations of 25-hydroxy vitamin D Shahraki et al. Main outcome measuresh BMC Women’s Health (2022) 22:431 Page 4 of 8 In addition, repeated-measures ANOVA revealed that the change in SUI severity scores and the improvement in SUI’s impact on life in both groups decreased signifi- cantly over time (P < 0.01). Table 2 displays that the effect of measurement time on the severity of SUI and the amelioration of SUI’s impact on life in premenopausal women was statis- tically significant (P < 0.001). Therefore, there was a significant difference in the average severity of SUI and the improvement of SUI’s impact on life at baseline, 4 weeks, 8 weeks, and 12 weeks after the intervention. Furthermore, the interaction effect of time and group was significant (P < 0.001). Indeed, there was a statisti- cally significant difference between the average sever- ity of SUI and the improvement of SUI’s impact on the lives of premenopausal women in the intervention and Fig. 1 CONSORT flowchart of the participants Table 1 Participants’ baseline characteristics by research group The data are expressed as percentages (%) or mean values [standard deviation] a Independent t-test b Chi-square for trend Variable Intervention (n = 30) Control (n = 30) P-value Age (years) 44.53 [2.5] 44.23 [2.4] 0.72a Type of delivery 0.5b Natural childbirth 15 (50) 14 (46.7) Cesarean section 15 (50) 16 (53.3) Number of deliveries 2.9 [1.1] 2.53 [1.1] 0.91a Weight (kg) 68.63 [5.9] 69.46 [5.5] 0.73a Height (cm) 161.56 [8.7] 162.33 [7.7] 0.80a BMI (kg/m2) 26.60 [2.5] 26.49 [2.8] 0.72a Serum levels of vitamin D (ng/ml) 25.86 [1.7] 25.73 [2.08] 0.08a Fig. 1 CONSORT flowchart of the participants Fig. Main outcome measuresh BMC Women’s Health (2022) 22:431 Page 5 of 8 Table 2 Comparison of severity of stress urinary incontinence and the impact of the severity of urinary incontinence on the life of premenopausal women in intervention and control groups Data are expressed as mean ± standard deviation a independent t-test between groups b repeated measures analysis of variance (Bonferroni correction) within groups Items Time Intervention (n = 30) Control (n = 30) P-valuea Time TimeGroup Group Severity of SUI Before intervention 13.93 ± 3.49 13.53 ± 3.33 0.652 < 0.001 < 0.001 0.001 Four weeks after intervention 13.13 ± 3.23 13.50 ± 3.30 0.666 Eight weeks after intervention 9.16 ± 2.27 12.93 ± 3.69 < 0.001 Twelve weeks after intervention 4.66 ± 0.71 12.23 ± 4.21 < 0.001 P-valueb < 0.001 0.010 The impact of the severity of urinary incontinence on life before intervention 7.90 ± 2.09 7.76 ± 2.06 0.804 < 0.001 < 0.001 < 0.001 4 weeks after intervention 7.36 ± 1.93 7.76 ± 2.06 0.442 8 weeks after intervention 5.06 ± 1.28 7.40 ± 2.29 < 0.001 12 weeks after intervention 2.23 ± 0.62 6.93 ± 2.79 < 0.001 P-valueb < 0.001 0.009 Table 2 Comparison of severity of stress urinary incontinence and the impact of the severity of urinary incontinence on the life of premenopausal women in intervention and control groups Data are expressed as mean ± standard deviation Table 3 Mean changes in stress urinary incontinence severity and the impact of the severity of urinary incontinence on life in control and intervention groups * Benferroni correction Variables time Intervention Control Mean different P Mean different P* Severity of SUI Before-4 weeks 0.800 < 0.001 0.03 1 Before-8 weeks 4.76 < 0.001 0.60 0.259 Before-12 weeks 9.26 < 0.001 1.30 0.084 4 weeks–8 weeks 3.96 < 0.001 0.56 0.153 4 weeks–12 weeks 8.46 < 0.001 1.26 0.063 8 weeks–12 weeks 4.50 < 0.001 0.70 0.095 The impact of the severity of urinary incontinence on life Before-4 weeks 0.53 < 0.001 0 1 Before-8 weeks 2.83 < 0.001 0.36 0.402 Before-12 weeks 5.66 < 0.001 0.83 0.056 4 weeks–8 weeks 2.30 < 0.001 0.36 0.265 4 weeks–12 weeks 5.13 < 0.001 0.833 0.053 8 weeks–12 weeks 2.83 < 0.001 0.46 0.066 As illustrated in Table 2, the effect of the group on SUI severity and improvement in SUI’s impact on life in pre- menopausal women was statistically significant (P < 0.001). Main outcome measuresh 1 CONSORT flowchart of the participants Table 1 Participants’ baseline characteristics by research group The data are expressed as percentages (%) or mean values [standard deviation] a Independent t-test b Chi-square for trend Variable Intervention (n = 30) Control (n = 30) P-value Age (years) 44.53 [2.5] 44.23 [2.4] 0.72a Type of delivery 0.5b Natural childbirth 15 (50) 14 (46.7) Cesarean section 15 (50) 16 (53.3) Number of deliveries 2.9 [1.1] 2.53 [1.1] 0.91a Weight (kg) 68.63 [5.9] 69.46 [5.5] 0.73a Height (cm) 161.56 [8.7] 162.33 [7.7] 0.80a BMI (kg/m2) 26.60 [2.5] 26.49 [2.8] 0.72a Serum levels of vitamin D (ng/ml) 25.86 [1.7] 25.73 [2.08] 0.08a Table 1 Participants’ baseline characteristics by research group significant difference in the average severity of SUI and the improvement of SUI’s impact on life at baseline, 4 weeks, 8 weeks, and 12 weeks after the intervention. Furthermore, the interaction effect of time and group was significant (P < 0.001). Indeed, there was a statisti- cally significant difference between the average sever- ity of SUI and the improvement of SUI’s impact on the lives of premenopausal women in the intervention and control groups at different times (P < 0.001). In addition, repeated-measures ANOVA revealed that the change in SUI severity scores and the improvement in SUI’s impact on life in both groups decreased signifi- cantly over time (P < 0.01). Table 2 displays that the effect of measurement time on the severity of SUI and the amelioration of SUI’s impact on life in premenopausal women was statis- tically significant (P < 0.001). Therefore, there was a Shahraki et al. Main outcome measuresh More specifically, there was a significant difference between the mean scores of SUI severity and the improve- ment of SUI’s impact on life among premenopausal women in the intervention and control groups. * Benferroni correction Discussion Vitamin D supplementation significantly improved SUI severity after 3 months of intervention in the experimen- tal group compared to the placebo group. Pelvic floor disorders are widespread, affecting one in every three women as they age [18]. Pelvic floor muscles are largely responsible for pelvic organ support and urinary reten- tion. Weakness of these muscles is linked with urinary incontinence, the most frequently reported symptom of pelvic floor disorders [2]. Urinary incontinence affects Table 4 depicts the frequency and relative percentage of urinary incontinence and urine leakage between premeno- pausal women in four stages as per the study groups. Shahraki et al. BMC Women’s Health (2022) 22:431 Page 6 of 8 Table 4 The frequency and relative percentage of urinary incontinence and urine leakage in premenopausal women in four stages by study groups a Chi-square test Item Severity of symptoms before intervention 4 weeks after 8 weeks after 12 weeks after Intervention Control Intervention Control Intervention Control Intervention Control Prevalence of inconti- nence 1 weekly – – – – 1 (3.3) 0 (0) 17 (56.7) 0 (0) 2 or 3 weekly 3 (10) 4 (13.3) 4(13.3) 4 (13.3) 14 (46.7) 6 (20) 13 (43.3) 7 (23.3) 1 daily 9 (30) 11 (36.7) 10(33.3) 11 (36.7) 12 (40) 10 (33.3) 0 (0) 11 (36.7) Several daily 11 (36.7) 11 (36.7) 12(40) 12 (40) 3 (10) 11 (36.7) 0 (0) 11 (36.7) Always 7 (23.3) 4 (13.3) 4(13.3) 3 (10) 0 (0) 3 (10) 0 (0) 1 (3.3) P-value a 0.762 0.979 0.018 < 0.001 Leakage rate Low 3 (10) 2 (6.7) 4 (13.3) 2 (6.7) 14 (46.7) 4 (13.3) 30 (100) 6 (20) Medium 15 (50) 18 (60) 15 (50) 18 (60) 16 (53.3) 17 (56.7) 0 (0) 15 (50) Hight 12 (40) 10 (33.3) 11 (36.7) 10 (33.3) 0 (0) 9 (30) 0 (0) 9 (30) P-valuea 0.721 0.610 0.001 < 0.001 with our findings, this study confirmed the association between vitamin D and SUI, even in pregnant women. more than 28% of the population, with SUI being the most prevalent type [19]. High body mass index (BMI) and pelvic organ prolapse [20] were found to have a strong correlation with urinary incontinence [21]. The mean BMI in our study was 26.5 (2.6), which is in the overweight range and a contributor to women’s urinary incontinence. Discussion These findings were compatible because vitamin D has receptors in musculoskeletal tissue that affect muscle strength and function. Indeed, it can be beneficial in alle- viating SUI by strengthening the detrusor muscles of the bladder and pelvic floor muscles. Although this type of incontinence increases with age, the condition does not merely affect the elderly; it also affects 30% of middle-aged women [22]. Vitamin D levels decline with age. Age also decreases calcium absorption, which corresponds to a decline in vitamin D. The mean age in this study was 44.38 ± 2.45 years. Among micro- nutrients, vitamin D is known to have a pandemic defi- ciency [23]. Vitamin D receptors are found in the bladder and the pelvic floor muscles’ striated muscles [8, 10]. While vitamin D deficiency exhibits the most apparent impact on musculoskeletal health [5], it is one of the pos- sible causes of urinary incontinence in older women [24]. In a study conducted by Vaughan et al. [13], prelimi- nary findings indicated a linkage between vitamin D and the prevalence of urinary incontinence in older individu- als living in the community, which is consistent with the present study’s findings. pl Markland et al. [24] investigated vitamin D con- sumption and the 10-year risk of urinary incontinence in elderly and middle-aged women. According to the authors, there was a link between moderate vitamin D consumption and the incidence of urinary inconti- nence in middle-aged women. Contrary to our study, the women in Markland et al.’s study did not have urinary incontinence at the start, and the serum level of vitamin D was not measured. Therefore, there may not be a cor- relation between these cases and urinary incontinence. This must be investigated. Alongside this, another study (2021) found that supplementation with moderate doses of vitamin D did not reduce urinary incontinence in older women [11]. This contradiction in results may be due to differences in the age range of the participants. Further- more, as demonstrated by Kamronrithisorn et al., the effects of vitamin D supplementation may depend on patients’ baseline vitamin D levels, type of vitamin D sup- plements, and duration of supplementation [25]. Although this type of incontinence increases with age, the condition does not merely affect the elderly; it also affects 30% of middle-aged women [22]. Vitamin D levels decline with age. Age also decreases calcium absorption, which corresponds to a decline in vitamin D. Availability of data and materials hypervitaminosis D include kidney damage, kidney stones, kidney failure, excessive bone loss, and calcifica- tion of arteries and soft tissues. In addition, high blood calcium levels can lead to abnormal heart rhythms [26]. The data are available upon request to the corresponding author after signing appropriate documents in line with ethical application and the decision of the Ethics Committee. Some strengths of this study include its random sam- ple allocation, double-blindness, and no participant attrition. Vitamin D serum levels were measured at baseline, and women with insufficient vitamin D serum levels were enrolled in the study and given vitamin D supplements to prevent drug overdose. Both groups were taught Kegel exercises as part of the intervention. However, the study was limited in certain respects. It did not mention the individuals’ diets in terms of vita- min D intake through daily food and did not follow up with them after vitamin D3 supplementation ceased. We did not consider the serum vitamin D levels of the participants again after the study’s conclusion. Also, at the beginning of the study, the participants were not separated based on the severity of their vitamin D deficiency. Indeed, each participant received a single dose of the supplement, regardless of the severity of their vitamin D deficiency, whereas the severity of this deficiency can affect the severity of incontinence. The research population consisted exclusively of women. Hence, the findings cannot be generalized to men. As a result, one cannot judge the consequences of drug withdrawal. Further research may be conducted with larger sample sizes. Discussion The mean age in this study was 44.38 ± 2.45 years. Among micro- nutrients, vitamin D is known to have a pandemic defi- ciency [23]. Vitamin D receptors are found in the bladder and the pelvic floor muscles’ striated muscles [8, 10]. While vitamin D deficiency exhibits the most apparent impact on musculoskeletal health [5], it is one of the pos- sible causes of urinary incontinence in older women [24]. In a study conducted by Vaughan et al. [13], prelimi- nary findings indicated a linkage between vitamin D and the prevalence of urinary incontinence in older individu- als living in the community, which is consistent with the present study’s findings. i Sharma et al. [5] studied vitamin D concentrations in women with SUI at a tertiary referral center in India, finding a significantly high rate of vitamin D deficiency in these women. Given the vitamin D shortage, vitamin D supplementation was employed in this investigation to determine the resultant effect, which is consistent with the present study’s findings. On the other hand, excessive vitamin D can increase blood calcium levels, potentially resulting in a condi- tion known as hypercalcemia. Fatigue, loss of appe- tite, weight loss, excessive thirst, excessive urination, dehydration, constipation, irritability, nervousness, ringing in the ear (tinnitus), muscle weakness, nausea, vomiting, dizziness, confusion, disorientation, high blood pressure, and heart arrhythmias are some of the symptoms. Long-term complications of untreated i Stafne et al. [2] studied the effect of vitamin D on SUI in pregnant women, indicating that serum vitamin D concentrations below 50 nmol/l were associated with an increased risk of incontinence, especially SUI. Consistent Shahraki et al. BMC Women’s Health (2022) 22:431 Shahraki et al. BMC Women’s Health (2022) 22:431 Page 7 of 8 hypervitaminosis D include kidney damage, kidney stones, kidney failure, excessive bone loss, and calcifica- tion of arteries and soft tissues. In addition, high blood calcium levels can lead to abnormal heart rhythms [26]. Acknowledgments The authors appreciate the gynecological clinic and all women who gener- ously participated in this study. We also wish to express our appreciation to the Student Research Committee of Kerman University of Medical Sciences and related facilities for their assistance with this study. 5. Sharma JB, Kakkad V, Kumar S, Roy K. Cross-sectional study on vitamin D levels in stress urinary incontinence in women in a tertiary referral center in India. Indian J Endocrinol Metab. 2019;23(6):623–7. 6. Farzinmehr A, Moezzi A, Koohpayezadeh J. A combination training of pelvic floor muscles and Core muscles in improvement of stress urinary incontinence. Razi J Med Sci. 2013;20(109):30–40 [In Persian]. Abbreviations SUI S i SUI: Stress urinary incontinence; ICIQ-SF: Incontinence Questionnaire -Urinary Incontinence Short; RCT: Randomized controlled trial. 4. Ahmadi B, Alimohammadian M, Golestan B, Mahjubi B, Janani L, Mirzaei R. The hidden epidemic of urinary incontinence in women: a population- based study with emphasis on preventive strategies. Int Urogynecol J. 2010;21(4):453–9. Conclusion In premenopausal women, vitamin D supplementation alleviated the severity of SUI. Hence, vitamin D sup- plements are recommended for premenopausal women to improve their SUI because they are readily available, cost-effective, and have no harmful effects. Author details 1 Department of Community Health Nursing, Razi Faculty of Nursing and Mid- wifery, Kerman University of Medical Sciences, Kerman, Iran. 2 Department of Midwifery, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 3 PhD student in Reproductive Health, Faculty of Nursing and Midwifery, Mash- had University of Medical Sciences, Mashhad, Iran. 4 Department of Reproduc- tive Health, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. 5 Student Research Committee, Kerman University of Medical Sciences, Kerman, Iran. Received: 13 June 2022 Accepted: 25 October 2022 Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. References References 1. Keller K, Engelhardt M. Strength and muscle mass loss with aging process. Age and strength loss. Muscles Ligaments Tendons J. 2013;3(4):346–50. 2. Stafne SN, Mørkved S, Gustafsson MK, Syversen U, Stunes AK, Salvesen KÅ, et al. Vitamin D and stress urinary incontinence in pregnancy: a cross- sectional study. BJOG. 2020;127(13):1704–11. 3. Kharaji G, Nikjooy A, Amiri A, Sanjari MA. Proprioception in stress urinary incontinence: a narrative review. Med J Islam Repub Iran. 2019;33:60. 4. Ahmadi B, Alimohammadian M, Golestan B, Mahjubi B, Janani L, Mirzaei R. The hidden epidemic of urinary incontinence in women: a population- based study with emphasis on preventive strategies. Int Urogynecol J. 2010;21(4):453–9. 5. Sharma JB, Kakkad V, Kumar S, Roy K. Cross-sectional study on vitamin D levels in stress urinary incontinence in women in a tertiary referral center in India. Indian J Endocrinol Metab. 2019;23(6):623–7. 6. Farzinmehr A, Moezzi A, Koohpayezadeh J. A combination training of pelvic floor muscles and Core muscles in improvement of stress urinary incontinence. Razi J Med Sci. 2013;20(109):30–40 [In Persian]. 7. Parker-Autry CY, Burgio KL, Richter HE. Vitamin D status: a review with implications for the pelvic floor. Int Urogynecol J. 2012;23(11):1517–26. 8. Crescioli C, Morelli A, Adorini L, Ferruzzi P, Luconi M, Vannelli GB, et al. Human bladder as a novel target for vitamin D receptor ligands. J Clin Endocrinol Metab. 2005;90(2):962–72. 9. Schwartz GG. Vitamin D in health and disease: vitamin D and the epi- demiology of prostate cancer. Seminars in dialysis. Wiley Online Library; 2005. 1. Keller K, Engelhardt M. Strength and muscle mass loss with aging process. Age and strength loss. Muscles Ligaments Tendons J. 2013;3(4):346–50. 2. Stafne SN, Mørkved S, Gustafsson MK, Syversen U, Stunes AK, Salvesen KÅ, et al. Vitamin D and stress urinary incontinence in pregnancy: a cross- sectional study. BJOG. 2020;127(13):1704–11. 3. Kharaji G, Nikjooy A, Amiri A, Sanjari MA. Proprioception in stress urinary incontinence: a narrative review. Med J Islam Repub Iran. 2019;33:60. Consent for publication Not applicable. Consent for publication Not applicable. Authors’ contributions SFE, SKS, MS, ZC, FT, and MA contributed to conceiving and designing the research. The data were collected, analyzed, and interpreted by SFE, SKS, MS, ZC, FT, and MA contributed equally to writing and revising the manuscript and approved the final manuscript. 7. Parker-Autry CY, Burgio KL, Richter HE. Vitamin D status: a review with implications for the pelvic floor. Int Urogynecol J. 2012;23(11):1517–26. 8. Crescioli C, Morelli A, Adorini L, Ferruzzi P, Luconi M, Vannelli GB, et al. Human bladder as a novel target for vitamin D receptor ligands. J Clin Endocrinol Metab. 2005;90(2):962–72. Ethics approval and consent to participate The protocol of this randomized controlled trial was approved by the Ethics Committee of Kerman University of Medical Sciences (ethics code: KMU. REC.1399.555) and registered with the Iranian Registry of Clinical Trials https:// fa.irct.ir/trial/53474 (identifier: IRCT20190724044318N2) on 11/02/2021. At the request of the ethics committee, the study was conducted in accordance with the Declaration of Helsinki and Ethics Publication on Committee (COPE). Other ethical criteria included written consent to enter the study and withdraw from the study whenever participants were willing. Written informed consent was obtained from each woman. Special codes were assigned to each participant to ensure information confidentiality. Lastly, the results were provided to the participants and authorities in the gynecological clinic affiliated upon their requests. Funding Th 9. Schwartz GG. Vitamin D in health and disease: vitamin D and the epi- demiology of prostate cancer. Seminars in dialysis. Wiley Online Library; 2005. This research received no specific grant from any funding agency in the pub- lic, commercial, or not-for-profit sectors. Page 8 of 8 Shahraki et al. BMC Women’s Health (2022) 22:431 Shahraki et al. BMC Women’s Health (2022) 22:431 10. Bischoff H, Borchers M, Gudat F, Duermueller U, Theiler R, Stähelin H, et al. In situ detection of 1, 25-dihydroxyvitamin D receptor in human skeletal muscle tissue. Histochem J. 2001;33(1):19–24. 10. Bischoff H, Borchers M, Gudat F, Duermueller U, Theiler R, Stähelin H, et al. In situ detection of 1, 25-dihydroxyvitamin D receptor in human skeletal muscle tissue. Histochem J. 2001;33(1):19–24. 11. Markland AD, Vaughan C, Huang A, Kim E, Bubes VY, Tangpricha V, et al. Effect of vitamin D supplementation on urinary incontinence in older women: ancillary findings from a randomized trial. Am J Obstet Gynecol. 2022;226(4):535.e1–535.e12. 12. Badalian SS, Rosenbaum PF. Vitamin D and pelvic floor disorders in women: results from the National Health and nutrition examination survey. Obstet Gynecol. 2010;115(4):795–803. y y 13. Vaughan CP, Tangpricha V, Motahar-Ford N, Goode PS, Burgio KL, Allman RM, et al. Vitamin D and incident urinary incontinence in older adults. Eur J Clin Nutr. 2016;70(9):987–9. 14. Parker-Autry CY, Markland AD, Ballard AC, Downs-Gunn D, Richter HE. Vitamin D status in women with pelvic floor disorder symptoms. Int Urogynecol J. 2012;23(12):1699–705. 15. Ghanbari Z, Karamali M, Mirhosseini N, Akbari M, Tabrizi R, Lankarani KB, et al. Vitamin D status in women with pelvic floor disorders: a meta- analysis of observational studies. J Midlife Health. 2019;10(2):57–62. 16. Dirks-Naylor AJ, Lennon-Edwards S. The effects of vitamin D on skeletal muscle function and cellular signaling. J Steroid Biochem Mol Biol. 2011;125(3–5):159–68. 17. Azami M, Shamloo MB, Nasirkandy MP, Veisani Y, Rahmati S, YektaKooshali MH, et al. Prevalence of vitamin D deficiency among pregnant women in Iran: a systematic review and meta-analysis. Koomesh. 2017;19(3):505–14 [In Persian]. 18. Rortveit G, Subak LL, Thom DH, Creasman JM, Vittinghoff E, Van Den Eeden SK, et al. Prevalence and risk factors. Female Pelvic Med Reconstr Surg. 2010;16(5):278–83. g 19. Falah-Hassani K, Reeves J, Shiri R, Hickling D, McLean L. The pathophysiol- ogy of stress urinary incontinence: a systematic review and meta-analysis. Int Urogynecol J. 2021;32(3):501–52. gy 20. Funding Th Hendrix SL, Clark A, Nygaard I, Aragaki A, Barnabei V, McTiernan A. Pelvic organ prolapse in the Women’s health initiative: gravity and gravidity. Am J Obstet Gynecol. 2002;186(6):1160–6. y 21. Buchsbaum G. Urinary incontinence and pelvic organ prolapse. Med Clin North Am. 2015;99(2):405–16. 21. Buchsbaum G. Urinary incontinence and pelvic organ prolapse. Med Clin North Am. 2015;99(2):405–16. 22. Ford AA, Taylor V, Ogah J, Veit-Rubin N, Khullar V, Digesu GA. Midurethral slings for treatment of stress urinary incontinence review. Neurourol Urodyn. 2019;38(Suppl 4):S70–5. y pp 23. Holick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences. Am J Clin Nutr. 2008;87(4):1080S–6S. 23. Holick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences. Am J Clin Nutr. 2008;87(4):1080S–6S. 24. Markland AD, Vaughan C, Huang A, Tangpricha V, Grodstein F. Vitamin D intake and the 10-year risk of urgency urinary incontinence in women. J Steroid Biochem Mol Biol. 2020;199:105601. 24. Markland AD, Vaughan C, Huang A, Tangpricha V, Grodstein F. Vitamin D intake and the 10-year risk of urgency urinary incontinence in women. J Steroid Biochem Mol Biol. 2020;199:105601. 25. Kamronrithisorn T, Manonai J, Vallibhakara SA, Sophonsritsuk A, Vallibha- kara O. Effect of vitamin D supplement on vulvovaginal atrophy of the menopause. Nutrients. 2020;12(9):2876. 25. Kamronrithisorn T, Manonai J, Vallibhakara SA, Sophonsritsuk A, Vallibha- kara O. Effect of vitamin D supplement on vulvovaginal atrophy of the menopause. Nutrients. 2020;12(9):2876. 26. Huizen J. Can too much vitamin D hurt you?; 2022 Available from https:// www.medicalnewstoday.com/articles/322602 26. Huizen J. Can too much vitamin D hurt you?; 2022 Available from https:// www.medicalnewstoday.com/articles/322602 • fast, convenient online submission • thorough peer review by experienced researchers in your ﬁeld • rapid publication on acceptance • support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year • At BMC, research is always in progress. Learn more biomedcentral.com/submissions Ready to submit your research Ready to submit your research ? Choose BMC and benefit from: ? Choose BMC and benefit from: Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. 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https://openalex.org/W2131380629	https://bmccomplementmedtherapies.biomedcentral.com/counter/pdf/10.1186/1472-6882-11-65	English	null	High catechin concentrations detected in Withania somnifera (ashwagandha) by high performance liquid chromatography analysis	BMC complementary and alternative medicine	2,011	cc-by	6,038	Abstract Background: Withania somnifera is an important medicinal plant traditionally used in the treatment of many diseases. The present study was carried out to characterize the phenolic acids, flavonoids and 1,1-diphenyl-2- picrylhydrazyl radical (DPPH) scavenging activities in methanolic extracts of W. somnifera fruits, roots and leaves (WSFEt, WSREt and WSLEt). Methods: WSFEt, WSREt and WSLEt was prepared by using 80% aqueous methanol and total polyphenols, flavonoids as well as DPPH radical scavenging activities were determined by spectrophotometric methods and phenolic acid profiles were determined by HPLC methods. Results: High concentrations of both phenolics and flavonoids were detected in all parts of the plant with the former ranging between 17.80 ± 5.80 and 32.58 ± 3.16 mg/g (dry weight) and the latter ranging between 15.49 ± 1.02 and 31.58 ± 5.07 mg/g. All of the three different plant parts showed strong DPPH radical scavenging activities (59.16 ± 1.20 to 91.84 ± 0.38%). Eight polyphenols (gallic, syringic, benzoic, p-coumaric and vanillic acids as well as catechin, kaempferol and naringenin) have been identified by HPLC in parts of the plant as well. Among all the polyphenols, catechin was detected in the highest concentration (13.01 ± 8.93 to 30.61 ± 11.41 mg/g). Conclusion: The results indicating that W. somnifera is a plant with strong therapeutic properties thus further supporting its traditional claims. All major parts of W. somnifera such as the roots, fruits and leaves provide potential benefits for human health because of its high content of polyphenols and antioxidant activities with the leaves containing the highest amounts of polyphenols specially catechin with strong antioxidant properties. Keywords: Withania somnifera, spectrophotometry, HPLC, polyphenols, antioxidant, free radical sca ania somnifera, spectrophotometry, HPLC, polyphenols, antioxidant, free radical scavenging activity High catechin concentrations detected in Withania somnifera (ashwagandha) by high performance liquid chromatography analysis Nadia Alam1, Monzur Hossain1, Md Ibrahim Khalil2, Mohammed Moniruzzaman2, Siti Amrah Sulaiman2 and Siew Hua Gan3 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Open Access © 2011 Alam 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: mibrahim12@yahoo.com 2Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia Full list of author information is available at the end of the article Background various parts of the plant have been reported to possess antiserotogenic, anticancer and anabolic properties and have shown beneficial effects in the treatment of arthri- tis, stress and geriatric problems [4]. W. somnifera is also made into dietary supplements with good nutri- tional properties and phytochemicals. Besides being used among the elderly to increase health vitality, a decoction of W. somnifera root is also used as nutrient and health restorative agent among postpartum ladies as it was purported to thicken and increase the nutritive value of the breastmilk when given to nursing mothers. The pharmacological effect of the roots of W. somnifera is attributed to its active ingredient, withanolides [5] which has a wide range of therapeutic applications [6]. Ashwagandha [Withania somnifera L. Dunal] (Solana- ceae) is an important medicinal plant, commonly-used as a domestic remedy for several diseases in India as well as other parts of the world [1]. It is described as an herbal tonic and health food in the famous book of Vedas and is considered as in ‘Indian Ginseng’ in tradi- tional Indian system of healing [2]. Several recent reports have demonstrated immunomodulator and anti- tumor effect of W. somnifera as well [3]. Moreover, * Correspondence: mibrahim12@yahoo.com 2Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia Full list of author information is available at the end of the article Page 2 of 8 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 There is a great deal of evidence indicating that excessive free radical production and lipid peroxida- tions are actively-involved in the pathogenesis of a wide number of chronic diseases, including athero- sclerosis [7], cardiac and cerebral ischemia [8], neuro- degenerative disorders [9], carcinogenesis [10], diabetes [11] and rheumatic disorders [12] and contributes a major role in the ageing process [13]. Plant-derived antioxidants such as vitamin E, vitamin C, polyphenols including phenolic acids, phenolic diterpenes, flavo- noids, catechins, procyanidins and anthocyanins are becoming increasingly important as dietary factors [14]. Supplementation with berry juice [15], flavones from skullcap, catechins from green tea, anthocyanins from chokeberry and condensed tannins from fava beans [16] are indices of oxidative stress protectant in rats. Furthermore, the growing interest in the substitu- tion of synthetic food antioxidants by natural chemi- cals has fostered research on plant sources and the screening of raw materials for identifying new antioxi- dants. Preparation of Plant Extracts p W. somnifera roots, fruits and leaves extract (WSREt, WSFEt and WSLEt) preparation was performed accord- ing to a modified method described by Kahkonen et al., [23]. Grinded dry plant materials (500 mg) were weighed into a test tube followed by the addition of a total of 10 ml of 80% aqueous methanol. The suspen- sion was then stirred slightly. The tubes were sonicated for 5 min and centrifugated for another 10 min at 1500 g and the resulting supernatants were collected. The extraction procedure was repeated and the supernatants were combined before being evaporated to a volume of approximately 1 ml. The concentrated extracts were further lyophilized and weighed. Determination of total flavonoids Determination of total flavonoids The total flavonoid contents of the W. somnifera extracts were determined according to the colorimetric assay method developed by Zhishen et al., [25]. Briefly, 1 ml of properly diluted (1 mg/ml) WSREt, WSFEt and WSLEt were mixed with 4 ml of distilled water. At base- line, 0.3 ml of (5% w/v) NaNO2 was added. After five minutes, 0.3 ml of (10% w/v) AlCl3 was added followed by the addition of 2 ml of NaOH solution (1 M) six minutes later. After that, the volume was immediately made up to 10 ml, with the addition of 2.4 ml of dis- tilled water. The mixture was shaken vigorously and the Background In this regard, polyphenols are being increas- ingly reported to exhibit antioxidant effects in foods [17]. Various plants have been analysed for the exis- tence of phenolic acids by HPLC [18]. Plant acids are known to have anticarcinogenic activity [19]. and phe- nolic compounds are believed to be an important part of the general defence mechanism of many plants against infections [20]. Therefore, it is useful to mea- sure the presence of phenolic compounds in natural substances. Chemicals and reagents Chemicals and reagents Reagents such as 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and Folin-Ciocalteu’s reactive were purchased from Sigma-Aldrich (St. Louis, USA). Sodium carbonate (Na2CO3), aluminium chloride (AlCl3), sodium nitrite (NaNO2) and sodium hydroxide (NaOH) were pur- chased from Merck (Darmstadt, Germany). All chemi- cals used were of analytical grades. Total Polyphenols Ph li Phenolic compounds in W. somnifera were estimated by a spectrophotometric determination using a modified Folin-Ciocalteu method Singleton et al., [24]. Briefly, 100 μl of sample extracts (1 mg/ml) was mixed with 1 ml of Folin and Ciocalteu’s phenol reagent (2 N). After 3 minutes, 1 ml of 10% Na2CO3 solution was added to the mixture and adjusted to 10 ml with distilled water. The reaction was kept in the dark for 90 min, after which the absorbance was read at 725 nm (T 80 UV/ VIS spectrophotometer, ChromoTek GmbH, Germany). Gallic acid was used to calculate the standard curve (20, 40, 60, 80 and 100 μg/ml, r2 = 0.993). Estimation of the phenolic compounds was carried out in triplicates. The results were mean values ± standard deviations and expressed as milligrams of gallic acid equivalents (GAEs) per g of W. somnifera dry weight (DW). Purification of phenolic acids is very difficult not only due to their isomeric similarities but also due to the influence of various effects such as acid-based treatment, temperature and light on their labile structures [21]. The determination of phenolic acids is important both for their characterization and to facilitate more efficient uses of important plant resources [22]. To date, very limited data exists on phenolic com- pounds reported in W. somnifera leaves, roots and fruits as well as their antioxidant effects to support their tradi- tional claims. Therefore, we aimed to undertake this task in the present study as W. somnifera is widely-used. If the presence of phenolic and flavonoid compounds present in W. somnifera can be confirmed, the plant can be used as a good possible source of antioxidant. Plant material The W. somnifera plant parts such as roots, fruits and leaves were collected from field grown plants after six months of cultivation in Botanical Garden, Rajshahi University, Bangladesh. The collected parts of medicinal plant were brought into the laboratory, cleaned and air- dried in shade and then grinded to a fine powder. Page 3 of 8 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 purchased from Sigma (St. Louis, MO, USA) and were used as reference standards. purchased from Sigma (St. Louis, MO, USA) and were used as reference standards. purchased from Sigma (St. Louis, MO, USA) and were used as reference standards. absorbance of the mixture was read at 510 nm. A cali- bration curve was prepared using a standard solution of catechin (20, 40, 60, 80 and 100 μg/ml, r2 = 0.996). The results were expressed as mg catechin equivalents (CEQ) per g of W. somnifera (DW). DPPH free radical-scavenging activity g g y The antioxidant capacity of the W. somnifera was also studied through the evaluation of the free radical- scavenging effect on the DPPH radical. The determina- tion was based on the method proposed by Ferreira et al., [26]. Briefly, 1 ml (1 mg/ml) of WSREt, WSFEt and WSLEt were mixed with 2.7 ml of methanolic solution containing DPPH radicals (0.024 mg/ml). The mixture was vigorously shaken and left to stand for 60 min in the dark (until their absorbance remained unchanged). The reduction of the DPPH radical was determined by measuring the absorbance at 517 nm [27]. The radical- scavenging activity (RSA) was calculated as a percentage of DPPH discolouration using the equation: % RSA = [(ADPPH-AS)/ADPPH] × 100, where AS is the absorbance of the solution when the sample extract has been added at a particular level and ADPPH is the absorbance of the DPPH solution. Flavonoids content The total contents of flavonoids of the three different W. somnifera extracts were also determined. Flavonoids were detected in high concentrations ranging from 15.49 ± 1.02 (WSREt) to 31.58 ± 5.07 (WSLEt) mg CEQ/g (Table 1). High Performance Liquid Chromatography (HPLC) High Performance Liquid Chromatography (HPLC) The HPLC method was based on the method published by Kaškonienė et al., [28]. Analysis of WSREt, WSFEt and WSLEt were performed by employing an HPLC sys- tem (Waters 2695, Milford, MA, USA) equipped with a Photodiode Array Detector (Waters 2996, Milford, MA, USA). The HPLC column was a Merck Purospher Star, RP-18e, (125 × 4 mm, 5 μm) fitted with a guard car- tridge packed with the same type of stationary phase (Merck, Darmstadt, Germany). The linear 76 gradient was used at a flow rate of 0.5 ml/min with total analyti- cal time of approximately 35 min. The binary mobile phase consisted of a solvent A (ultra pure water with 0.1% of phosphoric acid) and solvent B (pure methanol with 0.1% of phosphoric acid). Elution from the column was achieved with the following gradient: 0 min to 10 min of solvent B, increased from 35% to 55%; 10-25 min of solvent B, increased to 62%; 25-30 min of solvent B, increased to 85% and the final composition was kept constant till 35 min. All solvents used were of HPLC grade quality. The detection wavelength was done between 200 and 450 nm with specific monitoring at 265 nm. The identification of phenolic compounds was performed by comparing the retention time and UV absorption (lmax) of each peak of the analytes with the reference standards. Phenolic acids (gallic, syringic, caf- feic, vanillic, p-coumaric, benzoic and transcinnamic acids) as well as flavonoids (catechin, naringenin, luteo- lin, hesperetin, kaempferol, apigenin, naringin) were Statistical analysis All analyses were carried out in triplicates and the data was expressed as means ± standard deviations (SD). The data was analyzed using (Statistical Packages for Social Science 12.0) (SPSS Inc., USA) and MS Excel 2003. One-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test was used to compare the phenol contents, FRAP values, DPPH scavenging activities and colour parameters of WSREt, WSFEt and WSLEt (Table 1). The differences between means at 95% (p < 0.05) confidence level were considered statistically significant. Correlations were obtained by Pearson’s correlation coefficient (r) in bivariate linear correlations. Phenolic content The contents of total polyphenols (mg GAE/g) of WSREt, WSFEt and WSLEt were investigated using the modified Folin-Ciocalteu assay which is sensitive to phe- nol and polyphenols entities and other electron donating antioxidants such as ascorbic acid and vitamin E. The sources of the analysed WSREt, WSFEt as well as WSLEt were significantly different (p < 0.05), as shown in (Table 1). Among the three different W. somnifera extracts, the concentrations of polyphenols was found to be lowest in WSREt (17.80 ± 5.80 mg/g) and highest in WSLEt (32.58 ± 3.16 mg/g). Discussion To our knowledge, our study is the first to identify phe- nolic compounds present in W. somnifera. The present study confirmed the presence of phenolic compounds, flavonoids and antioxidant activities in WSREt, WSFEt and WSLEt. High concentrations of phenolic com- pounds were found in the different parts of W. somni- fera with significant variations in the amount. (Table 1) showed high content of phenolics and flavonoids in WSLEt (32.58 ± 3.16 and 31.58 ± 5.07 respectively) while that in WSREt was low (17.80 ± 5.80 and 15.49 ± 1.02 respectively). This indicates that the leaves of W. somnifera should be consumed for its antioxidant effects. Several reports have shown that different plant parts have variable polyphenols compositions, as shown by our findings [30]. W. somnifera extracts is directly related to its phenolic and flavonoids content. W. somnifera extracts is directly related to its phenolic and flavonoids content. syringic, benzoic, p-coumaric and vanillic acids as well as the flavonoids catechin, kaempferol and naringenin. (Figures 1, 2, 3) show the HPLC chromatograms obtained from WSREt, WSFEt and WSLEt. Six phenolic compounds were detected in WSLEt whereas, three compounds were identified in WSFEt and only two compounds were identified in WSREt. The unknown compounds that may have had similar flavonoid and phenolic acid spectra and chromatographic behaviours (shown as extra peaks in the figures) were also detected. However, they could not be fully identified due to lack of standard compounds. (Figure 4) compared the total phenolic compounds of sample extracts obtained when using spectrophotometric and HPLC methods. Overall, spectrophotometric methods tend to report higher levels of phenolics when compared to HPLC method. Correlations The correlations among the phenolic compounds, flavo- noids and DPPH radical scavenging activities are shown in (Table 2). The correlation matrix showed that signifi- cant linear correlation exists between the results of all three analytical methods employed indicating that the three measurements are reliable indicators of antioxi- dant activities. The lowest linear correlation value at r = 0.962 (p = 0.01) and the highest correlation value at r = 0.995. Both phenolic compounds and DPPH radical scavenging activity are strongly correlated (r = 0.995 and 0.983 respectively) with DPPH radical scavenging activities. The significant correlations existing between phenolic compounds and DPPH radical scavenging activities indicate the strong antioxidant properties of the tested WSREt, WSFEt and WSLEt. Similar to our findings, some literature also reported strong correlation between the antioxidant capacity and total phenolic con- tents [29] further suggesting that polyphenols are the major components responsible for the antioxidant effects of WSREt, WSFEt and WSLEt. DPPH radical scavenging activity There were significant differences in terms of their scavenging abilities present among the WSREt, WSFEt as well as WSLEt samples, expressed as percentage of inhibition on the DPPH radical (Table 1). Among the three extracts, the lowest scavenging activity was found in WSREt (59.16 ± 1.20%) while the highest activity was found in WSLEt (91.84 ± 0.38%). The DPPH radical scavenging test is one of the fastest tests available to investigate the overall hydrogen/electron donating activ- ity of single antioxidants and health-promoting dietary antioxidant supplements. The reasons behind the mark- edly higher radical scavenging capacity exhibited by the different types of W. somnifera extracts probably lie in their diverse botanical origin. Antioxidant potential of Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Page 4 of 8 Table 1 Spectrophotometric analysis of phenolics, flavonoids and antioxidant properties of W. somnifera roots, fruits and leaves. W. somnifera Phenolics mg GAE/g (DW) Flavonoids mg CEQ/g (DW) % of DPPH inhibition Roots 17.80 ± 5.80c 15.49 ± 1.02c 59.16 ± 1.20c Fruits 22.29 ± 1.99b 21.15 ± 5.32b 70.38 ± 0.84b Leaves 32.58 ± 3.16a 31.58 ± 5.07a 91.84 ± 0.38a In each column, values with different letters (superscripts) indicate significant differences (p < 0.05). DW = dry weight metric analysis of phenolics, flavonoids and antioxidant properties of W. somnifera roots, fruits Table 1 Spectrophotometric analysis of phenolics, flavonoids and antioxidant properties of W. so and leaves W. somnifera extracts is directly related to its phenolic and flavonoids content. HPLC analysis Sixteen phenolic and flavonoid standards were com- pared with the chromatograms produced by the unknown W. somnifera extracts. HPLC analysis of phe- nolic and flavonoids compounds in WSREt, WSFEt as well as WSLEt showed that only catechin is commonly found in all of the three extracts analyzed. Eight poly- phenols (five phenolic acids and three types of flavo- noids) have been identified and the phenolics patterns of all plant parts were confirmed to contain gallic, There is very poor data on analysis of phenolic com- pounds in W. somnifera. Udayakumar et al., [30] from India reported that the presence of total phenolic com- pounds in WSREt was 28.26 mg/g while that of flavo- noids was 17.32 mg/g. For WSLEt it was 5.4 mg/g total phenolic compounds and 5.1 mg/g flavonoids both of which were different from our study perhaps due to the different source of W. somnifera and polyphenols of plant parts which may also be related to the colour, maturity and environment. However, the spectrophoto- metric method tend to overestimate the phenolics con- tent with respect to the chromatographic method Table 2 Correlations matrix among phenolics, flavonoids content and free radical scavenging activities Correlations phenolics Flavonoids DPPH phenolics 1 0.995() 0.983() Flavonoids 0.995() 1 0.962() DPPH 0.983() 0.962() 1 ** Correlation is significant at the 0.01 level (2-tailed). Table 2 Correlations matrix among phenolics, flavonoids content and free radical scavenging activities Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Page 5 of 8 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Figure 1 HPLC chromatogram of W. somnifera roots. (1) catechin and (2) benzoic acid. Figure 1 HPLC chromatogram of W. somnifera roots. (1) catechin and (2) benzoic acid. in WSREt, WSFEt and WSLEt. The antioxidant capacity has been shown to be directly related with the total phe- nolic content (Table 2) which was in agreement with many previous reports [32-34]. In addition, the flavo- noids contributed to almost all of the total phenolic content (Table 1), which indicated that the flavonoids in W. somnifera are important constituents responsible for the bioactivities. perhaps due to the fact that non-phenolic materials pre- sent in the investigated extracts interfered in the spec- trophotometric analysis [31]. The scavenging ability of DPPH free radical is exten- sively used to screen the antioxidant potential of natu- rally-derived foods and plants. HPLC analysis Methanol was employed in this study to extract the low molecular weight and moderately polar substances because of its wide solubi- lity properties. We found that WSREt, WSFEt and WSLEt exhibited free radical DPPH scavenging abilities (Table 1). In this study, we attempted to isolate the active compounds responsible for antioxidant activities In HPLC analysis, six compounds were identified in WSLEt while three were identified in WSFEt and two were identified in WSREt. Out of the eight phenolic compounds catechin was found in the highest Figure 2 HPLC chromatogram of W. somnifera fruits. (1) catechin (2) naringenin and (3) kaempferol. Figure 2 HPLC chromatogram of W. somnifera fruits. (1) catechin (2) naringenin and (3) kaempferol. Figure 2 HPLC chromatogram of W. somnifera fruits. (1) catechin (2) naringenin and (3) kaempferol. Page 6 of 8 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Figure 3 HPLC chromatogram of W. somnifera leaves. (1) catechin, (2) gallic acid, (3) syringic acid, (4) vanillic acid, (5) p-coumaric acid and (6) benzoic acid. concentration compared to others amounting 12.82 mg/ g in WSREt, 19.48 in WSFEt and 28.38 mg/g in WSLEt (Table 3). This indicates that WSREt, WSFEt and WSLEt are rich sources of catechin. Catechin is one of the most important polyphenols that provide health benefits and is found in high quantities in green tea which is widely known for its strong antioxidant proper- ties. There are many reports on catechin which described its therapeutic role in human health. Modern studies have found that catechin is responsible for 0 10 20 30 40 50 60 Root Fruit Leave mg/g dry weight Spectrophotometric method HPLC method Figure 4 Comparison of total values of phenolic and flavonoid compounds (mg/g) of W. somnifera roots, fruits and leaves obtained by spectrophotometric and HPLC method (p < 0.05). Spectrophotometric method HPLC method Root Figure 4 Comparison of total values of phenolic and flavonoid compounds (mg/g) of W. somnifera roots, fruits and leaves obtained by spectrophotometric and HPLC method (p < 0.05). Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 Page 7 of 8 Page 7 of 8 Table 3 Phenolic acids and flavonoids compounds detected in Withania somnifera roots, fruits and leaves using high performance liquid chromatography analysis. References 1. Patwardhan B, Panse GT, Kulkarni PH: Ashwagandha a review. Journal of the National Integrated Medicine Association 1998, 30:7-11. 1. Patwardhan B, Panse GT, Kulkarni PH: Ashwagandha a review. Journal of the National Integrated Medicine Association 1998, 30:7-11. 2. Dhuley JN: Adaptogenic and cardioprotective action of ashwagandha in rats and frogs. J Ethnopharmacol 2000, 70(1):57-63. 2. Dhuley JN: Adaptogenic and cardioprotective action of ashwagandha in rats and frogs. J Ethnopharmacol 2000, 70(1):57-63. 3. Agarwal R, Diwanay S, Patki P, Patwardhan B: Studies on immunomodulatory activity of Withania somnifera (ashwagandha) extracts in experimental immune inflammation. J Ethnopharmacol 1999, 67(1):27-35. 4. Prakash J, Gupta SK, Kochupillai V, Gupta YK, Joshi S: Chemopreventive activity of Withania somnifera in experimentally induced fibrosarcoma tumors in Swiss albino mice. Phytother Res 2001, 15(3):240-244. 4. Prakash J, Gupta SK, Kochupillai V, Gupta YK, Joshi S: Chemopreventive activity of Withania somnifera in experimentally induced fibrosarcoma tumors in Swiss albino mice. Phytother Res 2001, 15(3):240-244. 5. Budhiraja RD, Sudhir S: Review of biological activity of withanolides. J Sci Ind Res 1987, 46:488-491. 5. Budhiraja RD, Sudhir S: Review of biological activity of withanolides. J Sci Ind Res 1987, 46:488-491. Author details 1 1Department of Botany, Rajshahi University, Bangladesh. 2Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 1Department of Botany, Rajshahi University, Bangladesh. 2Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. 3Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Kubang Kerian, Kelantan, Malaysia. 3Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. Authors’ contributions NA, MIK and MM have carried out the experimental parts of this investigation. MH, SAS and SHG supervised the work, evaluated the results and corrected the manuscript for publication. All authors read and approved the final manuscript. Conclusion Five phenolics (gallic, syringic, benzoic, p-coumaric and vanillic acids) and three flavonoids (catechin, kaemp- ferol, and naringenin) have been identified in WSREt, WSFEt and WSLEt and catechin was found in high con- centrations especially in the leaves part, confirming the antioxidant potential and health benefits of W. somnifera. 6. Udayakumar R, Kasthurirengan S, Mariashibu TS, Rajesh M, Ramesh Anbazhagan V, Kim SC, Ganapathi A, Choi CW: Hypoglycaemic and hypolipidaemic effects of Withania somnifera root and leaf extracts on alloxan-induced diabetic rats. Int J Mol Sci 2009, 10:2367-2382. 6. Udayakumar R, Kasthurirengan S, Mariashibu TS, Rajesh M, Ramesh Anbazhagan V, Kim SC, Ganapathi A, Choi CW: Hypoglycaemic and hypolipidaemic effects of Withania somnifera root and leaf extracts on alloxan-induced diabetic rats. Int J Mol Sci 2009, 10:2367-2382. 7. Parthasarathy S, Santanam N, Ange N: Oxidised low-density lipoprotein, a two-faced Janus in coronary artery disease. Biochem Pharmacol 1998, 56:279-284. 7. Parthasarathy S, Santanam N, Ange N: Oxidised low-density lipoprotein, a two-faced Janus in coronary artery disease. Biochem Pharmacol 1998, 56:279-284. 8. Keller JN, Kindly MS, Holtberg FW, St Clair DK, Yen HC, Germeyer A, Steiner SM, Bruce-Keller AJ, Hutchins JB, Mattson MP: Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite products, lipid peroxidation, and mitochondrial dysfunction. J Neurosci 1998, 18:687-697. 9. Perry G, Raine KA, Nunomura A, Watayc T, Sayre LM, Smith MA: How important is oxidative damage? Lessons from Alzheimer’s disease. Free Radic Biol Med 2000, 28:831-834. 8. Keller JN, Kindly MS, Holtberg FW, St Clair DK, Yen HC, Germeyer A, Steiner SM, Bruce-Keller AJ, Hutchins JB, Mattson MP: Mitochondrial manganese superoxide dismutase prevents neural apoptosis and reduces ischemic brain injury: suppression of peroxynitrite products, lipid peroxidation, and mitochondrial dysfunction. J Neurosci 1998, 18:687-697. Acknowledgements This study was financially supported by Universiti Sains Malaysia RU grant (grant no. 1001/PPSP/8120201 and 1001/PPSP/815058). The authors would like to acknowledge the Department of Botany, Rajshahi University, Bangladesh and the Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia for providing laboratory support and other facilities for this study. Competing interests The authors declare that they have no competing interests. Received: 22 May 2011 Accepted: 19 August 2011 Published: 19 August 2011 Received: 22 May 2011 Accepted: 19 August 2011 Published: 19 August 2011 Received: 22 May 2011 Accepted: 19 August 2011 Published: 19 August 2011 HPLC analysis SL No Standard compounds Retention time lmax (nm) Quantity of the identified compounds (mg/g DW) Roots Fruits Leaves 1 Catechin 3.36 278 12.82 19.48 28.38 2 Gallic acid 4.12 269, 216 ND ND 0.18 3 Syringic acid 8.10 268, 216 ND ND 0.30 4 Vanillic acid 8.61 224, 249, 269 ND ND 0.15 5 p-coumaric acid 10.97 264, 286, 310 ND ND 0.80 6 Benzoic acid 15.33 272, 241 0.19 ND 0.80 7 Naringenin 20.18 277, 292, 308 ND 0.50 ND 8 Kaempferol 25.59 361 ND 0.06 ND Total phenolic compounds 13.01 ± 8.93 20.04 ± 11.09 30.61 ± 11.41 (ND = Not detected), DW = dry weight Table 3 Phenolic acids and flavonoids compounds detected in Withania somnifera roots, fruits and leaves using high performance liquid chromatography analysis. Table 3 Phenolic acids and flavonoids compounds detected in Withania somnifera roots, fruits and leaves using high performance liquid chromatography analysis. (ND = Not detected), DW = dry weight antioxidant activity, anti-ageing properties and cardiac health maintenance [35]. Catechins’ beneficial effects are attributed to its ability to reduce oxidative stress, lipid peroxidation, free radical generation and unhealthy low density lipoprotein (LDL) cholesterol-oxidation [36]. There is also an evidence that suggests that catechins have a role in the protection against degenerative disor- ders [37]. Throughout the experiments, some catechins have also been shown to inhibit a key enzyme (squalene epoxidase) in the pathway of cholesterol biosynthesis [38]. The potent antioxidant properties of catechin reduce free radical damage to cells and prevent the oxi- dation of LDL cholesterol [39]. Besides catechin, other phenolic compounds found in the WSREt, WSFEt and WSLEt may also contribute to its medicinal and antioxi- dant properties [40]. Further studies to isolate individual active principles and antioxidant activity of individual extracts of roots, fruits as well as leaves through radical scavenging assay and their pharmacological validation in terms of modern medicine will be of great pharmacolo- gical importance in future which is under our consideration. 9. Perry G, Raine KA, Nunomura A, Watayc T, Sayre LM, Smith MA: How important is oxidative damage? Lessons from Alzheimer’s disease. Free Radic Biol Med 2000, 28:831-834. Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 The Open Nutraceuticals Journal 2010, 3:6-16. 17. Tang S, Kerry JP, Sheehan D, Buckley DJ, Morrissey PA: Antioxidative effect of added tea catechins on susceptibility of cooked red meat, poultry and fish patties to lipid oxidation. Food Res Int 2002, 35:651-657. Alam et al. BMC Complementary and Alternative Medicine 2011, 11:65 http://www.biomedcentral.com/1472-6882/11/65 11. Gorogawa SI, Kajimoto Y, Umayahara Y, Kaneto H, Watada H, Kuroda A, Kawamori D, Yasuda T, Matsuhisa M, Yamasaki Y, et al: Probucol preserves pancreatic-cell function through reduction of oxidative stress in type 2 diabetes. Diabetes Res Clin Pract 2002, 57:1-10. 34. Shukla S, Mehtaa A, Johna J, Singha S, Mehtaa P, Vyasb SP: Antioxidant activity and total phenolic content of ethanolic extract of Caesalpinia bonducella seeds. Food Chem Toxicol 2009, 47(8):1848-1851. 35. Lambert JD, Yang CS: Cancer chemopreventive activity and 35. Lambert JD, Yang CS: Cancer chemopreventive activity and bioavailability of tea and tea polyphenols. Mutat Res 2003, 523-524:201-208. 12. Hänninen O, Kaartinen K, Rauma AL, Nenenen M, Törrönen R, Häkkinen S, Adlercreutz H, Laakso J: Antioxidants in vegan diet and rheumatic disorders. Journal of Toxicology 2000, 155:45-53. p yp , 36. Coimbra S, Castro E, Rocha-Pereira P, Rebelo I, Rocha S, Santos-Silva A: The effect of green tea in oxidative stress. Clin Nutr 2006, 25:790-796. y 13. Khodr B, Khalil Z: Modulation of inflammation by reactive oxygen species: implications for aging and tissue repair. Free Radic Biol Med 2001, 30:1-8. 37. Cooper R, Morré DJ, Morré DM: Medicinal benefits of green tea: part I. Review of noncancer health benefits. J Alternative Compl Med 2005, 11:521-528. 14. Ferguson LR: Role of plant polyphenols in genomic stability. Mutat Res 2001, 475:89-111. 38. Abe I, Kashiwagi Y, Noguchi H, Tanaka T, Ikeshiro Y, Kashiwada Y: Ellagitannins and hexahydroxydiphenoyl esters as inhibitor of vertebrate squalene epoxidase. J Nat Prod 2001, 64:1010-1014. 38. Abe I, Kashiwagi Y, Noguchi H, Tanaka T, Ikeshiro Y, Kashiwada Y: Ellagitannins and hexahydroxydiphenoyl esters as inhibitor of vertebrate squalene epoxidase. J Nat Prod 2001, 64:1010-1014. 15. Netzel M, Strass G, Kaul C, Bitsch I, Dietrich H, Bitsch R: In vivo antioxidative capacity of composite berry juice. Food Res Int 2002, 35:213-216. q p 39. Cooper R, Morré DJ, Morré DM: Medicinal benefits of green tea: part II. Review of anticancer properties. J Alternative Compl Med 2005, 11:639-652. 39. Cooper R, Morré DJ, Morré DM: Medicinal benefits of green tea: part II. Review of anticancer properties. J Alternative Compl Med 2005, 11:639-652. 16. Zdunczyk Z, Frejnajel S, Wróblewska M, Juskiewicz J, Oszmianski J, Estrella I: Biological activity of polyphenols extracts from different plant sources. Food Res Int 2002, 35:183-186. 40. Khalil MI, Sulaiman SA, Boukraa L: Antioxidant Properties of Honey and Its Role in Preventing Health Disorder. Pre-publication history Th bli i hi 18. Nuutila AM, Kammiovirta K, Caldentey KMO: Comparison of methods for the hydrolysis of flavonoids and phenolic acids from onion and spinach for HPLC analysis. Food Chem 2002, 76:519-525. The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1472-6882/11/65/prepub doi:10.1186/1472-6882-11-65 Cite this article as: Alam et al.: High catechin concentrations detected in Withania somnifera (ashwagandha) by high performance liquid chromatography analysis. BMC Complementary and Alternative Medicine 2011 11:65. 19. Shahrzad S, Bitsch I: Determination of some pharmacologically active phenolic acids in juices by high-performance liquid chromatography. J Chromatogr A 1996, 741:223-231. 20. Friend J: Phenolic compounds are believed to be an important part of the general defence mechanism of many plants. Phytochemisty soc 1985, 25:367-372. 21. Keefer JF, Schuster SM: Separation of citric acid cycle intermediates by high-performance liquid chromatography with ion pairing. J Chromatogr A 1986, 383(2):297-305. 22. Scorska M, Matawska I, Gowniak K, Zgorka G: Qualitative and quantitative analysis of phenolic acids in Asclepias syriaca L. Acta Pol Pharm 2000, 57(1):69-72. 23. Kahkonen MP, Hopia AI, Vuorela HJPRJ, Pihlaja K, Kujala TS, Heinonen M: Antioxidant Activity of Plant Extracts Containing Phenolic Compounds. J Agric Food Chem 1999, 47:3954-3962. 24. Singleton VL, Orthofer R, Lamuela-Raventos RM: Analysis of total phenols and other oxidation substrates and antioxidant by means of Folin- Ciocalteu reagent. Meth Enzymol 1999, 299:152-178. 25. Zhishen J, Mengcheng T, Jianming W: The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 1999, 64:555-559. 26. Ferreira ICFR, Aires E, Barreira JCM, Estevinho LM: Antioxidant activity of Portuguese honey samples: Different contributions of the entire honey and phenolic extract. Food Chem 2009, 114:1438-1443. 27. Hatano T, Kagawa H, Yashura T, Okuda T: Two new flavonoids and other constituents in licorice root: Their relative astringency and radical scavenging effects. Chemical & Pharmaceutical Bulletin 1988, 36:2090-2097. 28. Kaškonienė V, Maruška A, Kornyšova O: Quantitative and qualitative determination of phenolic compounds in honey. Cheminė Technologija 2009, 52(3):74-80. 29. Piljac-Žegarac J, Stipčević T, Belščak A: Antioxidant properties and phenolic content of different floral origin honeys. J ApiProd ApiMed Sci 2009, 1(2):43-50. 30. Udayakumar R, Kasthurirengan S, Vasudevan A, Mariashibu TS, Rayan JJS, Choi CW, Ganapathi A, Kim SC: Antioxidant Effect of Dietary Supplement Withania somnifera L. Reduce Blood Glucose Levels in Alloxan-Induced Diabetic Rats. Plant Foods Hum Nutr 2010, 65:91-98. Acknowledgements h d f 9. Perry G, Raine KA, Nunomura A, Watayc T, Sayre LM, Smith MA: How important is oxidative damage? Lessons from Alzheimer’s disease. Free Radic Biol Med 2000, 28:831-834. This study was financially supported by Universiti Sains Malaysia RU grant (grant no. 1001/PPSP/8120201 and 1001/PPSP/815058). The authors would like to acknowledge the Department of Botany, Rajshahi University, Bangladesh and the Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia for providing laboratory support and other facilities for this study. 10. Kamat JP, Devasagayam TPA: Oxidative damage to mitochondria in normal and cancer tissues, and its modulation. Toxicology 2000, 155:73-82. 10. Kamat JP, Devasagayam TPA: Oxidative damage to mitochondria in normal and cancer tissues, and its modulation. Toxicology 2000, 155:73-82. Page 8 of 8 Page 8 of 8 Pre-publication history Th bli i hi Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 31. Escarpa A, González MC: Approach to the content of total extractable phenolic compounds from different food samples by comparison of chromatographic and spectrophotometric methods. Anal Chim Acta 2001, 427(1):119-127. 32. Azlim Almey AA, Ahmed Jalal Khan C, Syed Zahir I, Mustapha Suleiman K, Aisyah MR, Kamarul Rahim K: Total phenolic content and primary antioxidant activity of methanolic and ethanolic extracts of aromatic plants’ leafs. International Food Research Journal 2010, 17:1077-1084. 33. Liu SC, Lin JT, Wang CK, Chen HY, Yang DJ: Antioxidant properties of various solvent extracts from lychee (Litchi chinenesis Sonn.) flowers. Food Chem 2009, 114:577-581.
https://openalex.org/W2060356117	https://europepmc.org/articles/pmc4369842?pdf=render	English	null	Improvement of HAART in Brazil, 1998–2008: a nationwide assessment of survival times after AIDS diagnosis among men who have sex with men	BMC public health	2,015	cc-by	6,881	* Correspondence: mmalta2@jhsu.edu 1Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street E7152, Baltimore, MD 21205, USA 2Oswaldo Cruz Foundation - FIOCRUZ, Sergio Arouca National School of Public Health, Department of Social Sciences, Rua Leopoldo Bulhoes, 1480 suite 905 Manguinhos, Rio de Janeiro, RJ 21041-210, Brazil Full list of author information is available at the end of the article Improvement of HAART in Brazil, 1998–2008: a nationwide assessment of survival times after AIDS diagnosis among men who have sex with men Monica Malta1,2, Cosme M F P da Silva3, Monica MF Magnanini4, Andrea L Wirtz5, André R S Perissé6, Chris Beyrer1, Steffanie A Strathdee7 and Francisco I Bastos8 Malta et al. BMC Public Health (2015) 15:226 DOI 10.1186/s12889-015-1530-y Malta et al. BMC Public Health (2015) 15:226 DOI 10.1186/s12889-015-1530-y Open Access Methods This study utilized four databases comprising different longitudinal information of all people living with HIV/ AIDS (PLWHA) under treatment and care through the Brazilian public health system. These databases comprise all patients receiving any antiretroviral medicine in Brazil since such medicines are not procured, purchased, or delivered by private health facilities or pharmacies for patients who are not hospitalized. However, AIDS-related deaths continue to occur, even in high-income countries, and the emergence of drug- resistant HIV variants and drug toxicities remain major barriers and challenges to successful long-term anti- retroviral efficacy [8,9]. Although stable, on average, the epidemic in Central and South America is currently concentrated in specific groups, mainly among MSM [1,10]. In Brazil MSM accounted for more than 32% of the cumulative reported cases of AIDS from 1980 to 2010 among men older than 13 years of age [11]. Data from a Brazilian meta-analysis indicated a pooled HIV prevalence of 13.6% (95% CI:8.2- 20.2) among MSM [12]. National data from sexually transmitted infections’ clinics in Brazil demonstrated a 1.7% overall prevalence for HIV among clinic attendees [11]. However, when stratified for subgroups defined by sexual exposure, prevalence among MSM was found to be 5.7%, compared to 1.1% among heterosexual men. Data from three voluntary counseling and testing ser- vices reported 24.8% prevalence among MSM and 4.3% among heterosexual men [13]. HIV incidence at VCT units in Rio de Janeiro, Brazil, indicated incidence up to 11 times higher among MSM when compared to hetero- sexual men [13,14]. These databases contain the core information of Brazil’s surveillance system. Their basic characteristics are briefly summarized as follows: i. Socio-demographic and basic clinical (for the sake of diagnosis) information on AIDS cases (SINAN-AIDS - Information System for Notifiable Diseases/AIDS); ii. Data from exams conducted in the network of accredited public laboratories, particularly TCD4+/TCD8 lymphocyte counting and HIV viral load (SISCEL - National Database for Laboratory Tests); iii. Information about monthly ARV refills and therapeutic regimen changes over time (SICLOM - Logistics Control System of ARV Medicines); and iv. The date and cause/s of death (SIM - National Mortality Database). Background eligible patient, since 1996 [15]. As of June 2014, ap- proximately 400,000 patients were receiving HAART in Brazil [11], making it the most comprehensive HIV treatment initiative implemented thus far in a middle- income country, worldwide [16]. By the end of 2013, 35 million [33.2 million-37.2 million] people were living with HIV worldwide and 1.5 million [1.4 million-1.7 million] AIDS-related deaths were re- ported in the latest Epidemiological Bulletin released by UNAIDS. The introduction of combination antiretroviral therapies has led to dramatic improvements in AIDS- related morbidity and mortality in countries where this treatment is available [1]. Recent Brazilian observational studies have brought essential information about mortality patterns among HIV-positive patients under follow-up in Brazil’s referral centers [17,18]. However, clinical cohorts from selected referral centers do not assess real-life conditions found in busy, understaffed public health clinics scattered all over Brazil, a country where social and regional hetero- geneities are very relevant in the field of AIDS care [19,20], as well as health care in a broad sense [21]. Highly active antiretroviral therapy (HAART) signifi- cantly improves the prognosis of HIV-infected persons by reducing HIV viral load, increasing CD4+ lymphocyte counts and delaying progression to AIDS, ultimately re- ducing mortality rates [2,3]. Herein, we report differences in survival from AIDS diagnosis according to period of AIDS diagnosis, within the unique Brazilian setting. The study evaluated all MSM receiving treatment in the country between 1998 and 2008, therefore avoiding selection bias present in analyses from specific subsets (such as referral centers from major southeastern metropolitan areas, particularly Rio de Janeiro and Sao Paulo) of the country’s popula- tion living with HIV/AIDS. Pre-exposure prophylaxis (PrEP) for HIV infection has greatly improved the field of HIV prevention [4], and a recent systematic review suggests its efficacy among high-risk groups such as men who have sex with men – MSM [5]. Early detection and management of HIV infection are key challenges worldwide, but a growing body of evi- dence supports the immediate use of HAART to main- tain CD4 count and functionality, limit the size of the HIV reservoir, and reduce the risk of onward viral trans- mission [6]. Following such groundbreaking results, Brazil has recently announced plans to adopt the “Treat- ment as Prevention” strategy to curb HIV and AIDS [7]. Abstract Background: In 1996, Brazil became the first developing country to provide free, universal access to HAART, laboratory monitoring, and clinical care to any eligible patient. As of June 2014, approximately 400,000 patients were under treatment, making it the most comprehensive HIV treatment initiative implemented thus far in a middle-income country, worldwide. The Brazilian epidemic is highly concentrated among men who have sex with men (MSM). Methods: Four national information systems were combined and Cox regression was used to conduct retrospective cohort analysis of HAART availability/access on all-cause mortality among MSM diagnosed with AIDS reported to the information systems between 1998–2008, adjusting for demographic, clinical, and behavioral factors and controlling for spatially-correlated survival data by including a frailty effect. Multiple imputation by chained equations was used to handle missing data. Results: Among 50,683 patients, 10,326 died during the 10 year of period. All-cause mortality rates declined following introduction of HAART, and were higher among non-white patients and those starting HAART with higher viral load and lower CD4 counts. In multivariable analysis adjusted for race, age at AIDS diagnosis, and baseline CD4 cell count, MSM diagnosed in latter periods had almost a 50% reduction in the risk of death, compared to those diagnosed between 1998–2001 (2002–2005 adjHR: 0.54, 95% CI:0.51-0.57; 2006–2008 adjHR: 0.51, 95% CI:0.48-0.55). After controlling for spatially correlated survival data, mortality remained higher among those diagnosed in the earliest diagnostic cohort and lower among non-white patients and those starting HAART with higher viral load and lower CD4 lymphocyte counts. Conclusions: Universal and free access to HAART has helped achieve impressive declines in AIDS mortality in Brazil. However, after a 10-years follow-up, differential AIDS-related mortality continue to exist. Efforts are needed to identify and eliminate these health disparities, therefore improving the Brazilian response towards HIV/AIDS epidemic. Keywords: HIV, AIDS, HAART (Highly active antiretroviral therapy), Men who have sex with men (MSM), Survival analysis, Brazil © 2015 Malta et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Malta et al. BMC Public Health (2015) 15:226 Page 2 of 8 Methods Whereas patients may seek private medical care and perform laboratory exams in private facilities, all PLWHA receive their antiretroviral drugs through SUS (Brazil’s Unified Public Health System), the single Brazil was the first middle-income country to provide full access to HAART, laboratory monitoring and clinical care at no cost at the point of health care delivery to any Malta et al. BMC Public Health (2015) 15:226 Page 3 of 8 following: age at AIDS diagnosis, race, period of AIDS diagnosis (1998–2001; 2002–2005; 2006–2008), first CD4+ lymphocyte count, HAART uptake, and first HIV- RNA viral load exam. The likelihood ratio test was used to select variables to be entered in the Cox models, adjusting for potential confounders. The assumption of proportional hazards was evaluated through the examin- ation of Schoenfeld residuals [22]. accredited source of ARVs in Brazil, and should be in- cluded in SICLOM database. At the time we conducted our analysis, SICLOM data have been updated to im- prove the comprehensiveness of collected data, which tend to be limited to the core information which is es- sential for medicines dispensing at the several health ser- vices belonging to the network of accredited public health units. Thus, to utilize data from all regions, we could not include additional information such as mea- sures of adherence, side effects, drug regimen shifts, etc. as these data were not yet available for all regions and services. Hazard rates are strongly influenced by selection ef- fects operating in the population (e.g. higher availability of referral centers in a given locality), and individuals surviving up to a certain time will on average be less frail than the original population, therefore unobserved indi- vidual heterogeneity (or frailty) was also taken into ac- count [23]. Data from the entire population of PLWHA with AIDS diagnosis between 1998 and 2008, whose trans- mission category was homo/bisexual contact were ex- tracted from those databanks and merged. The databank linkage procedures have been described in detail else- where [15]. With 8.5 million square kilometers and over 202 million inhabitants according to the National Census, 2010, up- dated as of July 2013 [24] and regular updates issued by the Brazilian Institute for Geography and Statistics (IBGE), Brazil is the largest and most populous country in Latin America, and faces deep health and socio-economic differ- ences between and within each region. Statistical analyses The primary endpoint of interest in the present study was all-cause mortality during 1998–2008. Survival was calculated as the time elapsed from date of AIDS diag- nosis until the date of death. Individuals who remained alive were censored at the end of data collection period (December 31, 2008). Time from AIDS diagnosis was transformed from days to years since diagnosis. Since analyses were based on secondary data, we did not have access to information on clinical visits; therefore we were not able to calculate losses to follow-up. Creating multiple imputations, as opposed to single imputations, accounts for the statistical uncertainty in the imputations. In addition, the chained equations ap- proach is very flexible and can handle variables of vary- ing types (e.g., continuous or binary), and has been reported as very reliable when used in different datasets [27,28]. All analyses were performed using R software version 3.0.2 (library survival) and Stata 13.0 (College Station, TX) [29]. Kaplan-Meier curves were fitted to compare probabil- ities of survival according to selected period of diagnosis and CD4 count at diagnosis using the log-rank test. Cox proportional hazard regression modeling was used to examine covariates associated with survival among the Methods In an attempt to account for the heterogeneity between and within differ- ent Brazilian regions and localities, Cox regression ana- lyses were repeated with the introduction of a random effect term to assess the potential association between survival times within a cluster – state or municipality of residency – using gamma-distributed frailty [23]. A frailty indicator >1 denotes a faster event (death) rate, while groups will frailty <1 survival tend to be longer [25]. For this analysis, participants were included if they were diagnosed as having AIDS between January 1, 1998 and December 31, 2008, and if their most probable route of HIV infection was male-male unprotected anal inter- course. Those patients were included because we aimed to analyze differences in survival from AIDS diagnosis among the MSM population, for which higher rates of initiation and better adherence to HAART during three different AIDS diagnosis periods: 1998–2001, 2002– 2005, and 2006–2008 have been documented in the lit- erature and by our own exploratory analyses. The final merged database comprised the following co- variates: date of birth, race, date of AIDS diagnosis, dates of initiation of HAART, CD4 counts and viral load at diagnosis, cause(s) and date of death. CD4 counts at initial diagnosis were classified into three categories: < 200 cells/ mm3, 200–350 cells/mm3, and >350 cells/mm3. Viral load at initial diagnosis was classified as <4.00 log10 cop- ies, 4.00-5.00 log10 copies, and >5.00 log10 copies. To estimate confidence intervals for Cox's propor- tional hazards models, robust methods were used. Max- imum penalized likelihood estimation was used to fit frailty models. To handle missing data, we implemented multiple imputation by chained equations using classifi- cation and regression trees (MICE-CART) as the condi- tional models for imputation [26]. This method can result in more plausible imputations, and hence more reliable inferences, in complex settings than the naive application of standard sequential regression imputation techniques. Characteristics of the patients Between January 1 1998 and December 31 2008, 50,683 MSM were diagnosed with AIDS in Brazil. Of these, 20,702 (40.9%) were diagnosed between 1998–2001, 19,280 (38.0%) in 2002–2005, and 10,701 (21.1%) in the last period under analysis, 2006–08. The overall mean age at AIDS diagnosis was 35.4 years (SD: 9.5). Data on race from 39.0% of our sample was missing. Although all patients had a confirmed AIDS (clinical) diagnosis, there were no information respecting 29.5% of the CD4 counts and 42.5% HIV-1 RNA viral load determinations in the In the unadjusted analysis, the risk of death was lower for both periods 2002–2005 (HR: 0.74; 95% CI:0.71-0.77; p < 0.001) and 2006–2008 (HR: 0.81; 95% CI:0.76-0.86; p < 0.001), compared to the first period of diagnosis (1998–2001). Ethical approval d f bl No identifiable personal data were used for this study. The dataset used in the study is not openly available. Page 4 of 8 Malta et al. BMC Public Health (2015) 15:226 Page 4 of 8 Permission to use non-identifiable, individual data ex- tracted from HIV/AIDS administrative databases was granted by the Brazilian Ministry of Health, Department of STDs, AIDS and Viral Hepatitis. Ethical approval for use of encrypted and aggregated data was also obtained from the Oswaldo Cruz Foundation Research Ethics Committee (Protocol CEP/ENSP 179/08) and the Brazilian National Research Ethics Committee (CONEP 15308). merged database (Table 1). Due to the significant amount of missing data, MICE-CART multiple imputation was conducted before running survival analysis. Permission to use non-identifiable, individual data ex- tracted from HIV/AIDS administrative databases was granted by the Brazilian Ministry of Health, Department of STDs, AIDS and Viral Hepatitis. Ethical approval for use of encrypted and aggregated data was also obtained from the Oswaldo Cruz Foundation Research Ethics Committee (Protocol CEP/ENSP 179/08) and the Brazilian National Research Ethics Committee (CONEP 15308). There were 10,326 deaths during the 10 year period. The median follow-up times for patients in three diagnosis periods are: period 1998–2001: 8.36 years; period 2002– 2005: 4.68 years; and period 2006–2008: 1.44 years. In Kaplan-Meier analyses stratified by diagnostic period, those diagnosed in the first period had significantly lower survival since AIDS diagnosis (p < 0.0001), with visible improvements among patients from the second and third cohort period (p < 0.0001). Taking into consider- ation the short follow-up time for participants in diag- nostic period 2006–2008, there appeared to be little difference in survival between the last two diagnostic periods (Figure 1). Characteristics of the patients The unadjusted HR for the period 2006–2008 Table 1 Sociodemographic and clinical characteristics of the study patients Table 1 Sociodemographic and clinical characteristics of the study patients Table 1 Sociodemographic and clinical characteristics of the study patients Diagnostic period Total 1998 −2001 2002 −2005 2006 −2008 Subjects (N, %) 50,683 20,702 19,280 10,701 Follow-up time (person-years) 249,656.30 149,494.24 84,776.07 15,385.97 Age at diagnosis (years) Mean ± SD 35.4 ± 9.5 35.3 ± 9.2 35.3 ± 9.5 35.8 ± 9.9 Range 18 – 89 18 – 89 18 – 86 18 – 85 Ethnicity Caucasian 18,399 (36.3) 3,087 (14.9) 9,454 (49.0) 5,858 (54.8) Mulatto (mixed white and black) 9,290 (18.3) 1,408 (6.8) 4,701 (24.4) 3,181 (29.7) Black 2,912 (5.8) 478 (2.3) 1,520 (7.9) 914 (8.5) Others (Asian, Indigenous…) 307 (0.6) 64 (0.3) 132 (0.7) 111 (1.0) Unspecified 19,775 (39.0) 15,666 (75.7) 3,473 (18.0) 637 (6.0) Deathsa 10.326 5.791 3.258 1.277 Have at least one CD4 exam available on SISCEL No 14,934 (29.5) 8.081 (39.0) 4.082 (21.2) 2,771 (25.9) Yes 35,749 (70.5) 12.621 (61.0) 15.198 (78.8) 7,930 (74,1) CD4 lymphocytes, cells/mm3 (first exam available)b Mean ± SD 298.1 ± 185.3 324.8 ± 179.3 289.5 ± 191.7 261.9 ± 177.0 Range 0 – 999 0 – 998 0 – 998 0 – 999 Have at least one HIV-1 RNA Viral load exam available on SISCEL No 21,562 (42.5) 11,097 (53.6) 6,601 (34.2) 3,864 (36.1) Yes 29,121 (57.5) 9.605 (46.4) 12,679 (65.8) 6,837 (63.9) HIV RNA in plasma, log10 copies/ml (first exam available)c Mean ± SD 4.46 ± 1.01 4.38 ± 1.04 4.46 ± 1.02 4.60 ± 0.91 Range 1.70 – 6.69 1.70 – 6.67 1.70 – 6.69 1.70 – 6.69 aAll causes of death; bAmong those with at least one CD4 exam; cAmong those with at least one viral load exam; SISCEL: National Database for Laboratory. Page 5 of 8 Malta et al. BMC Public Health (2015) 15:226 2005 adjHR: 0.54, 95CI:0.51-0.57; Period 2006–2008 adjHR: 0.51, 95%CI:0.48-0.55). There was no difference between the cohort diagnosed in 2006–2008 compared to the cohort diagnosed in 2002–2005, after adjustment in the Cox model (adjHR: 0.95; 95% CI:0.89-1.02). Add- itional predictors of longer survival included younger age at diagnosis and higher CD4 counts at diagnosis. Patients with an HIV-RNA viral load >5log10 copies/ml at diagnosis had a higher risk of death, as well as non- White patients (Table 2). Characteristics of the patients Figure 1 Overall Survival Among MSM in Brazil from 1998–2009 After AIDS Diagnosis, by Period of Diagnosis. Upon multivariate Cox frailty regression that accounted for a random effect associated with the Brazilian state where patients lived (Table 2), the increased risk of death observed in patients diagnosed in the first period of time persisted. Both higher CD4 cells counts and lower HIV-1 RNA viral load remained as significant pre- dictors of survival. Non-White patients remained with a higher risk of death; however, there was no longer a dif- ference between white participants and those with un- specified race. Figure 1 Overall Survival Among MSM in Brazil from 1998–2009 After AIDS Diagnosis, by Period of Diagnosis. was higher (HR: 1.09; 95% CI:1.02-1.17; p < 0.007) when compared to the reference of 2002–2005 to assess dif- ference between latter periods (data not shown). The estimated frailty variance was significantly differ- ent from zero, suggesting that the risk of death after AIDS diagnosis was heterogeneous among subjects liv- ing in different states (Table 2). We adjusted the final model with and without MICE-CART imputation Hazard Ratio (95% Confidence Interval); p-value < 0.001; Cox proportional hazards model adjusted for all variables listed in the table. tComparing period of diagnosis in 2006–2008 to the reference of 2002–2005: adjusted HR: 0.95 (95% CI: 0.89 - 1.02; p = 0.144); model adjusted for state frailty effect: HR: 0.94 (95% CI: 0 88 - 0 01; p = 0 085) Discussion Figure 2 Frailty estimates of relative risks of state showing the point estimate (circle) and a confidence interval of 95%. Both the interval and the distribution are skewed because the relative risks in an exponential function of the gamma estimate. Abbreviations: Acre (AC); Alagoas (AL); Amapá (AP); Amazonas (AM); Bahia (BA); Ceará (CE); Distrito Federal (DF); Espírito Santo (ES); Goiás (GO); Maranhão (MA); Mato Grosso (MT); Mato Grosso do Sul (MS); Minas Gerais (MG); Pará (PA); Paraíba (PB); Paraná (PR); Pernambuco (PE); Piauí (PI); Rio de Janeiro (RJ) ; Rio Grande do Norte (RN); Rio Grande do Sul (RS); Rondônia (RO); Roraima (RR); Santa Catarina (SC); São Paulo (SP); Sergipe (SE); Tocantins (TO). Predictors of survival Changes in the main causes of death from AIDS to non-AIDS related conditions have also been identified [18,32], however, those studies were conducted in a pool of referral centers or in a single refer- ral center, and none of them assessed nationwide data, taking in consideration their pronounced heterogeneity. American countries [31]. Changes in the main causes of death from AIDS to non-AIDS related conditions have also been identified [18,32], however, those studies were conducted in a pool of referral centers or in a single refer- ral center, and none of them assessed nationwide data, taking in consideration their pronounced heterogeneity. Greater frailty effects (and therefore greater mortality) were found in larger states located in the Tropical Rain Forest region and in states located in the Northeast, Brazil’s poorest region. On the other hand, industrialized areas from the Southern and Southeastern regions accounted for the smallest frailty effect (Figure 2). The observed improvements in the latter periods of diagnosis compared to those diagnosed between 1998– 2001 are favorable. No difference, however, has yet been detected between those diagnosed in 2006–2008 com- pared to those diagnosed in 2002–2005. This finding may be attributed to barriers and problems in the current treatment program and the concrete operations of the respective network of public health services. Among the most relevant of such challenges, some are highlighted as follows: i) the increasing pressures over costs [33], challenging the very sustainability of universal access over time; ii) the deep heterogeneity of the Brazil- ian network of health services, either in the specific field of AIDS management and care [20] or from the perspec- tive of the health system in a broad sense [21]; and, iii) last but not the least, the late presentation of a substan- tial fraction of patients [19]. Nonetheless, it is important to note that the median follow-up time for patients diag- nosed in 2006–2008 is substantially shorter and future research may observe improvements after longer dura- tions of follow-up of this cohort. Discussion This is the first Brazilian nationwide analysis of survival after AIDS diagnosis among MSM. Our study compared different time periods after the full introduction of HAART over the whole country, following the approval of the federal legislation regulating the right to the uni- versal access to antiretroviral medicines for people living with AIDS, as of the end of 1996 [29]. Improvements in survival were observed for latter periods of diagnosis, with almost 50% reduction in risk of death when com- pared to the earliest cohort. Protective effects were also observed with lower age and higher CD4 classifications at initial diagnosis. Previous papers documented the dramatic improve- ment of survival before and after HAART [30], and identified lower mortality rates among PLWHA from Brazil, when compared with patients from other Latin Figure 2 Frailty estimates of relative risks of state showing the point estimate (circle) and a confidence interval of 95%. Both the interval and the distribution are skewed because the relative risks in an exponential function of the gamma estimate. Abbreviations: Acre (AC); Alagoas (AL); Amapá (AP); Amazonas (AM); Bahia (BA); Ceará (CE); Distrito Federal (DF); Espírito Santo (ES); Goiás (GO); Maranhão (MA); Mato Grosso (MT); Mato Grosso do Sul (MS); Minas Gerais (MG); Pará (PA); Paraíba (PB); Paraná (PR); Pernambuco (PE); Piauí (PI); Rio de Janeiro (RJ) ; Rio Grande do Norte (RN); Rio Grande do Sul (RS); Rondônia (RO); Roraima (RR); Santa Catarina (SC); São Paulo (SP); Sergipe (SE); Tocantins (TO). Other problems to be tackled by clinicians and health managers include the progressive, albeit moderate, in- crease of secondary resistance [34] and the permanent challenge posed by side effects of different antiretroviral medications, such as lipodystrophy and metabolic syn- dromes [35]. However, despite all such caveats and diffi- culties, the present paper documents a progressive increase of survival times among gay men over succes- sive periods of the decade 1998–2008. Similar findings were identified by a multi-site study comprising data from several North American cohorts. According to the study conducted by Samji and colleagues [29], who ana- lyzed data from almost 23,000 treatment-naive ART pa- tients from the MSM population, the life-expectancy at age 20 years increased with calendar time, from 53.3 in 2000–2002 to 69.3 in 2006–2007. Future Brazilian stud- ies should include more recent timeframes, as well as additional information about HAART uptake and treat- ment adherence. Predictors of survival Controlling for other variables, the risk of death among latter cohorts of diagnosis was lower when compared to the cohort diagnosed between 1998–2001 (Period 2002– Table 2 Hazard ratios of mortality according to baseline variables among MSM patients diagnosed with AIDS in Brazil, 1998–2008 Adjusted Adjusted by state frailty effect Predictor HR 95% CI HR 95% CI Age (per 10 year increase) 1.13* (1.11 - 1.16) 1. 13* (1.11 - 1.16) Race** White Reference Reference Non-white 1.15* (1.09 - 1.21) 1.29* (1.22 - 1.37) Unspecified 0.89* (0.84 - 0.94) 1.02 (0.96 - 1.08) Period of diagnosist 1998-2001 Reference Reference 2002-2005 0.54* (0.51 - 0.57) 0.55* (0.52 - 0.58) 2006-2008 0.51* (0.48 - 0.55) 0.52* (0.49 - 0.56) CD4 cell count at diagnosis (cells/mm3) < 200 Reference Reference 200 - 350 0.65* (0.63 - 0.68) 0.67* (0.65 - 0.70) >350 0.20* (0.18 - 0.21) 0.19* (0.17 - 0.20) HIV-1 RNA (log10 copies) <4.00 Reference Reference 4.00-5.00 1.28* (1.19 - 1.37) 1.22* (1.14 - 1.31) >5.00 4.68* (4.40 - 4.98) 5.26* (4.94 - 5.61) Estimated frailty variance — 0.099* Hazard Ratio (95% Confidence Interval); p-value < 0.001; Cox proportional hazards model adjusted for all variables listed in the table. tComparing period of diagnosis in 2006–2008 to the reference of 2002–2005: adjusted HR: 0.95 (95% CI: 0.89 - 1.02; p = 0.144); model adjusted for state frailty effect: HR: 0.94 (95% CI: 0.88 - 0.01; p = 0.085). of mortality according to baseline variables among MSM patients diagnosed with AIDS in Brazil, Table 2 Hazard ratios of mortality according to baseline variables among MSM patients diagnosed with AIDS in Brazil, 1998–2008 Hazard Ratio (95% Confidence Interval); p-value < 0.001; Cox proportional hazards model adjusted for all variables listed in the table. tComparing period of diagnosis in 2006–2008 to the reference of 2002–2005: adjusted HR: 0.95 (95% CI: 0.89 - 1.02; p = 0.144); model adjusted for state frailty effect: HR: 0.94 (95% CI: 0.88 - 0.01; p = 0.085). Malta et al. BMC Public Health (2015) 15:226 Page 6 of 8 Page 6 of 8 procedure; however there were no relevant influence in the results (data not shown). American countries [31]. Authors’ contributions MM designed the study, organized the dataset, coordinated the statistical analysis and drafted the manuscript. CMFPS and MMM participated in the design of the study and performed the statistical analysis. AW contributed to statistical analysis and to the draft of the methods and results. AP reviewed the statistical analysis and helped to draft the manuscript. FIB, SAS and CB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. The improvement in survival from the first period of HAART is undeniable and should be commended. How- ever, results should be viewed in light of some study lim- itations. First, the process of merging four national databases resulted in missing data among key variables, such as CD4+ lymphocyte counts and viral load measures. Such limitations forced us to conduct multiple imputation (MICE-CART) of several variables before running the sur- vival analysis. We did not conduct MICE-CART for race, a key variable with a significant proportion of missing in- formation. This was a methodological and operational de- cision: patients with private health insurance usually have scarce information available on national datasets and our MICE-CART could not be accurately conducted with the available information. This problem calls for an urgent need to provide better training of professionals and clerks in charge of monitoring the Brazilian AIDS epidemic and to increase the accountability and consistency of such sys- tems. A second limitation lies in the comparison of sur- vival across time periods of initial diagnosis. Because our dataset is limited to 1998 to 2008, length of follow-up is shorter for those diagnosed in 2002–2005 and 2006–2008, limiting comparisons in survival after six to ten years fol- lowing diagnosis. Nonetheless, these data are sufficient to allow for comparisons of trends in survival across different periods of diagnosis and CD4 count to AIDS diagnosis during the early years of HAART. Acknowledgements The authors would like to thank the Brazilian National STD/AIDS and Viral Hepatitis Program for authorizing our access to their Nation-wide datasets The authors would like to thank the Brazilian National STD/AIDS and Viral Hepatitis Program for authorizing our access to their Nation-wide datasets Author details 1 1Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street E7152, Baltimore, MD 21205, USA. 2Oswaldo Cruz Foundation - FIOCRUZ, Sergio Arouca National School of Public Health, Department of Social Sciences, Rua Leopoldo Bulhoes, 1480 suite 905 Manguinhos, Rio de Janeiro, RJ 21041-210, Brazil. 3Department of Epidemiology and Quantitative Methods in Health, Oswaldo Cruz Foundation - FIOCRUZ, Sergio Arouca National School of Public Health, Rua Leopoldo Bulhoes, 1480- suite 802 Manguinhos, Rio de Janeiro 21041-210, Brazil. 4Federal University of Rio de Janeiro, Institute of Public Health Studies, Praça da Prefeitura Universitária Cidade Universitária, Rio de Janeiro 21941-598, Brazil. 5Department of Emergency Medicine, Johns Hopkins Bloomberg School of Public Health Department of Epidemiology, Johns Hopkins Medical Institute, 615 N. Wolfe St, E 7143, Baltimore, MD 21205, USA. 6Department of Biological Sciences, Rua Leopoldo Bulhoes, Oswaldo Cruz Foundation- FIOCRUZ, National School of Public Health, 1480, suite 21 Manguinhos, Rio de Janeiro RJ 21041-210, Brazil. 7School of Medicine, University of California, UCSD 9500 Gilman Drive La Jolla, San Diego, CA 92093-0507, USA. 8Oswaldo Cruz Foundation - FIOCRUZ, Biblioteca de Manguinhos suite 229 Av. Brasil 4365, Rio de Janeiro 21045-900, Brazil. Received: 18 February 2014 Accepted: 13 February 2015 Received: 18 February 2014 Accepted: 13 February 2015 Competing interests D S hd k l Dr. Strathdee acknowledges support from the Fogarty International Center, grant R25-TW007500. Such barriers and caveats must be progressively over- come by policymakers, managers, health professionals and the civil society in a continental-sized, deeply het- erogeneous country, housing the most comprehensive network of ARV dispensing units worldwide. With in- creasing costs and the pressing need to switch thera- peutic regimens in order to minimize both HIV resistance and different adverse effects of ARVs, substan- tial improvement of information systems is mandatory and urgently needed, as already highlighted by Grangeiro and colleagues [36]. Dr. Malta acknowledges support from INOVA/ENSP FIOCRUZ, University of California Center for AIDS Research (NIAID 5 P30 AI 036214), PAPES-VI/CNPq and CAPES (Proc. No BEX 2582/13-9). Dr. Beyrer acknowledges support of the Johns Hopkins University Center for AIDS Research (P30AI094189). Dr. Beyrer acknowledges support of the Johns Hopkins University Center for AIDS Research (P30AI094189). The authors declare no conflict of interest. All analyses are based on public available databases, stripped of personal identification. The authors declare no conflict of interest. All analyses are based on public available databases, stripped of personal identification. Conclusions Our statistical modeling incorporated frailty effects and demonstrated a pronounced effect of geographic, social and/or health care quality upon survival times. It is not possible to disentangle the independent effects of each one of these factors; however, our study highlighted the negative impact on survival among patients living in less Malta et al. BMC Public Health (2015) 15:226 Page 7 of 8 Page 7 of 8 treatment for PLWHA at no cost at the point of deliv- ery. Notwithstanding, the concerted effort from across different sectors of government, community leadership, and the civil society at large may be seriously compro- mised in the absence of a comprehensive and accurate monitoring system. Although our analyses documented some progress since the early years of HAART, current levels of missing information remain unacceptably high - a critical challenge as we enter in the third decade of the HIV/AIDS epidemic, with more complex and costly treatments, more patients under follow-up and more data being collected. industrialized and most deprived regions of Brazil. Patients living in such areas are likely to suffer from an adverse and synergic effect of poverty and lack of social support, receiving less than optimal care in settings where health services are scarce, overburdened, less accessible and of lower quality [21]. As in any analysis of large secondary datasets, there is a trade-off between the augmented comprehensiveness of patient data under analysis and the deficiencies in terms of coverage and quality of databases. In this sense, the missing information identified in the current study constitutes serious limitations, not only in terms of the resulting analyses, but also from the broader perspective of monitoring and policymaking. Malta et al. BMC Public Health (2015) 15:226 The global north: HIV epidemiology in high-income countries. Curr Opin HIV AIDS. 2014;9(2):199–205. y y y 8. Sullivan PS, Jones JS, Baral SD. The global north: HIV epidemiology in high-income countries. Curr Opin HIV AIDS. 2014;9(2):199–205. 32. Pacheco AG, Tuboi SH, May SB, Moreira LF, Ramadas L, Nunes EP, et al. Temporal changes in causes of death among HIV-infected patients in the HAART era in Rio de Janeiro. Brazil J Acquir Immune Defic Syndr. 2009;51(5):624–30. 9. Hosseinipour MC, Gupta RK, Van Zyl G, Eron JJ, Nachega JB. Emergence of HIV drug resistance during first- and second-line antiretroviral therapy in resource-limited settings. J Infect Dis. 2013;207 Suppl 2:S49–56. 33. Nunn AS, Fonseca EM, Bastos FI, Gruskin S, Salomon JA. Evolution of antiretroviral drug costs in Brazil in the context of free and universal access to AIDS treatment. PLoS Med. 2007;4:e305. 10. Bastos FI, Cáceres C, Galvão J, Veras MA, Castilho EA. AIDS in Latin America: assessing the current status of the epidemic and the ongoing response. Int J Epidemiol. 2008;37(4):729–37. 34. Westin MR, Biscione FM, Fonseca M, Ordones M, Rodrigues M, Greco DB, et al. Resistance-associated mutation prevalence according to subtypes B and Non-B of HIV type 1 in antiretroviral-experienced patients in minas gerais. Brazil AIDS Res Hum Retroviruses. 2011;27(9):981–7. 11. Brazilian Ministry of Health. HIV/AIDS and Viral Hepatitis Department. HIV/ AIDS and STI Epidemiological Bulletin. Available at http://www.aids.gov.br/ publicacao/2013/boletim-epidemiologico-aids-e-dst-2013 12. Malta M, Magnanini MM, Mello MB, Pascom AR, Linhares Y, Bastos FI. HIV prevalence among female sex workers, drug users and men who have sex with men in Brazil: a systematic review and meta-analysis. BMC Public Health. 2010;10:317. 35. Signorini DJ, Netto AM, Gabbay S, Monteiro MC, Signorini DH, Andrade MD, et al. A comparison of sonographic assessments and clinical questionnaire in the diagnosis of HIV-associated lipodystrophy. J Int Assoc Physicians AIDS Care (Chic). 2011;10(6):351–6. 13. Velasco-de-Castro CA, Grinsztejn B, Veloso VG, Bastos FI, Pilotto JH, Fernandes N, et al. HIV-1 diversity and drug resistance mutations among people seeking HIV diagnosis in voluntary counseling and testing sites in Rio de Janeiro. Brazil PLoS One. 2014;9(1):e87622. 36. Grangeiro A, Escuder MM, Menezes PR, Alencar R. Ayres De Castilho E. Late entry into HIV care: estimated impact on AIDS mortality rates in Brazil, 2003–2006. PLos One. 2011;6(1):14585. 14. Barroso PF, Harrison LH, de Fatima Melo M, Batista SM, da Silva Bastos M, da Rosa Faulhaber JC, et al. Malta et al. BMC Public Health (2015) 15:226 Identification of a high-risk heterosexual cohort for HIV vaccine efficacy trials in Rio de Janeiro, Brazil, using a sensitive/less- sensitive assay: an update. J Acquir Immune Defic Syndr. 2004;36(3):880–1. 15. Malta M, Bastos FI, da Silva CM, Pereira GF, Lucena FF, Fonseca MG, et al. Differential survival benefit of universal HAART access in Brazil: a nation-wide comparison of injecting drug users versus men who have sex with men. J Acquir Immune Defic Syndr. 2009;52(5):629–35. 16. Greco DB, Simão M. Brazilian policy of universal access to AIDS treatment: sustainability challenges and perspectives. AIDS. 2007;21 Suppl 4:S37–45. 17. Oliveira MT, Latorre Mdo R, Greco DB. The impact of late diagnosis on the survival of patients following their first AIDS-related hospitalization in Belo Horizonte. Brazil AIDS Care. 2012;24(5):635–41. 18. Grinsztejn B, Luz PM, Pacheco AG, Santos DV, Velasque L, Moreira RI, et al. Changing mortality profile among HIV-infected patients in Rio de Janeiro, Brazil: shifting from AIDS to non-AIDS related conditions in the HAART era. PLoS One. 2013;8(4):e59768. 19. Grangeiro A, Escuder MM, Pereira JC. Late entry into HIV care: lessons from Brazil, 2003 to 2006. BMC Infect Dis. 2012;12:99. 20. Nemes MI, Alencar TM, Basso CR, Castanheira ER, Melchior R, Alves MT, et al. Assessment of outpatient services for AIDS patients, Brazil: comparative study 2001/2007. Rev Saude Publica. 2013;47(1):137–46. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Malta et al. BMC Public Health (2015) 15:226 Malta et al. BMC Public Health (2015) 15:226 Page 8 of 8 Page 8 of 8 26. White IR, Royston P, Wood AM. Multiple imputation using chained equations: Issues and guidance for practice. Stat Med. 2011;30(4):377–99. asymptomatic HIV-infected patients approaches that of uninfected individ- uals. AIDS. 2010;24(10):1527–35. 27. Eisemann N, Waldmann A, Katalinic A. Imputation of missing values of tumour stage in population-based cancer registration. BMC Med Res Methodol. 2011;11:129. 3. Kimmel AD, Charles M, Deschamps MM, Severe P, Edwards AM, Johnson WD, et al. Lives saved by expanding HIV treatment availability in resource-limited settings: the example of Haiti. J Acquir Immune Defic Syndr. 2013;63(2):e40–8. 28. R Core Development Team [computer software] version 2.13.0. Vienna: R Foundation for Statistical Computing. Available at http://www.r-project.org/ 4. Person AK, Hicks CB. Pre-exposure prophylaxis–one more tool for HIV prevention. Curr HIV Res. 2012;10(2):117–22. 29. Samji H, Cescon A, Hogg RS, Modur SP, Althoff KN, Buchacz K, et al. Cohort collaboration on research and design (NA-ACCORD) of IeDEA. Closing the Gap: increases in life expectancy among treated HIV-positive individuals in the united states and Canada. PLoS One. 2013;8(12):e81355. 5. Okwundu CI, Uthman OA, Okoromah CA. Antiretroviral pre-exposure prophylaxis (PrEP) for preventing HIV in high-risk individuals. Cochrane Database Syst Rev. 2012;7, CD007189. the united states and Canada. PLoS One. 2013;8(12):e81355. 30. Marins JR, Jamal LF, Chen SY, Barros MB, Hudes ES, Barbosa AA, et al. Dramatic improvement in survival among adult Brazilian AIDS patients. AIDS. 2003;17(11):1675–82. 6. Smith MK, Rutstein SE, Powers KA, Fidler S, Miller WC, Eron Jr JJ, et al. The detection and management of early HIV infection: a clinical and public health emergency. J Acquir Immune Defic Syndr. 2013;63 Suppl 2:S187–99. health emergency. J Acquir Immune Defic Syndr. 2013;63 Suppl 2:S187–99. 7. Brazil to adopt B.C.’s Treatment as Prevention strategy as country’s national HIV/AIDS policy. http://cfenet.ubc.ca/news/releases/brazil-adopt-bc’s- treatment-prevention-strategy-country’s-national-hivaids-policy 8. Sullivan PS, Jones JS, Baral SD. The global north: HIV epidemiology in high-income countries. Curr Opin HIV AIDS. 2014;9(2):199–205. 31. Tuboi SH, Schechter M, McGowan CC, Cesar C, Krolewiecki A, Cahn P, et al. Mortality during the first year of potent antiretroviral therapy in HIV-1- infected patients in 7 sites throughout Latin America and the Caribbean. J Acquir Immune Defic Syndr. 2009;51(5):615–23. y y 7. Brazil to adopt B.C.’s Treatment as Prevention strategy as country’s national HIV/AIDS policy. http://cfenet.ubc.ca/news/releases/brazil-adopt-bc’s- treatment-prevention-strategy-country’s-national-hivaids-policy HIV/AIDS policy. http://cfenet.ubc.ca/news/releases/brazil-adopt-bc’s- treatment-prevention-strategy-country’s-national-hivaids-policy 8. Sullivan PS, Jones JS, Baral SD. References 1. UNAIDS. The GAP Report. Geneva: Joint United Nations Programme on HIV/AIDS; 2014. 1. UNAIDS. The GAP Report. Geneva: Joint United Nations Programme on HIV/AIDS; 2014. Brazil has been a worldwide leader in the global efforts to curb AIDS and to provide a standard of care and 2. Van Sighem AI, Gras LA, Reiss P, Brinkman K, de Wolf F. ATHENA national observational cohort study. Life expectancy of recently diagnosed Submit your next manuscript to BioMed Central and take full advantage of: 21. Victora CG, Barreto ML, Do Carmo Leal M, Monteiro CA, Schmidt MI, Paim J, et al. Lancet brazil series working group. Health conditions and health-policy innovations in brazil: the way forward. Lancet. 2011;377(9782):2042–53. • Convenient online submission 22. Schoenfeld D. Partial residuals for the proportional hazards regression model. Biometrika. 1982;69:239–41. • Thorough peer review 23. Aalen OO. Effects of frailty in survival analysis. Stat Methods Med Res. 1994;3:227–43. 24. Brazilian Institute of Geography and Statistics - IBGE. National Population Estimates, July 2013. Available at http://www.ibge.gov.br/home/estatistica/ populacao/estimativa2013/estimativa_dou.shtm 25. Dias SS, Andreozzi V, Martins MO, Torgal J. Predictors of mortality in HIV-associated hospitalizations in Portugal: a hierarchical survival model. BMC Health Serv Res. 2009;9:125. 25. Dias SS, Andreozzi V, Martins MO, Torgal J. Predictors of mortality in HIV-associated hospitalizations in Portugal: a hierarchical survival model. BMC Health Serv Res. 2009;9:125.
https://openalex.org/W2966607380	https://europepmc.org/articles/pmc6722885?pdf=render	English	null	Analysis of Bn<scp>MTL</scp>, a novel metallothionein‐like protein in the bast fiber crop Ramie (<i>Boehmeria nivea</i>)	FEBS open bio	2,019	cc-by	5,391	FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 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. Analysis of BnMTL, a novel metallothionein-like protein in the bast ﬁber crop Ramie (Boehmeria nivea) Gang Gao , Aiguo Zhu Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Changsha, China Ramie (Boehmeria nivea) is a perennial herb that is highly tolerant of heavy metals. In the present study, we cloned a novel metallothionein-like gene from ramie; this gene, termed BnMTL, encodes a putative 46 amino acid protein with a molecular mass of 4.38 kDa. Analysis using quantita- tive RT-PCR revealed that cadmium (Cd2+) treatment results in elevated expression of BnMTL in the roots. We heterologously overexpressed BnMTL in Escherichia coli cells to examine its binding to Cd2+ and its pos- sible role in homeostasis. Recombinant E. coli cells expressing BnMTL exhibited a high tolerance of Cd2+ stress up to a concentration of 1 mM, and the observed accumulation of Cd2+ was almost eight-fold higher than the control. These results demonstrate that BnMTL (i) is highly expressed in the root following exposure to Cd2+ and (ii) encodes a typical metalloth- ionein-like protein with high cadmium-binding activity. Correspondence Correspondence C. Yu and A. Zhu, Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Xianjiahu West Road No. 348, Changsha 410205, China Tel: +86 0731 88998507 E-mails: zhuaiguo@caas.cn (AZ); yuchunming@caas.cn (CY) Correspondence C. Yu and A. Zhu, Institute of Bast Fiber Crops, Chinese Academy of Agricultural Sciences, Xianjiahu West Road No. 348, Changsha 410205, China Tel: +86 0731 88998507 E-mails: zhuaiguo@caas.cn (AZ); yuchunming@caas.cn (CY) (Received 6 May 2019, revised 18 July 2019, accepted 26 July 2019) doi:10.1002/2211-5463.12705 Plants have developed a suitable mechanism for con- trolling and responding to the uptake and accumula- tion of heavy metals that are considered as critical environmental contaminants of great concern with respect to the ecological environment and also pose nutritional and evolutionary threats [1]. Cadmium (Cd2+) is one of such heavy metal, although it occurs in trace quantities, yet causes toxic effects to both plants and animals [2–4]. Growing interest in molecu- lar genetics and transgenic plants has increased our understanding of mechanisms of heavy metal tolerance in plants and opens new possibilities with respect to phytoremediation. to curb heavy metal pollution has focused on the screen- ing of suitable plant species that are potential heavy metal accumulators, such as Solanum nigrum [7], Helianthus annuus [8], Sorghum bicolor [9], Zea mays [10] and Boehmeria nivea [11]. Ramie (Boehmeria nivea) is conisidered to have excellent qualities for enduring heavy metal pollution and is considered as an ideal eco- nomic crop for the phytoremediation of mild or moder- ately heavy metal polluted areas as a result of several ramie varieties performing hyperaccumulative charac- teristics on heavy metals [12]. Ramie is a perennial ﬁber crop with high biomass and strong root system. Moreover, the ramie ﬁber is mainly produced as textile raw material, and this may help minimize the potential hazards of bringing toxic metals into the food chain. Some wild geno- types were dominant in the smelter tailings, high- lighting their hyperaccumulation properties. Ramie genome [13] and genome-wide expression proﬁles [11] will aid the identiﬁcation, quantiﬁcation and Phytoremediation as a perspective technology of soil clean-up has been intensively studied as a result of its low cost, environmental aesthetics and in situ effective treatment. The phytoremediation technique for treating heavy metal contaminated soil includes phytoextraction, phytostabilization, rhizoﬁltration and phytovolatiliza- tion [5,6]. Recent progress in phytoremediation aiming IPTG, isopropyl-D-thiogalactoside; qRT-PCR, quantitative RT-PCR. Keywords BnMTL; Cd2+ tolerance; expression pattern; heterologous expression; ramie Plant growth and Cd2+ stress treatment Ramie plants were cultured in hydroponic system, as described previously [21]. Non-ligniﬁed tender shoots (12– 15 cm long) with two or three leaves were cut and soaked in 0.1% carbendazim for 5 min. The sterilized shoots were then transferred to a hydroponic apparatus, with distilled water being used as the solution to induce aquatic root ger- mination, which was later replaced with nutrient solution. The set-up was placed in greenhouse under a 14 : 10 h light/dark photocycle at 25/20 °C, with a light intensity of 100–170 Wm2 and 60% relative humidity. Sequence and structure analysis of ramie metallothionein-like protein annotation of key genes related to heavy metal toler- ance. This will also be helpful for target discovery and pathway studies. DNA sequence analysis and comparison were performed using LASERGENE (https://www.dnastar.com) and BLAST (http://www.ncbi.nlm.nih.gov/) and the open reading frames of the sequences were identiﬁed using ORF-FINDER (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Amino acid sequences alignment and phylogenetic analysis were per- formed using CLUSTAL W (www.phylogeny.fr) and MEGA 6.0 respectively. Predictions of functional motif were performed via the Expasy proteomics server (http://www.expasy.org). Metallothioneins are small proteins that appear to play key role in heavy metal homoeostasis [14]. Several plant metallothioneins have been overexpressed and heterologously expressed in microbial hosts aiming to examine the metal binding properties of these proteins and their ability to exert heavy metal tolerance [15– 17]. Such studies have provided important evidence indicating that plant metallothioneins are capable of providing a biological function and a metal tolerance ability in nonplant systems. Plant metallothioneins are cysteine-rich polypeptides with a cysteine content vary- ing between 10 and 17 residues. The large number of cysteine residues binds a variety of metals by mercap- tide bonds. Although many metallothionein and metal- lothionein-like proteins have been identiﬁed in plants [18,19], there are still some difﬁculties with respect to the functional characterization of these proteins because of the instability of metallothionein in the presence of oxygen [20]. Abbreviations IPTG, isopropyl-D-thiogalactoside; qRT-PCR, quantitative RT-PCR. 1632 G. Gao et al. A metallothionein-like protein in Ramie Quantitative RT-PCR (qRT-PCR) analysis of BnMTL expression under Cd2+ stress Tissue samples were collected and saved in a liquid nitro- gen container. Until all of the samples from different treat- ments and time points (0 h, 6 h, 12 h, 24 h, 3 days and 5 days) were collected, the total RNA were extracted using a Trizol kit (Invitrogen, Carlsbad, CA, USA) and quanti- ﬁed using a NanoDrop (Gene Co., Beijing, China) for the independent qRT-PCR analysis. The ﬁrst-strand cDNA synthesis was performed with 1 lg of total RNA using the Marathon TM cDNA Ampliﬁcation Kit (Clontech, Palo Alto, CA, USA) in accordance with the manufacturer’s recom- mendations and the qRT-PCR analysis was performed using gene-speciﬁc primers and SYBR Green (Invitrogen) dye detection on a CFX96 system (Bio-Rad, Hercules, CA, USA). The speciﬁc primers were designed using OLIGO 5 (https://www.oligo.net) and the 18s rRNA gene was used as a reference gene. The primers used to amplify 18s rRNA and BnMTL were: 18s rRNA, forward: TGACGGAGA ATTAGGGTTCGA; 18s rRNA, reverse: CCGTGTCAG GATTGGGTAATTT; BnMTL, forward: ATGGGTTGC CCTTGTGGAAAC; BnMTL, reverse: TTGATTGCAA GAGCAGCTTGAG. The present study therefore aimed to (i) clone and identify the putative metallothionein-like protein encoding gene; (ii) determine its regulation under Cd2+ stress in the ramie; and (iii) assay its expression pat- terns at various Cd2+ concentration levels. Its Cd2+ binding properties and possible roles in detoxiﬁcation were also evaluated by heterologous expression in E. coli cells. FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Results and Discussion Expression proﬁling of Cd2+ response genes in root of ramie has been reported previously, with 36 unigenes from the cysteine and methionine metabolic pathway being up-regulated [11]. In the present study, the BnMTL gene cloned from ramie encodes a putative 46 amino acid protein with a molecular mass of 4.38 kDa. The ramie BnMTL is a typical micro-molec- ular metallothionein-like protein with a low molecular weight [1,2,24,25], with its cysteine residues organized in two rich domains. Phylogenetic analysis suggested the BnMTL gene to be a type I metallothionein pro- tein as a result of the equal distribution of six C-X-C motifs on both the N- and C-terminal ends of the pro- tein separated by a Cys-poor linker. It is interesting to note that the length of the Cys-poor linker region in BnMTL varies among different plant species (Fig. 1). These structural characteristics suggested the possible involvement of BnMTL in heavy metal detoxiﬁcation and also that these residues may serve as primary chelating sites. Nucleotide sequence accession number Western blot analysis of target protein was performed according to the standard protocol. Brieﬂy, the recombi- nant protein was separated on a 12% SDS/PAGE gel, which was semi-dry transferred at 15 V for 30 min to 0.45 mm poly(vinylidene diﬂuoride) membrane (Bio-Rad), immunoblotted with anti-His Tag mouse monoclonal anti- body (dilution 1 : 5000; BOSTER, Wuhan, China). Next, the IgG goat anti-mouse antibody conjugated with horse- radish peroxidase was used as a secondary antibody (dilu- tion 1 : 5000) and a diaminobenzidine kit was used for the visualization of the protein band. The nucleotide sequence data of the ramie metallothionein- like gene (BnMTL) have been submitted to the nucleotide sequence databases (GenBank) under accession number MH481283. Expression and western blotting analyses of BnMTL Using the speciﬁc primers BnMTLF (50-GGAATT CATGGGTTGCCCTTGTGGAAAC-30) and BnMTLR (50-CAAGCTTTTGATTGCAAGAGCAGCTTGAG-30), the ORF fragment encoding the mature peptide (MGCPCGNNCQCGSSCACGGNSHTATEPSGCNCGP NCSCGSSCSCNQ) was obtained. It was puriﬁed using agarose gel electrophoresis, digested with EcoRI and Hin- dIII enzymes and ligated into the EcoRI-/HindIII-digested expression vector pET-30a (Novagen, Madison, WI, USA). The constructed plasmid was transformed into competent BL21 (DE3) cells for expression of the TRX (thioredoxin)- 6His-BnMTL fusion protein and induced with 1 mM isopropyl-D-thiogalactoside (IPTG) for 6 h at 30 °C. The bacterial pellets were harvested by centrifugation and lysed At 5 weeks, the plants were treated with different con- centrations of cadmium chloride (50, 100 and 200 lM) and 1 mg samples (roots, stems, leaves) from the same plants were collected at time intervals of 0 h, 6 h, 12 h, 24 h, 3 days and 5 days. At each treatment, samples from three different plants were collected for replicates. All of the sam- ples were quickly frozen in liquid nitrogen for total RNA preparation or stored at 70 °C until use. 1633 FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. A metallothionein-like protein in Ramie G. Gao et al. by the lysis solution (50 mM Tris–HCl, pH 8.0, 50 mM NaCl, 0.5% Triton X-100, 2 mgmL1 lysozyme). After sonication, the supernatants were recovered by centrifuga- tion and subjected to Ni2+-NTA column chromatography for puriﬁcation of the recombinant fusion protein. The puriﬁed recombinant fusion protein was dialyzed and dis- solved in PBS (pH 7.4) to a ﬁnal concentration of 1 mgmL1. The fractions containing BnMTL were col- lected, concentrated with poly(ethylene glycol) 2000, dia- lyzed in double-distilled water for desalination and ﬁnally lyophilized [22]. Analysis of the puriﬁed recombinant BnMTL was carried out using Tricine-SDS/PAGE. 0.5 nm and the measurements carried out in an air/acety- lene ﬂame. The Cd2+ binding ability assay was performed in tripli- cate and differences between the treatments were examined for statistical signiﬁcance using Duncan’s test (P < 0.05, ANOVA). HCl solution was used as blank. The limit of detection was calculated as the analyte concentration equal to three times the SD of the blank signal divided by the slope of the calibration curve. FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Assay of the Cd2+ tolerance and accumulation in pET30-BnMTL/BL21 Amino acid sequence alignment of metallothionein-like protein for Boehmeria nivea with other plants: Brassica napus (ADP37975), Arabidopsis thaliana (NP_172240), Phytolacca acinosa (AEP14524), Arabidopsis lyrata (XP_020870902) and Eutrema salsugineum (XP_006417787). Identical amino acid residues are indicated in blue and purple. The arrows indicate the conserved cysteine residues. G. Gao et al. A metallothionein-like protein in Ramie G. Gao et al. A metallothionein-like protein in Ramie Fig. 1. Amino acid sequence alignment of metallothionein-like protein for Boehmeria nivea with other plants: Brassica napus (ADP37975), Arabidopsis thaliana (NP_172240), Phytolacca acinosa (AEP14524), Arabidopsis lyrata (XP_020870902) and Eutrema salsugineum (XP_006417787). Identical amino acid residues are indicated in blue and purple. The arrows indicate the conserved cysteine residues. p Fig. 1. Amino acid sequence alignment of metallothionein-like protein for Boehmeria nivea with other plants: Brassica napus (ADP37975), Arabidopsis thaliana (NP_172240), Phytolacca acinosa (AEP14524), Arabidopsis lyrata (XP_020870902) and Eutrema salsugineum (XP_006417787). Identical amino acid residues are indicated in blue and purple. The arrows indicate the conserved cysteine residues. Fig. 2. The expression proﬁles of BnMTL and the growth status of hydroponic ramie plant treated with various levels of Cd2+ stress. BnMTL expression in the roots (A), steams (B) and leaves (C) of hydroponic ramie cultured in the presence of different concentration of Cd2+ for the indicated periods were measured by qRT-PCR. (D) Growth status of ramie plant treated with various levels of Cd2+ after 2 weeks. The value represents the mean SD of three biological replicate and three technical replicates were conducted for each organ. Fig. 2. The expression proﬁles of BnMTL and the growth status of hydroponic ramie plant treated with various levels of Cd2+ stress. BnMTL expression in the roots (A), steams (B) and leaves (C) of hydroponic ramie cultured in the presence of different concentration of Cd2+ for the indicated periods were measured by qRT-PCR. (D) Growth status of ramie plant treated with various levels of Cd2+ after 2 weeks. The value represents the mean SD of three biological replicate and three technical replicates were conducted for each organ. Table 1. The total Cd content and growth status of ramie treated with different concentrations of Cd2+. Table 1. The total Cd content and growth status of ramie treated with different concentrations of Cd2+. Assay of the Cd2+ tolerance and accumulation in pET30-BnMTL/BL21 The cells of E. coli strain BL21 (DE3) that transformed with pET30a-BnMTL and pET30a (control) were cultured in Luria–Bertani medium, and then the cell concentration was using the D600 measurements. When D600 of the bac- terium liquid reached 0.2, the transformed E. coli cells con- taining pET30a-BnMTL cells were induced with 1 mM IPTG in a 100 mL ﬂask and simultaneously treated with different types of Cd2+. The D600 values were measured every 1 h to determine the growth rate and tolerance of cells in Cd2+ treatment. To assess the Cd2+ binding capacity of the BnMTL, the ﬂasks were supplemented with CdCl2 at concentration of 0.2 mM because the recombinant cells can growth normally in this concentration. Following the induction and Cd2+ treating, the accumulated Cd2+ (g1 by dry weight) in E. coli cells was measured in accordance with the method described by Pan et al. [23]: Cell samples (0.05 g) were placed into 50 mL porcelain crucibles and heated in a muf- ﬂe furnace at 500 25 °C for 8 h and then 10 mL of mixed acid (HNO3:HClO4 = 3 : 4) was added to each cru- cible. The porcelain crucibles were cooled at room temper- ature and then heated again under gentle heat until no carbon residues were visible. Subsequently, 10 mL of 8.3% HCl was added to dissolve remaining residues. The liquid solutions were then analyzed by ﬂame atomic absorption spectrometry. A Cd2+ hollow cathode lamp was used as light source operated at 3.5 mA. The wavelength was set at 228.8 nm resonance line, the spectral bandpass at The BnMTL genes were dramatically up-regulated in ramie roots when exposed to various concentrations of cadmium chloride (Fig. 2). Similar results were found in several different plants such as Avicennia ger- minnas [26] and Arachis hypogaea [27]. Studies on expression patterns of metallothionein in tomato demonstrated the best expression at an approximately 50 lM Cd2+ concentration [28]. The results obtained in the present study, however, indicated signiﬁcant up- regulation of the gene at doses of 100–200 lM Cd2+ mainly induced in the roots (Fig. 2). On the other hand, a high Cd2+ concentration above 200 lM revealed an extremely harmful effect (Fig. 2) to ramie plants, which is in consistent with the studies 1634 FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Fig. 1. FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Assay of the Cd2+ tolerance and accumulation in pET30-BnMTL/BL21 Cd2+ concentration (lM) Cd content Growth status Roots (mgkg1) Stems (mgkg1) Leaves (mgkg1) Plant height (cm) Stem width (mm) Dry weight (g) 0 NA NA NA 30.15 1.50 5.170 0.200 9.30 0.25 50 2560 115 115 8 6.08 0.95 29.63 1.15 4.985 0.135 8.90 0.20 100 3550 150 183 15 8.25 1.21 25.15 1.20 4.555 0.121 5.80 0.12 200 3490 163 190 11 8.63 1.00 23.75 1.23 4.330 0.129 5.55 0.10 The value are shown as the mean SD. NA, not available. the same studies also reported decreased expression at even lower doses. The different responses may be asso- ciated with physiological functions in ramie such as conducted in tomato by Tombuloglu et al. [29], who reported the expression of tomato metallothionein gene to be decreased at higher Cd2+ doses, although FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 1635 G. Gao et al. A metallothionein-like protein in Ramie the hormonal status of tissues tissue type and heavy Comprehensive consideration of the growth status Fig. 3. SDS/PAGE and western blot analysis of recombinant BnMTL. Lanes: M, standard protein molecular mass markers; 1, BnMTL from supernatant of E. coli BL21 cell lysates; 2, puriﬁed BnMTL; 3, western blotting of BnMTL from supernatant of E. coli BL21 cell lysates; The target bands of BnMTL are indicated by the arrows. Fig. 4. Cd2+ tolerance analysis of recombinant E. coli cells expressing BnMTL. The growth status of pET30a-BnMTL/BL21 (A) and pET30a / BL21(B) in different Cd2+ concentrations. The cell concentration was calculated from the D600 measurements. Data represent the means of three experiments and error bars represent the SD. Fig. 3. SDS/PAGE and western blot analysis of recombinant BnMTL. Lanes: M, standard protein molecular mass markers; 1, BnMTL from supernatant of E. coli BL21 cell lysates; 2, puriﬁed BnMTL; 3, western blotting of BnMTL from supernatant of E. coli BL21 cell lysates; The target bands of BnMTL are indicated by the arrows. Fig. 3. SDS/PAGE and western blot analysis of recombinant BnMTL. Lanes: M, standard protein molecular mass markers; 1, BnMTL from supernatant of E. coli BL21 cell lysates; 2, puriﬁed BnMTL; 3, western blotting of BnMTL from supernatant of E. Assay of the Cd2+ tolerance and accumulation in pET30-BnMTL/BL21 coli BL21 cell lysates; The target bands of BnMTL are indicated by the arrows. Fig. 4. Cd2+ tolerance analysis of recombinant E. coli cells expressing BnMTL. The growth status of pET30a-BnMTL/BL21 (A) and pET30a / BL21(B) in different Cd2+ concentrations. The cell concentration was calculated from the D600 measurements. Data represent the means of three experiments and error bars represent the SD. the hormonal status of tissues, tissue type and heavy metal uptake. The regulation of gene expression repre- sents the ﬁrst level of integration between environmen- tal stress and the genome [30]. Ramie is a perennial herb plant with developed underground roots, a high accumulation of heavy metal Cd2+ and BnMTL expression in roots rather than stems or leaves, consis- tent with the fact that the roots are the main organ for this species to adapt to a stress environment [31]. Comprehensive consideration of the growth status, biomass accretion and total Cd content (Table 1) shows that these will provide an advantage for using ramie as a bast ﬁber or in some other multi-purpose use, including as a candidate plant for phytoremedia- tion of Cd polluted soil. In an effort to characterize the Cd2+ binding proper- ties, the BnMTL was heterologously overexpressed in E. coli cells. Analysis of protein expression using 1636 FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. Fig. 5. The accumulation of Cd2+ ions in the pET30a-BnMTL/BL21 and the control E. coli cells (BL21 and pET30a/BL21). The value represents the mean SD of three biological replicates. Statistical signiﬁcance was based on Duncan’s test. Signiﬁcant difference between E. coli cell samples at P < 0.05. G. Gao et al. A metallothionein-like protein in Ramie G. Gao et al. accumulation of Cd2+ ions, with signiﬁcantly higher levels compared to those of controls (pET30a/BL21 and BL21 strains), being almost eight-fold greater than the controls (Fig. 5). The expression patterns of the recombinant BnMTL suggested that the cells trans- formed with the recombinant plasmids pET30a- BnMTL had a high tolerance to Cd2+ stress and can be grown well in the concentration of Cd2+ under 1000 lM. This is in accordance with an enhanced toler- ance to Cd2+ in recombinant strains expressing metal- lothionein being demonstrated in Musca domestica [34], biofuel plant Jatropha curcas [35], Anabaena sp. [36] and bacterial metagenome [37]. Acknowledgements This work was supported by the Agricultural Science and Technology Innovation Project of Chinese Acad- emy of Agricultural Sciences (CAAS-ASTIP-2018), the China Agricultural Research System (CARS-16) and the National Key Research and Development Program of China (2017YFD0800900). The accumulation of Cd2+ (lmolg1 dry weight) in E. coli cells was also investigated. The highest levels of Cd2+ were detected in E. coli cells expressing BnMTL (82.16 lmolg1 dry weight). The recombinant E. coli cells expressing BnMTL exhibited the highest Conclusions In the present study, we have cloned and identiﬁed a low weight metallothionein-like protein gene (BnMTL) from the potential phytoremediation plant ramie. Tis- sue-speciﬁc expression analysis showed the expression of BnMTL to be regulated by Cd2+ treatment and induced in roots. As a result of difﬁculty in isolating native metallothionein protein because of its low molecular weight and susceptibility to proteolysis, we heterologously overexpressed BnMTL in E. coli cells. The Cd2+ tolerance and accumulation analysis demon- strated that BnMTL improved the Cd2+ tolerance of the recombinant E. coli cells. Such work lays a foun- dation for deﬁning the roles of BnMTL in Cd chela- tion and detoxiﬁcation. The recombinant cells grown in the Luria–Bertani medium supplemented with different concentrations of cadmium chloride (0, 200, 500 and 1000 lM) showed no signiﬁcant difference with respect to the growth rate of pET30a-BnMTL/BL21 recombinant cells cul- tured under 0 and 200 lM Cd2+. After 6 h of culture, they reached the stationary phase with a OD600 value of 1.1. The growth rate of recombinant cells was inhib- ited at a higher concentration of cadmium ions (500 and 1000 lM), with the cells attaining a stationary phase under the 1000 lM Cd2+ ions stress when the OD600 value was only 0.6. The growth of control cells (pET-30a/BL21) was also found to be signiﬁcantly inhibited in the Cd2+ concentration of 200 and 500 lM (Fig. 4). Assay of the Cd2+ tolerance and accumulation in pET30-BnMTL/BL21 There was a direct relationship between increased metallothionein gene expression and survival of the recombinant E. coli cells. Furthermore, a high level of cadmium ions accu- mulated in the recombinant E. coli cells harboring pET30a-BnMTL, indicating that expression of BnMTL could enhance tolerance in cells to the Cd2+ ion con- centration and promote the accumulation of Cd2+. The results obtained in the present study may help to con- front to Cd2+ pollution using the overexpression of the metallothionein gene in recombinant bacteria. Fig. 5. The accumulation of Cd2+ ions in the pET30a-BnMTL/BL21 and the control E. coli cells (BL21 and pET30a/BL21). The value represents the mean SD of three biological replicates. Statistical signiﬁcance was based on Duncan’s test. Signiﬁcant difference between E. coli cell samples at P < 0.05. Tricine-SDS/PAGE showed puriﬁed homogenous recombinant BnMTL (fusion protein included the tags from the pET30a vector) with a molecular mass of approximately 14 kDa (Fig. 3, lane 1). The low molec- ular weight of metallothionein protein and its suscepti- bility to proteolysis were ascribed to be the cause of difﬁculty in native metallothionein protein isolation in plants, in addition to the difﬁculties with respect to its puriﬁcation as a result of instability in the presence of oxygen [20,32,33]. The expression of BnMTL was fur- ther conﬁrmed by western blotting analysis using His- tag antibodies (Fig. 3, lane 3). References 14 Lane B, Kajioka R and Kennedy T (1987) The wheat- germ Ec protein is a zinc-containing metallothionein. Biochem Cell Biol 65, 1001–1005. 1 Cobbett C and Goldsbrough P (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxiﬁcation and homeostasis. Annu Rev Plant Biol 53, 159–182. 15 Tommey AM, Shi J, Lindsay WP, Urwin PE and Robinson NJ (1991) Expression of the pea gene PSMTA in E. coli. Metal-binding properties of the expressed protein. FEBS Lett 292, 48–52. 2 Bourdineaud JP, Baudrimont M, Gonzalez P and Moreau JL (2006) Challenging the model for induction of metallothionein gene expression. Biochimie 88, 1787– 1792. 16 Zhou J and Goldsbrough PB (1994) Functional homologs of fungal metallothionein genes from Arabidopsis. Plant Cell 6, 875–884. 3 Rehman MZ, Rizwan M, Ghafoor A, Naeem A, Ali S, Sabir M and Qayyum MF (2015) Effect of inorganic amendments for in situ stabilization of cadmium in contaminated soils and its phyto-availability to wheat and rice under rotation. Environ Sci Pollut Res 22, 16897–16906. 17 Dundar E, Sonmez GD and Unver T (2015) Isolation, molecular characterization and functional analysis of OeMT2, an olive metallothionein with a bioremediation potential. Mol Genet Genomics 290, 187–199. 4 Huang Y, He C, Shen C, Guo J, Mubeen S, Yuan J and Yang Z (2017) Toxicity of cadmium and its health risks from leafy vegetable consumption. Food Func 8, 1373–1401. 18 Jin S, Cheng Y, Guan Q, Liu D, Takano T and Liu S (2006) A metallothionein-like protein of rice (rgMT) functions in E. coli and its gene expression is induced by abiotic stresses. Biotech Lett 28, 1749–1753. 5 Salt DE (1997) Phytoremediation of metals: using plants to remove pollutants from the environment. Curr Opin Biotechnol 8, 221–226. 19 Yookongkaew N (2013) Functional characterization of metallothioneins in Arabidopsis and barley. Dissertations & Theses – Gradworks. 6 Ali H, Khan E and Sajad MA (2013) Phytoremediation of heavy metals—Concepts and applications. Chemosphere 91, 869–881. 20 Singh G, Tripathi S, Shanker K and Sharma A (2018) Cadmium-induced conformational changes in type 2 metallothionein of medicinal plant Coptis japonica: insights from molecular dynamics studies of apo, partially and fully metalated forms. J Biomol Struct Dyn 37, 1520–1533. 7 Ji P, Tang X, Jiang Y, Tong YA, Gao P and Han W (2015) Potential of gibberellic acid 3 (GA3) for enhancing the phytoremediation efﬁciency of Solanum nigrum L. Bull Environ Contam Toxicol 95, 810–814. Conﬂict of interest The authors declare no conﬂict of interest. 1637 FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. G. Gao et al. A metallothionein-like protein in Ramie Author contributions 11 She W, Zhu S, Jie Y, Xing H and Cui G (2015) Expression proﬁling of cadmium response genes in ramie (Boehmeria nivea L.) root. Bull Environ Contam Toxicol 94, 453–459. GG and ZA conceived the study and, together with YC, supervised its conduct. CJ and CK performed the gene cloning experiment and qRT-PCR analysis. CP, GG and ASA performed the heterologously overex- press of BnMTL and the cadmium-binding activity assay. All authors analyzed and discussed the data and contributed to writing the manuscript. 12 Wang WH, Luo XG, Liu L, Zhang Y and Zhao HZ (2018) Ramie (Boehmeria nivea)’s uranium bioconcentration and tolerance attributes. J Environ Radioact 184, 152–157. 13 Luan MB, Jian JB, Chen P, Chen JH, Chen JH, Gao Q, Gao G, Zhou JH, Chen KM, Guang XM et al. (2018) Draft genome sequence of ramie, Boehmeria nivea (L.) Gaudich. Mol Ecol Resour 18, 639–645. References 21 Gao G, Xiong HP, Chen JK, Chen KM, Chen P, Yu CM and Zhu AG (2018) Hydroponic method for ramie and removal of nitrogen and phosphorus from livestock wastewater. Int J Phytorem 20, 545–551. 8 Rizwan M, Ali S, Rizvi H, Rinklebe JR, Tsang DC, Meers E and Ishaque W (2016) Phytomanagement of heavy metals in contaminated soils using sunﬂower: a review. Crit Rev Environ Sci Technol 46, 1498–1528. 22 Jin FL, Sun Q, Xu XX, Li LM, Gao G, Xu YJ, Yu XQ and Ren SX (2012) cDNA cloning and characterization of the antibacterial peptide cecropin 1 from the diamondback moth, Plutella xylostella L. Protein Expr Purif 85, 230–238. 9 Feng J, Jia W, Lv S, Bao H, Miao F, Zhang X and Zhu C (2017) Comparative transcriptome combined with morpho-physiological analyses revealed key factors for differential cadmium accumulation in two contrasting sweet sorghum genotypes. Plant Biotechnol J 16, 558–571. 23 Pan Y, Pan Y, Zhai J, Xiong Y, Li J, Du X, Su C and Zhang X (2016) Cucumber metallothionein-like 2 (CsMTL2) exhibits metal-binding properties. Genes 7, 106. 10 Rehman MZ, Rizwan M, Ali S, Fatima N, Yousaf B, Naeem A and Ok YS (2016) Contrasting effects of biochar, compost and farm manure on alleviation of nickel toxicity in maize (Zea mays L.) in relation to plant growth, photosynthesis and metal uptake. Ecotoxicol Environ Saf 133, 218–225. 24 Domenech J, Tinti A, Capdevila M, Atrian S and Torreggiani A (2007) Structural study of the zinc and cadmium complexes of a type 2 plant (Quercus suber) 1638 FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. G. Gao et al. A metallothionein-like protein in Ramie 31 Liu F, Liu Q, Liang X, Huang H and Zhang S (2005) Morphological, anatomical, and physiological assessment of ramie [Boehmeria nivea(L.) gaud.] tolerance to soil drought. Genet Resour Crop Evol 52, 497–506. metallothionein: insights by vibrational spectroscopy. Biopolymers 86, 240–248. 25 Samardzic JT, Nikolic DB, Timotijevic GS, Jovanovic ZS, Milisavljevic MD and Maksimovic VR (2010) Tissue expression analysis of FeMT3, a drought and oxidative stress related metallothionein gene from buckwheat (Fagopyrum esculentum). J Plant Physiol 167, 1407–1411. 32 Zhou J and Goldsbrough PB (1995) Structure, organization and expression of the metallothionein gene family in Arabidopsis. Mol Gen Genet 248, 318–328. 33 Freisinger E (2011) Structural features speciﬁc to plant metallothioneins. FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. References J Biol Inorg Chem 16, 1035–1045. 26 Gonzalez-Mendoza D, Moreno AQ and Zapata-Perez O (2007) Coordinated responses of phytochelatin synthase and metallothionein genes in black mangrove, Avicennia germinans, exposed to cadmium and copper. Aquat Toxicol 83, 306–314. 34 Tang T, Huang DW, Zhang D, Wu YJ, Murphy RW and Liu FS (2011) Identiﬁcation of two metallothionein genes and their roles in stress responses of Musca domestica toward hyperthermy and cadmium tolerance. Comp Biochem Physiol Biochem Mol Biol 160, 81–88. 27 Quan XQ, Shan L and Bi YP (2007) Cloning of metallothionein genes from Arachis hypogaea and characterization of AhMT2a. Russ J Plant Physiol 54, 669–675. 35 Mudalkar S, Golla R, Sengupta D, Ghatty S and Reddy AR (2014) Molecular cloning and characterisation of metallothionein type 2a gene from Jatropha curcas L., a promising biofuel plant. Mol Biol Rep 41, 113–124. 28 Kısa D, €Ozt€urk L and Tekin S (2016) Gene expression analysis of metallothionein and mineral elements uptake in tomato (Solanum lycopersicum) exposed to cadmium. J Plant Res 129, 1–7. 36 Divya TV, Chandwadkar P and Acharya C (2018) NmtA, a novel metallothionein of, Anabaena, sp. strain PCC 7120 imparts protection against cadmium stress but not oxidative stress. Aquat Toxicol 199, 152–161. 29 Tombuloglu H, Semizoglu N, Sakcali S and Kekec G (2012) Boron induced expression of some stress-related genes in tomato. Chemosphere 86, 433–438. 37 Mori T, Iwamoto K, Wakaoji S, Araie H, Kohara Y, Okamura Y, Shiraiwa Y and Takeyama H (2016) Characterization of a novel gene involved in cadmium accumulation screened from sponge-associated bacterial metagenome. Gene 576, 618–625. 30 Cha^abene Z, Rorat A, Rekik Hakim I, Bernard F, Douglas GC, Elleuch A and Mejdoub H (2018) Insight into the expression variation of metal-responsive genes in the seedling of date palm (Phoenix dactylifera). Chemosphere 197, 123–134. 1639 FEBS Open Bio 9 (2019) 1632–1639 ª 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd. 1639
https://openalex.org/W2810298060	https://inria.hal.science/hal-01920743/document	English	null	Exploring the Cloud Computing Loop in the Strategic Alignment Model	IFIP advances in information and communication technology	2,018	cc-by	3,598	Exploring the Cloud Computing Loop in the Strategic Alignment Model Belitski, Maksim1; Fernandez, Valerie2; Khalil, Sabine3; Li, Weizi1 and Liu, Kecheng1 1 Henley Business School, University of Reading, Whiteknights campus, Reading, RG6 6UD, UK 2 Department of Economic and Social Sciences, Telecom ParisTech, 46 Rue Barrault, 75013 Paris, France 3 ICD – International Business School, 12 rue Alexandre Parodi, 75010, Paris, France m.belitski@reading.ac.uk, valerie.fernandez@telecom- paristech.fr, skhalil@groupe-igs.fr, weizi.li@henley.ac.uk, k.liu@henley.ac.uk 1 Henley Business School, University of Reading, Whiteknights campus, Reading, RG6 6UD, UK 2 Department of Economic and Social Sciences, Telecom ParisTech, 46 Rue Barrault, 75013 Paris, France 3 ICD – International Business School, 12 rue Alexandre Parodi, 75010, Paris, France m.belitski@reading.ac.uk, valerie.fernandez@telecom- paristech.fr, skhalil@groupe-igs.fr, weizi.li@henley.ac.uk, k.liu@henley.ac.uk 1 Henley Business School, University of Reading, Whiteknights campus, Reading, RG6 6UD, UK 2 Department of Economic and Social Sciences, Telecom ParisTech, 46 Rue Barrault, 75013 Paris, France 3 ICD – International Business School, 12 rue Alexandre Parodi, 75010, Paris, France m.belitski@reading.ac.uk, valerie.fernandez@telecom- paristech.fr, skhalil@groupe-igs.fr, weizi.li@henley.ac.uk, k.liu@henley.ac.uk Abstract. Since its emergence, Cloud Computing (CC) has revolutionized or- ganization through offering them a large range of easily accessible, scalable, and non-expensive services. As CC has been gaining popularity, it has an impact on the strategic and operational level of every organization. Thus, wondering about its impact on the organizational strategic alignment is a must. This study develops the Cloud Computing framework of Strategic Alignment Model, where strategic fit between operational and strategic levels in organization is achieved through cloud-enabled multiple iterative processes. The study aims to understand whether cloud computing increases operational and strategic efficiencies of organization and if yes then how. After presenting the theoretical background, the hypotheses, and the built cloud framework, we discuss the way strategic alignment theory helps us to better understand how information flows within the strategic fit of business and IT and what is the role of CC in it. This study addresses the strategic alignment as well as the cloud computing literature. Keywords: Cloud Computing, Strategic Alignment, Operational Management, Validation Keywords: Cloud Computing, Strategic Alignment, Operational Management, Validation 1 Introduction Although an intense research has been done about the strategic value of information technology adoption using resource-centered view [1] the contingency-based view [2- 4], semiotic framework [5] and leadership and management view [6-8], there is a pau- city of research assessing a value created by cloud computing technology (CC) [9] and the mechanism of CC adoption in organization [5]. Cloud computing, referring to in- formation technologies enabling convenient, on-demand network access to a shared pool of configurable computing resources and has fundamentally changed the way companies operate and co-create value [10-13]. As a transformative technology, CC has changed various aspects of business and social interactions in the way businesses operate, exchange data and engage with customers [9]. To better understand a value creation process enabled by CC, more theoretical underpinning and practical evidence is required regarding the role that CC plays in improvement of the organization’s oper- ations, innovation, efficiencies as well as used to design a robust IT and business strat- egy. It is important to operationalize previous findings and results that are focused on issues associated with information technology adoption and a framework to analyze it [4, 14]. has changed various aspects of business and social interactions in the way businesses operate, exchange data and engage with customers [9]. To better understand a value creation process enabled by CC, more theoretical underpinning and practical evidence is required regarding the role that CC plays in improvement of the organization’s oper- ations, innovation, efficiencies as well as used to design a robust IT and business strat- egy. It is important to operationalize previous findings and results that are focused on issues associated with information technology adoption and a framework to analyze it [4, 14]. CC continues revolutionizing the ways business collect, process, analyze, review and manage information [15-16] with a special attention on consistent measurements [17] of returns associated with CC by organization [18]. We use the term organization in this study to further emphasize the vast and far-reaching impact of cloud technology investments which goes beyond a separate business unit such as organization or busi- ness. Although interest of IS scholars and practitioners on the cloud’s impact on organi- zational operations and performance has been growing, the prior research related to CC and efficiencies has primarily focused on adoption or operations, cost reduction, ex- ploiting the IT resource mobility offered by the CC as well as other supplementary technologies. 2.1 Strategic Alignment Lens in Cloud Computing Framework Cloud computing is as a strategic asset and tool rather than a service [9]. In order to assess the impact of CC, managers need to be aware that it has become another strategic asset embedded in strategic alignment of IT and business [5, 9, 19, 20, 23]. Being at- tributed to an improvement in data sharing, technology standardization and infrastruc- ture, administering and operations, CC is further associated with the ability in deliver- ing new projects and applications within budget, time and scope, changing strategy and engaging different stakeholders [9]. It changed the paradigm of IT investment moving from operational expenditure (OPEX) to capital expenditure (CAPEX) and decreased usage of hardware [12, 16, 31, 32, 33]. g Furthermore, CC is related to greater scalability, flexibility and operation ability. This enables organizations to improve organizational infrastructure and business ser- vices [31, 32]. The ubiquitous nature of CC [31, 32, 34, 35] enables greater alignment of business and IT strategies, where different business units can integrate and use cloud solutions anywhere and anytime. On the one hand, CC is thought to be a reliable source when organizations acquire standardized solutions from trusted providers, therefore once adopted by organisational and IT infrastructure it affects strategic choices made by executives and related to investment in IT and business [31, 34]. To build our theoretical framework on the role of cloud technology adoption in or- ganization we use Strategic Alignment lens [30, 36]. First, we conducted a detailed literature review to identify relevant theories of alignment and validated learning. Our extensive literature search failed to unearth theories addressing strategic alignment be- tween strategy and technology with the important stage of validated learning. This is important when investigating the fit and integration between strategy and operations [21, 23, 30]. 1 Introduction There has been little attention paid to CC as an asset but also a strategic tool, creating more agile and flexible IT infrastructure, business operations, skills, ad- ministrative infrastructure and IT architecture. CC is likely to result in changes in ad- ministering and operations; higher rate of new product development, higher engage- ment of executives in IT investment and management decision-making, business growth, meticulous design of IT and business strategy within budget, time and scope. These and other important issues related to CC have not been explicitly discussed within the Strategic Alignment (SA) framework [14, 19, 20] with a lack of evidence on how information flows to achieve strategic fit and integration [21]. Neither has been investigated the interplay between CC, strategic operations and strategic performance of organization with the published work being fragmented and incomplete [20]. CC can play a major role in SA in organization described in [14] as the difficulties of achieving alignment for professional organizations; the limitations, organizations have in being agile; the rationale for acquiring technology and determining IT skills; the imperative meaning that CIOs attribute to IS alignment. To demonstrate the role that CC plays ion IS in organization we build on Information Systems (IS) [4, 22], Strategic Alignment [23-27] and organizational performance literature [28, 29]. This study makes the following contribution. We develop and test the “cloud-ena- bled mechanism of validated learning” embedded into IS alignment model. More spe- cifically we develop the Cloud Computing framework of SA, where strategic fit be- tween operational and strategic levels in organization is achieved through cloud-ena- bled multiple iterative process. This study offers practical implications for managers to better understand the com- plementary nature of new technologies and CC’s embeddedness in Strategic Alignment Model. Understanding how information is collected, assessed, distributed and analyzed should enable decision-makers to design more effective and robust business and IT strategy. We argue for the need to study the continuous organizational adaptation of evolving CC because of the challenges such technologies pose for users, as well as the operational capabilities and strategic skills they demand. 2.2 Stages of Theoretical Framework Our theoretical framework consists of three stages associated with Apprehension of Information, Unitization of Information, and Validated Learning. All three stages rely on ‘continuous adaptation and change’ within SA model [23]. Our theoretical frame- work illustrates various steps in information gathering, conceptualization, optimization, implementation and validated learning. At the first stage, apprehension of Information is done through generating insights, gathering information and a process of conceptualization. It is associated with people gaining knowledge and experience through using CC for operations and as a part of IT infrastructure development. Employees who access CC may not immediately under- stand CC solutions until they have experienced them. Apprehension of information stage is about ‘finding the answer’ where ‘finding’ is something more than mere re- trieval of information. It is about adoption and use of CC in daily processes. This changes the cognition of users, focused on pure knowledge acquisition by experiencing and absorbing it [28, 37]. In addition, it requires generating and conceptualizing. Gen- erating involves getting CC in place for operational and IT infrastructure. Generative thinking involves imagining possibilities where CC could be applied to automate pro- cesses and increase efficiencies. It further requires, questioning, sensing new opportu- nities and viewing IT and business processes from different perspectives and gathering information through experience and validated learning from previous iterations. The process of decision-making at this stage starts with exploring options (divergence), se- lecting and continues with exploiting and applying solutions with conceptualizing (con- vergence). The ambiguity of CC on alignment-operational should be pinned down. Conceptualizing results in putting new ideas together on how to use CC more effi- ciently. y At the second stage, Utilization of Information features the outcomes of cloud adop- tion and performance. At this stage, IT and business strategy can be designed reflecting on information received at stage one through optimizing and implementing strategy. It demands decision makers to apply knowledge obtained at the first stage to design strat- egy. They may either design a brand-new strategy, or update the existing strategy in light of changes in business processes and IT infrastructure triggered by CC. At this stage optimisation and implementation are required. Optimisation gains understanding of CC for organizational and IT strategy. This results in developing practical solutions and plans from abstract ideas, trends and insights. 2.2 Stages of Theoretical Framework Given a well-defined solution, deci- sion makers should be able to sort through large amounts of information to pinpoint the critical factors and processes where cloud is required. They should be confident in their ability to make a sound, logical evaluation of transformative impact of CC and integrate those processes. Finally, implementation is execution of designed strategy. Implemen- tation of strategy requires complete understanding of how business operations and IT infrastructure are going to be affected by the CC. In case of strategy complete or partial failure or as a result of changes in external environment (e.g. technology, institutions, market competition, etc.), decision makers need to be agile and respond quickly. This requires transition from the second to the third stage of “Validated Learning”. At the third stage, Validated Learning measures the strategic efficiencies on both sides of IT and business. At this stage the performance is assessed and the application of CC is redesigned adjusting to environmental changes or addressing gaps in perfor- mance outcomes. Validated learning offers two choices: first, either retaining IT and business strategy or revising the way CC is implemented. At this stage the use of CC will be reshaped and redesigned to feedback to management in order to improve the efficiency of the next round of iteration of the cloud-enabled loop. 3 The Model When an organization learns through development, implementation, measurement and feedback, the impact of CC could become more pronounced and ubiquitous [9]. This permits us to measure its value more distinctively. In particular, we can measure the transformative impact of CC by analysing its relation to innovation, strategy, develop- ment of new products and services, standardizing and sharing data, management oper- ations, changes in processes. Fig. 1 illustrates the place of CC within SA model which can be considered in terms of three interdependent dimensions of cloud-enabled validated learning loop. The first stage in a loop represents the Apprehension of Information through cloud adoption to improve organisational and IT infrastructure and processes. The second stage repre- sents the Utilization of Information received from operations. Both stages are precon- ditioners to the third stage of Validated Learning which enables the assessment of CC efficiencies and advises action. The theoretical framework supports the interplay between operations, strategic effi- ciencies and use of CC. The mechanism is iterative and can be described in stages as adopt – measure – learn – rebuild. The loop changes the way SA works, Strategic align- ment is seen a process of continuous adaptation and change which could be achieved both through iterations and technology-enabled experimental learning [14, 23]. 4 Discussion and Conclusion Cloud computing marks a paradigm shift in the way business is done and is greatly associated with delivering high quality and timely service to customers. The major benefits of CC found in this study are as follows. First, investing in CC enables rapidly easing the administering and operating processes within the organiza- tion and with external partners; second, it reduces cost of network maintenance, infor- mation exchange; third, CC decreases time solution and new products are developed; fourth, it improves security and compliance; and finally, it enables global deployments of solutions and faster decision making. Building on the extent literature [4, 13, 23, 27] this study develops the Cloud Com- puting Framework of Strategic Alignment Theory by theoretically discussing and em- pirically validating the cloud-enabled mechanism embedded into business and IT align- ment within SA model of organization [23]. More specifically, we established and de- scribed three distinctive stages of cloud’s transformative impact on organizations: first, Apprehension of Information through cloud adoption to improve organisational and IT infrastructure and processes; second, Utilization of Information received from opera- tions and deciding on business and IT strategy; third, Validated Learning which enables to assess the role that CC plays in operations and strategic fit and advise changes. When an organization learns through generalization, development, optimisation implementa- tion and validated learning, the impact of CC could become more pronounced and ubiq- uitous facilitating both operational and strategic levels of organization. Our contribution in the scholarship of IS and SA is as follows. First, we applied Strategic Alignment lens to demonstrate how each of three stages of cloud-enabled val- idated learning process contribute to business operations and IT infrastructure, business and IT strategy. Each of three stages forms a cloud-enabled loop of validated learning used by organizations as part of their “adopt – measure – learn – rebuild” strategy. Second, our methodological contribution is in applying a multi-level mixed method and used various sources of data to test our theoretical framework. The future research will need to focus on evaluating to what extent CC changes stra- tegic integration between IT infrastructure and business operations as well as between IT and business governance. Scholars would also like to know how investment in CC will to affect strategic fit between IT and business of economic agents when their per- formance and operations are interconnected, such as digital ecosystems. References 1. Barney, J.: Frim resources and sustained competitive advantage. Journal of Management 17, 99–120 (1991). 2. Fry, L. W., Smith, D. A.: Congruence, contingency, and theory building. Academy of Man- agement Review 12(1), 117–132 (1987). 3. Tosi, H. L., Slocum, J. W.: Contingency theory: Some suggested directions. Journal of Man- agement 10(1), 9–26 (1984). 4. Oh, W., Pinsonneault, A.: On the Assessment of the Strategic Value of Information Tech- nologies: Conceptual and Analytical Approaches. MIS Quarterly 31(2), 239–265 (2007). 5. Mingers, J., Willcocks, L.: An integrative semiotic methodology for IS research. Infor- mation and Organization 27(1), 17–36 (2017). 6. Wang, P., Ramiller, N. C.: Community Learning in Information Technology Innovation. MIS Quarterly 33(4), 709–734 (2009). 7. LEAD: E-Leadership Skills for Small and Medium Sized Enterprises project. European Commission, Directorate-General for Enterprise and Industry online publication 15 July (2014). 8. Li, W., Liu, K., Belitski, M., Ghobadian, A., O’Regan, N.: e-Leadership through strategic alignment: an empirical study of small and medium sized enterprises in the digital age. Jour- nal of Information Systems (2016). 9. Willcocks, L., Venters, W., Whitley, E.: Moving to the cloud corporation. Palgrave, London (2014). 10. Gold, J.: Protection in the cloud: risk management and insurance for cloud computing. Jour- nal of Internet Law 15(12), 24–28 (2012). 11. Juels, A., Oprea, A.: New approaches to security and availability for cloud data. Communi- cations of the ACM 56(2), 64–73(2013). 12. Yeboah-Boateng, E. O., Essandoh, K. A.: Factors influencing the adoption of cloud compu- ting by small and medium enterprises in developing economies. International Journal of Emerging Science and Engineering 2(4), 13–20 (2014). 13. Winkler, T. J., Benlian, A., Piper, M., Hirsch, H.: Bayer HealthCare Delivers a Dose of Reality for Cloud Payoff Mantras in Multinationals. MIS Quarterly Executive, 13(4) (2014). 14. Silva, L., Figueroa, E., González-Reinhart, J.: Interpreting IS alignment: A multiple case study in professional organizations. Information and Organization 17(4), 232–265 (2007). 15. Noor, T. H., Sheng, Q. Z., Zeadally, S., Yu, J.: Trust management of services in cloud envi- ronments: Obstacles and solutions. ACM Computing Surveys (CSUR) 46(1), 12 (2013). 16. Garrison, G., Kim, S., Wakefield, R. L.: Success factors for deploying cloud computing. Communications of the ACM 55(9), 62–68 (2012). 17. Brynjolfsson, E.: The productivity paradox of information technology. Communications of the ACM 36(12), 66–77 (1993). 18. Barua, A., Konana, P., Whinston, A. B., Yin, F.: An empirical investigation of net-enabled business value. 4 Discussion and Conclusion It may appear that strategic alignment of organization is not any more dominant unit of analysis, and strategic alignment of an ecosystem could be viewed as an alternative unit of analysis. In a new digitized economy, when operations and efficiencies of ecosystem players are complementary, CC is likely to benefit the entire ecosystem of inter-connected organi- zations, rather than a focal organization. This is because CC makes access, processing, sharing and transforming data faster, easier and more secure. Future research needs to offer theory and implications on how CC will support operations and efficiencies of inter-connected organizations. 1. Theoretical Framework of Cloud enabled Validated Learning in Strateg Fig. 1. Theoretical Framework of Cloud enabled Validated Learning in Strategic References MIS quarterly 28(4), 585–620 (2004). 19. Preston, D. S., Karahanna, E.: Antecedents of IS strategic alignment: a nomological net- work. Information Systems Research 20(2), 159–179 (2009). 20. Gerow, J. E., Grover, V., Thatcher, J. B., Roth, P. L.: Looking Toward the Future of IT- Business Strategic Alignment through the Past: A Meta-Analysis. MIS Quarterly, 38(4), 1059–1085 (2014). 9 21. Coltman, T. R., Tallon, P. P., Sharma, R., Queiroz, M.: Strategic IT alignment: twenty-five years on. Journal of Information Technology, 30(2), 91–100 (2015). 22. Sabherwal, R., Chan, Y. E.: Alignment between business and IS strategies: A study of pro- spectors, analyzers, and defenders. Information systems research 12(1), 11–33 (2001). p y y 23. Henderson, J. C., Venkatraman, N.: Strategic alignment: leveraging information technology for transforming organizations. IBM Systems Journal 32(1), 4–16 (1993). 24. Hirschheim, R., Sabherwal, R.: Detours in the path toward strategic information systems alignment. California Management Review 44(1), 87–108 (2001). 25. Peppard, J., Campbell, B.: The Co-evolution of Business/Information Systems Strategic Alignment: An Exploratory Study. Journal of Information Technology (2014). xploratory Study. Journal of Information Technology (201 26. Avison, D., Jones, J., Powell, P., Wilson, D.: Using and validating the strategic alignment model. The Journal of Strategic Information Systems 13(3), 223–246 (2004). 27. Aanestad, M., Jensen, T.: Collective mindfulness in post-implementation IS adaptation pro- cesses. Information and Organization 26(1), 13–27 (2016). 28. Dehning, B., Richardson, V. J., Zmud, R. W.: The value relevance of announcements of transformational information technology investments. MIS Quarterly, 637–656 (2003). 29. Devaraj, S., Kohli, R.: Performance impacts of information technology: Is actual usage the missing link? Management science 49(3), 273–289 (2003). 30. Henderson, J. C., Venkatraman, N.: Strategic alignment: a framework for strategic infor- mation technology management. Working Paper No. 190, Cambridge, MA: Center for In- formation Systems Research, MIT (1989). 31. Dutta, A., Peng, G. C. A., Choudhary, A.: Risks in enterprise cloud computing: the perspec- tive of IT experts. Journal of Computer Information Systems 53(4), 39–48 (2013). 32. Armbrust, M., Fox, A., Griffith, R., Joseph, A.D., Katz, R., Konwinski, A., Lee, G., Patterson, D., Rabkin, A., Stoica, I., Zaharia, M.: A view of cloud compu- ting. Communications of the ACM 53(4), 50–58 (2010). 33. Buyya, R., Yeo, C. S., Venugopal, S., Broberg, J., Brandic, I.: Cloud computing and emerg- ing IT platforms: Vision, hype, and reality for delivering computing as the 5th utility. Future Generation computer systems 25(6), 599–616 (2009). 34. References Tiers, G., Mourmant, G., Leclercq-Vandelannoitte, A.: L'envol vers le Cloud: un phénomène de maturations multiples. Systèmes d'Information & Management 18(4), 7–42 (2014). 35. Leavitt, N.: Is cloud computing really ready for prime time. Growth 27(5), 15–20 (2009). 36. Ciborra, C. U.: De profundis? Deconstructing the concept of strategic alignment. Scandina- vian Journal of Information Systems 9(1), 67–82 (1997). 37. Harvey, O.J., Hunt, D., Schroeder, H.: Conceptual systems and personality organization. John Wiley, New York (1961).
https://openalex.org/W2744701100	https://europepmc.org/articles/pmc5543559?pdf=render	English	null	Estimation of the sensitivity and specificity of two serum ELISAs and one fecal qPCR for diagnosis of paratuberculosis in sub-clinically infected young-adult French sheep using latent class Bayesian modeling	BMC veterinary research	2,017	cc-by	10,043	* Correspondence: f.corbiere@envt.fr 1IHAP, Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, F-31076 Toulouse Cedex 3, France Full list of author information is available at the end of the article Mathevon et al. BMC Veterinary Research (2017) 13:230 DOI 10.1186/s12917-017-1145-x Mathevon et al. BMC Veterinary Research (2017) 13:230 DOI 10.1186/s12917-017-1145-x Open Access Estimation of the sensitivity and specificity of two serum ELISAs and one fecal qPCR for diagnosis of paratuberculosis in sub- clinically infected young-adult French sheep using latent class Bayesian modeling Yoann Mathevon1, Gilles Foucras1, Rémy Falguières2 and Fabien Corbiere1* © 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. * Correspondence: f.corbiere@envt.fr 1IHAP, Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, F-31076 Toulouse Cedex 3, France Full list of author information is available at the end of the article © 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. Background field studies [16, 15]. Furthermore, a diagnostic test’s sensi- tivity may also vary between species, age and possibly MAP strains [2], while its specificity may be influenced by the presence of environmental mycobacterial [21]. Surveillance and control of paratuberculosis are largely hampered by the lack of sensitivity of available diagnostic tests, especially for the detection of sub-clinically infected (i.e., clinically healthy) animals. Historically, the evaluation of diagnostic test accuracy for the diagnosis of paratuber- culosis has been based on cases confirmed by histopatho- logical examination, fecal or tissue culture or repeated fecal culture for the detection of Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of paratuberculosis. However, due to the long and complex physiopathology of the disease, these cases do not include all latent cases of infection, generally leading to biased estimates of sensibility of diagnostic tests [1, 2]. Fecal quantitative PCR (qPCR) has been widely devel- oped in the last two decades as an alternative to fecal culture for the detection of animals. It is less time con- suming, especially for the detection of S-strains (sheep strains) of MAP that grow slowly in vitro compared to C- strains (cattle strains) [22]. There is also growing evidence that fecal qPCR might be at least as sensitive as, or even more sensitive than, fecal culture [23, 24]. However, its analytical sensitivity depends on several factors, including sample quality, DNA extraction methods, DNA target and qPCR systems [25, 26]. Furthermore, from an epidemio- logical point of view, Bayesian latent class models have seldom been applied to evaluate the diagnostic accuracy of fecal PCR [12], and estimates for sheep are scarce [14]. In the last few decades, however, special attention has been given to the evaluation of diagnostic test accuracy in sub-clinically infected animals. Because of the unknown true disease status of the study subjects, due to the ab- sence of a perfect reference test, latent class models have been increasingly used. These non-gold standard methods were first introduced by Hui and Walter (1980) [3] for 2 conditionally independent tests and two populations and were further extended to take account of conditional de- pendence between tests [2–6]. Bayesian modeling has been extensively developed to tackle non-identifiability issues that might arise in such models by incorporating prior knowledge of test performances [7, 8]. Background In this context, it would be unwise to simply extrapo- late already published estimates of diagnostic test accur- acy to any situation without utmost caution. In this study, we used a latent class approach in a Bayesian framework to estimate the diagnostic accuracy of two serum ELISAs and one fecal qPCR for the diagnosis of paratuberculosis in sub-clinically infected young-adult meat sheep, focusing on a narrow age range. Special attention was paid to the possibility of conditional dependence between tests under evaluation. When erroneously assumed, the assumption of condi- tional independence between tests can seriously bias par- ameter estimations [9, 10]. Conditional dependence has been taken into account in most cases when evaluating two or more tests based on the same biological process (i.e., two fecal culture methods or two serological tests) for the diagnosis of paratuberculosis in cattle or in small ruminants [11–14]. Conversely, the a priori assumption of conditional independence between tests based on the identification of MAP (i.e., fecal culture, Ziehl-Neelsen stained fecal smear or fecal PCR) and those targeting the immune response (i.e., serum ELISA or AGID) has often been made [12, 15] but has been explicitly evaluated in only a few studies [16, 17]. Abstract Background: The objective was to evaluate the diagnostic accuracy of two serum ELISAs and one quantitative PCR on feces for the diagnosis of paratuberculosis in sub-clinically infected young-adult sheep. A cross-sectional study was performed to collect 1197 individual blood and fecal samples from 2- to 3-year-old sub-clinically infected ewes in 14 closed meat sheep flocks in France. Fecal excretion was determined using qPCR based on IS900 sequence detection, and serology was performed on serum samples using two commercial ELISAs. Data were analyzed in a 3-test multiple-population Bayesian latent class model accounting for potential dependence between the three tests fitted in OpenBUGS. Separate analyses were performed according to whether doubtful ELISA results were handled as positive or negative and based on two thresholds for fecal qPCR (Ct ≤42 or Ct ≤40). Results: The best fit to the data was provided by accounting for a pairwise dependence between the two ELISAs on sensitivity and pairwise dependence between the three tests on specificity. Under this model, the estimated ELISA sensitivities were 17.4% (95% PCI: 10.6 – 25.9) and 17.9% (95% PCI 11.4 – 25.6), with estimated specificities of 94.8% (95% PCI: 93.1 – 96.3) and 94.0% (95% PCI: 92.2 – 95.7). Fecal qPCR demonstrated significantly higher sensitivity (47.5%; 95% PCI: 29.3 – 69.9) and specificity (99.0%; 95% PCI: 97.9 – 99.9) than the ELISAs. Assumptions regarding doubtful ELISA results and qPCR thresholds had only a slight impact on test accuracy estimates. Models not accounting for pairwise dependence between ELISA and fecal qPCR results yielded higher sensitivity and specificity estimates but always provided a worse fit to the data. Conclusions: Although the overall sensitivity of serum ELISAs and fecal qPCR remains low, the higher diagnostic performances of fecal qPCR make it more suitable for paratuberculosis diagnosis in sub-clinically infected sheep. Our results also illustrate that all dependence structures should be investigated when evaluating diagnostic test accuracy and selection based on a rigorous statistical approach. Keywords: Paratuberculosis, Sheep, Elisa, Fecal quantitative PCR, Sensitivity, Specificity, Bayesian latent class model Mathevon et al. BMC Veterinary Research (2017) 13:230 Page 2 of 11 Sample collection and handling A handful of feces was sampled from the rectum of selected animals using single-use gloves without lubricant and was placed in an individually identified sterile plastic bag for transportation. In parallel, a five-milliliter blood sample was also collected from the jugular vein in vacuum tubes with- out anticoagulant (Vacutainer® System). Feces and blood samples were frozen at −20 °C prior to analysis. Animal handling was performed in compliance with the European Commission Directive 2010/63/EU. All farmers gave writ- ten consent for their animals to be used in this study. Fecal real-time PCR First, fecal samples underwent a concentration procedure using the ADIAFILTER system (BioX, Rochefort, Belgium) following the manufacturer’s instructions. Ten grams of feces were rehydrated overnight in 70 mL of bidistilled sterile water. The top 10 mL of the supernatant were then filtered and centrifuged using the ADIAFILTER® disposal. Pellets were then resuspended in 500 μL of bi-distilled water and mixed with 300 mg of 150-250 μm silica beads (Silibeads, Sigmund Lindner, Warmensteinach, Germany) for 30 s at 6800 rpm three times in a bead beater (Precellys 24®, Bertin Technologies, Montigny-le- Bretonneux, France). A magnetic bead-based DNA extrac- tion was performed on a Kingfisher Flex® magnetic particle processor (Thermo Fisher Scientific, Courtaboeuf, France) following the NucleoMag 96 tissue protocol (Macherey-Nagel, Hoerdt, France), with addition of an ex- traction control (ADIAVET™PARATB REAL TIME, BioX, Rochefort, Belgium) in each plate well. Samples were sub- jected to qPCR (ADIAVET™PARATB REAL TIME, BioX, Flock and animal selection Forty-five amplifi- cation cycles were performed on a LightCycler 480 (Roche Life Science, Meylan, France), and fluorescent signals were recorded in two channels, with FAM detecting IS900 and VIC detecting the extraction control. Due to the overlap- ping spectra of the two dyes, a color compensation step was applied. Raw fluorescence data were obtained from the LightCycler 480 and modeled using the qpcR package [27] in R software [28]. Cycle thresholds were determined using second derivative maximum (CpD2). According to the manufacturer’s recommendations, samples that reached fluorescence with a cycle count (Ct) below 40 were considered positive. A higher threshold (Ct ≤42) was also considered. Indeed, careful examination of late fluorescence curves indicated that they were associated with low but unambiguously positive results up to 42 Ct, while non-specific amplification results could not be ruled out beyond this threshold. time of sampling, animals were excluded and the next one fulfilling the inclusion criteria was substituted. Depending on flock size, the target sample size ranged between 60 and 150 ewes per flock. Flock and animal selection Fourteen meat flocks with a size ranging from 290 to 1400 adult ewes (median 610) were selected for the study. They all belonged to the same breeders’ association located in the Lot administrative region of France. Inclusions criteria were (i) Causse du Lot purebred closed flocks with no introduction of replacement ewes for at least 4 years, (ii) history of positive serological results and/or of clinical cases of paratuberculosis, and (iii) no history of vaccination against paratuberculosis. Sampling was performed from March 2014 to March 2015, avoiding the month before and after lambing as well as the month after artificial insemination or mating. Although it has been shown that the sensitivity of serological testing may be enhanced in early and late lactation in cattle [15, 21], this sampling scheme was applied to fulfill breeders’ requests to reduce animal stress. Only 2- to 3-year-old ewes were included, using their eartag as an indicator of their birth cohort. Individual ages at sampling were calculated based on birth date available from the French Systeme National d’Infor- mation Génétique (SNIG) database. Ewes showing obvious clinical signs of paratuberculosis, if any, were excluded because the target population was sub-clinically infected animals. If no feces could be retrieved intra-rectum at the One other assumption underlying latent class models is that the accuracy of tests is constant across all populations, or in other words, that the various infection stages among different populations are homogeneously distributed [2]. This assumption may, however, be difficult to stick to in practice, especially when sampling without controlling for factors that influence test accuracy [18]. For paratubercu- losis, the increasing test sensitivity with the course of infection at the individual level would advocate for an age- specific evaluation of test accuracy [19]. This may lead to wide confidence or credible intervals of accuracy estimates, especially when prevalence is low and sample size is lim- ited, as shown by simulation studies [20] and experience in Mathevon et al. BMC Veterinary Research (2017) 13:230 Page 3 of 11 Page 3 of 11 Page 3 of 11 Rochefort, Belgium), following the manufacturer’s instruc- tions. Each sample was also tested for amplification of the internal control. Bi-distilled water and synthetic IS900 DNA provided in the amplification kit were used as nega- tive and positive controls, respectively. Target conditions The purpose of this evaluation was to provide an accur- ate appraisal of sensitivity and specificity of two ELISAs and one fecal qPCR for the diagnosis of paratuberculosis in sub-clinically infected 2- to 3-year-old ewes. The target condition for this evaluation was MAP-infected animals that shed enough bacteria in their feces to potentially test positive on fecal PCR at the time of sampling, that mounted an antibody response towards MAP that could be detected by ELISA, or both. Following the Nielsen and Toft (2008) definition [29], this target condition included both infected and infectious animals but probably only few affected ones, as ewes showing obvious clinical signs of paratuberculosis were excluded on farms. Note that animals passively shedding MAP in their feces [30, 31] as a result of heavy environmental con- tamination were also included in our target conditions. Laboratory testing Serological tests Two commercial ELISA tests were applied to serum samples using an overnight incubation protocol following the manufacturer’s instructions: ELISA A (ID Screen Paratuberculosis Indirect®, batch 602, IDVet, Montpellier, France) and ELISA B (IDEXX paratuberculosis screening® kit, batch 5074, IDEXX, Montpellier, France). Negative and positive controls provided by the manufacturers were included on each ELISA plate, and manufacturer’s guide- lines were strictly followed for interpretation of sample to positive (S/P) ratio results: for ELISA A serum, samples with S/P values <60%, between 60 and 70%, and ≥70% were considered negative, doubtful, and positive for MAP antibodies, respectively. For ELISA B, the negative and positive thresholds were 45% and 55%, respectively. All tests were performed blind for other test outcomes. Serial and parallel testing The accuracy of serial and parallel testing for the combi- nations of one ELISA and fecal qPCR was finally evalu- ated. For two conditionally dependent tests, namely, Test 1 and Test 2, the sensitivity (Seser) and specificity (Spser) of serial testing are given by Seser ¼ Se1Se2 þ CovSe12 Spser ¼ 1−1−Sp1 ð Þ 1−Sp2 ð Þ þ CovSp12 ð Þ; Seser ¼ Se1Se2 þ CovSe12 Spser ¼ 1−1−Sp1 ð Þ 1−Sp2 ð Þ þ CovSp12 ð Þ; where CovSe12 and CovSp12 denote the covariance terms for the pairwise dependence of sensitivities and specific- ities, respectively. Sensitivity (Separ) and specificity (Sppar) of parallel testing were given by P Tþ 1 ; T þ 2 ; T þ 3 ¼ πjSe1Se2Se3 þ 1−πj 1−Sp1 ð Þ 1−Sp2 ð Þ 1−Sp3 ð Þ Separ ¼ 1−1−Se1 ð Þ 1−Se2 ð Þ þ CovSe12 ð Þ Sppar ¼ Sp1Sp2 þ CovSp12 Separ ¼ 1−1−Se1 ð Þ 1−Se2 ð Þ þ CovSe12 ð Þ Sppar ¼ Sp1Sp2 þ CovSp12 The probability of other combinations of test out- comes can be easily derived analogously. The assump- tion of conditional independence between tests may, however, not hold in practice and should be challenged against models allowing for the conditional dependence between tests [2]. We considered the approach proposed by Dendikuri and Joseph (2001) [4], where pairwise de- pendence of sensitivities and specificities of tests are ex- plicitly modeled by covariance terms (Covse and Covsp). In the fully dependent case, the probability of all three tests being positive on a sample within population j is then given by Statistical analysis Separate analyses were performed for the four scenarios according to whether doubtful ELISA results were han- dled as positive or negative and on the choice of the positive cut-off for fecal qPCR (Ct ≤42 or Ct ≤40). Based on previous serological results, history of paratu- berculosis clinical cases and judgment of practicing veterinarians and technicians supervising the flocks, flocks were grouped into 4 sub-populations according to the within-flock suspected prevalence of infection: very low (3 flocks, 287 sampled ewes), low (5 flocks, 299 Page 4 of 11 Mathevon et al. BMC Veterinary Research (2017) 13:230 sampled ewes), moderate to high (6 flocks, 447 sampled ewes) and very high (2 flocks, 164 samples ewes). sampled ewes), moderate to high (6 flocks, 447 sampled ewes) and very high (2 flocks, 164 samples ewes). [34]. The DIC evaluates the model fit while penalizing the number of parameters, and it is generally accepted that models with smaller DIC are better supported by the data. Prior distributions Uniform distributions in the range from 0 to 1 were used as priors for sensitivity and prevalence model parameters. Based on previous published estimates in sheep [16, 35–37], the specificity of ELISAs and fecal qPCR was set at 0.95, with 95% certainty to be greater than 0.80. The corresponding Beta distribution Beta (21.20, 2.06) was generated using the epi.betabuster function embedded in the epiR package in R software [38] and was used as prior distribution for all specificity parameters. P T þ 1 ; T þ 2 ; Tþ 3 ¼ πjðSe1Se2Se3 þ Covse23Se1 þ Covse13Se2 þCovse12Se3 þ Covse123Þ þ 1−πj ð1−Sp1 ð Þ 1−Sp2 ð Þ 1−Sp3 ð Þ þ Covsp23 1−Sp1 ð Þ þ Covsp13 1−Sp2 ð Þ þ Covsp12 1−Sp3 ð Þ−Covsp123Þ Constraints were defined for covariance terms so that each of the 8 probabilities of combinations of test out- comes was between 0 and 1 [4], and uniform distributions between the lower and upper constraint bounds were used as non-informative priors. Starting from the fully saturated model below, covari- ance terms were removed one-by-one following a step- wise backward selection procedure using the Deviance Information Criterion (DIC) as the selection criterion Comparing diagnostic test accuracies The Bayesian posterior probability of difference (PPD) in sensitivity and specificity between tests was estimated using the Boolean step function in OpenBUGS [12, 16]. If PPD <0.05 or >0.95, we concluded that the sensitiv- ities (or specificities) of two compared tests were signifi- cantly different. The models were defined following the approach by Dendikuri and Joseph (2001) [4] that uses a multinomial distribution to model the frequency of the 8 observed combinations of test outcomes. The simplest model assumes conditional independence between tests (i.e., given the true disease state of a sample, the outcome of one test does not have any influence on the probability of a positive or negative outcome in a second test). Under this assumption, the probability of a combination of test outcomes in a given population only depends on the true prevalence within this population and the sensi- tivities and specificities of diagnostic tests, which are assumed constant across all populations [3]. If Ti + de- notes the event of a positive outcome for test i, i = 1, …, 3, Sei and Spi denote the sensitivity and specificity of test i, respectively, and πj, the true prevalence in a given population j, j = 1…4, then the probability of all three test being positive on a sample in this population is given by Model definition We applied multiple populations Bayesian Latent Class models [32, 33] to estimate the diagnostic accuracy of the two ELISAs and the fecal qPCR in the absence of gold standard. Test results The cross-tabulated counts of the dichotomous outcome of the three tests are given in Table 1 for the 1197 sampled animals when assuming a fecal qPCR positive threshold of Ct ≤42. The proportion of concordant test results was greater between the two ELISAs (1137/1197 = 95%) than between fecal qPCR and ELISA A (1047/1137 = 87%) or ELISA B (1051/1197 = 88%). Both ELISAs yielded fewer positive test results (n = 85 for ELISA A, n = 93 for ELISA B) than fecal qPCR (n = 105). Doubtful results were few for both ELISAs tests and significantly fewer for ELISA A (n = 8) compared to ELISA B (n = 23, Fisher’s Exact test p = 0.0109). Setting the positive cut-off at Ct ≤42 for fecal qPCR, rather than Ct ≤40, yielded 32 more positive samples. The aggregated data sets supporting the results of this article and the R2OpenBUGS code used are provided as additional files (Additional files 1 and 2). Sensitivity analysis and model assumption checking To assess the influence of prior information on the esti- mates of model parameters, poorly informative uniform distributions in the range of 0.5 to 1 were also consid- ered for specificities. These truncated distributions were chosen to avoid convergence issues of single MCMC chains due to label switching [44]. To verify the assumption of constant test accuracy across all populations, we first excluded each of the 4 populations and subsequently each of the 14 flocks, one at a time, and re-ran all investigated models. Implementation Computations were performed with OpenBUGS [39] embedded in R software using the R2OpenBUGS package [40]. Posterior estimates for test sensitivity and specificity Page 5 of 11 Page 5 of 11 Mathevon et al. BMC Veterinary Research (2017) 13:230 Page 5 of 11 were generated using the Markov Chain Monte Carlo (MCMC) sampling method and the Gibbs algorithm. Three simulation chains of 200,000 iterations were run with different starting values, with the first 10,000 itera- tions discarded as the burn-in period. The chains were then thinned, taking every tenth sample to reduce auto- correlation among the samples. The convergence of the chains following the initial burn-in period was assessed visually by examining the traces, histories, Monte Carlo errors and the Gelman-Rubin diagnostic plots [41, 42]. The posterior distribution of each parameter was summa- rized using the mean and the 95% posterior credible inter- val (95% PCI). Analysis and graphing of the MCMC output were conducted using the coda package in R [43]. Model selection Doubtful ELISA results and moving the positive cut-off from 40 to 42 for fecal qPCR had no influence on model selection. Based on DIC, the best fitting model (model 1) was the one with a pairwise dependence between ELISA A and ELISA B on sensitivity and pairwise dependence be- tween the three tests on specificity (Table 2). This model always outperformed the one assuming a conditional inde- pendence between fecal qPCR and both ELISAs on sensi- tivity and specificity (model 2). The difference in the DIC of model 1 and model 2 was always greater than 12.5, sug- gesting that including covariance terms between the fecal qPCR and both ELISAs provides a better fit to the data, although this was only significant for specificity. As expected, the assumption of conditionally independent ELISAs was not supported by the data, as shown by the high DIC values of model 3 (Table 2). Results Complete tests results were available for 1197 animals fulfilling the inclusion criteria, with a median sample size per flock of 89 (minimum 59, maximum 147). The median age at sampling was 2.5 years (lower quartile 2.3, upper quartile 2.7). Doubtful ELISA and fecal qPCR Ct ≤42 results were treated as positive. When doubtful ELISA and fecal qPCR 40 ≤Ct ≤42 results were treated as negative, counts are shown in brackets Estimated accuracy of diagnostic tests The fully conditional independent model (model 3) yielded unrealistic significantly higher es- timated sensitivity and specificity for ELISA A (Se = 70.0%, Sp = 98.7%) and ELISA B (Se = 80.0%, Sp = 98.9%) than for fecal qPCR (Se = 31.3%, Sp = 93.2%). From model 1, ELISA A and ELISA B appeared posi- tively correlated for sensitivity and specificity (Covse me- dian of 0.108 and 95% PCI between 0.068 and 0.153; Covsp median 0.029 and 95% PCI between 0.018 and 0.033). No evidence of correlation was found between ELISAs and fecal qPCR for sensitivity. In model 1, co- variance terms for specificity between the fecal qPCR and ELISA A (Covsp median 0.001 and 95% PCI be- tween 0.0009 and 0.00529) and ELISA B (Covsp median 0.00472 and 95% PCI between 0.00029 and 0.01179) were very small, although significantly different from 0. Table 3 in form-of-point estimates (mean) and 95% Bayesian posterior density credible intervals (95% PCI). For comparison purposes, the results from model 2 and model 3 are also shown. The estimated sensitivity and specificity were similar for ELISA A and ELISA B (Se ≈17%, PPD = 0.121; Sp ≈95%, PPD = 0.401) (Table 3, model 1). The fecal qPCR was found to be more sensitive (47.5%) and specific (99.0%) than ELISA tests, with PPD > 0.999 and posterior 95% credible interval excluding zero. Under the assumption of complete independence between the fecal qPCR and both ELISA tests (model 2), higher estimated sensitivities were obtained, especially for No substantial differences in estimated sensitivity and specificity were observed when analyzing the three other datasets (Table 4). Treating doubtful ELISA results as negative mostly induced a slightly lower estimated sensi- tivity of ELISA B (14.7%), which was expected from the larger number of doubtful results obtained with this test compared to ELISA A. Estimated accuracy of diagnostic tests Under the assumption of complete indepe between the fecal qPCR and both ELISA tests (m higher estimated sensitivities were obtained, espec Table 2 Bayesian Deviance Information Criterion (DIC) fo 1 to 3 under different scenarios Deviance Information Crite Doubtful ELISA results Fecal qPCR positive cut-off Model 1a Model 2b Positive Ct ≤42 120.4 134.2 Positive Ct ≤40 114.3 130.1 Negative Ct ≤42 115.6 128.2 Negative Ct ≤40 114.3 127.5 aModels with pairwise dependence between ELISA A and ELISA B for s and pairwise dependence between the three tests for specificity bModels with pairwise dependence between ELISA A and ELISA B for s and specificity and assuming conditional independence of fecal qPCR cModels assuming fully conditional independence between the thre Table 3 Mean and 95% posterior credible intervals (PCI qPCR and true prevalence (Ps) of MAP in sub-population Me Mo DIC 12 ELISA A Se 17 Sp 94 ELISA B Se 17 Sp 94 Fecal qPCR Se 47 Sp 99 Sub-pop. 1 P1 0.0 Sub-pop. 2 P2 5.4 Sub-pop. 3 P3 21 Sub-pop. 4 P4 57 ΔELISA A – ELISA B Se 0.5 Sp 0.8 ΔFecal PCR – ELISA A Se 29 Sp 4.2 Δ Fecal PCR – ELISA B Se 30 Sp 5.0 Doubtful ELISA and fecal qPCR Ct ≤42 results were treated as positi aModels with pairwise dependence between ELISA A and ELISA B fo bModels with pairwise dependence between ELISA A and ELISA B fo cModels assuming fully conditional independence between the thre Table 2 Bayesian Deviance Information Criterion (DIC) for model 1 to 3 under different scenarios Deviance Information Criterion Doubtful ELISA results Fecal qPCR positive cut-off Model 1a Model 2b Model 3c Positive Ct ≤42 120.4 134.2 227.9 Positive Ct ≤40 114.3 130.1 197.5 Negative Ct ≤42 115.6 128.2 222.1 Negative Ct ≤40 114.3 127.5 194.5 aModels with pairwise dependence between ELISA A and ELISA B for sensitivity and pairwise dependence between the three tests for specificity bModels with pairwise dependence between ELISA A and ELISA B for sensitivity and specificity and assuming conditional independence of fecal qPCR cModels assuming fully conditional independence between the three tests Table 2 Bayesian Deviance Information Criterion (DIC) for model 1 to 3 under different scenarios fecal qPCR (56.3%), without substantial changes for esti- mated specificities. Doubtful ELISA and fecal qPCR Ct ≤42 results were treated as positive aModels with pairwise dependence between ELISA A and ELISA B for sensitivity and pairwise dependence between the three tests for specificity bModels with pairwise dependence between ELISA A and ELISA B for sensitivity and specificity and assuming conditional independence of fecal qPCR cModels assuming fully conditional independence between the three tests Estimated accuracy of diagnostic tests The posterior distributions for sensitivity and specificity of the three tests and prevalence are summarized in Table 1 Cross-classified positive (+) and negative (−) results of two serum ELISAs and one fecal PCR in sub-populations 1 to 4 for sub-clinically infected 2- to 3-year-old French Causse du Lot sheep ELISA A ELISA B Fecal qPCR Subpop. 1 Subpop. 2 Subpop. 3 Subpop. 4 Total + + + 0 2 5(4) 8 (5) 15 (11) + + − 9 (7) 11 (7) 14 (14) 10 (6) 44 (34) + − + 0 1 2 2 5 (5) + − − 6 (7) 5 (8) 9 (6) 1 (6) 21 (27) − + + 1 3 6 (4) 1 11 (9) − + − 9 (5) 6 (4) 7 (6) 1 23 (16) − − + 0 4(3) 37 (17) 33 (28) 74 (48) − − − 262 (267) 267 (271) 367 (394) 108 (115) 1004 (1047) Total 287 299 447 164 1197 Doubtful ELISA and fecal qPCR Ct ≤42 results were treated as positive. When doubtful ELISA and fecal qPCR 40 ≤Ct ≤42 results were treated as negative, counts are shown in brackets tive (+) and negative (−) results of two serum ELISAs and one fecal PCR in sub-populations 1 to 4 for -year-old French Causse du Lot sheep Table 1 Cross-classified positive (+) and negative (−) results of two serum ELISAs and one fecal PCR in sub-p sub-clinically infected 2- to 3-year-old French Causse du Lot sheep Doubtful ELISA and fecal qPCR Ct ≤42 results were treated as positive. When doubtful ELISA and fecal qPCR 40 ≤Ct ≤42 results were treated as negative, counts are shown in brackets Mathevon et al. BMC Veterinary Research (2017) 13:230 Page 6 of 11 Table 3 in form-of-point estimates (mean) an Bayesian posterior density credible intervals (95 For comparison purposes, the results from mode model 3 are also shown. The estimated sensitiv specificity were similar for ELISA A and EL (Se ≈17%, PPD = 0.121; Sp ≈95%, PPD = 0.401) ( model 1). The fecal qPCR was found to be more s (47.5%) and specific (99.0%) than ELISA test PPD > 0.999 and posterior 95% credible interval ex zero. Doubtful ELISA and fecal qPCR Ct ≤42 results were treated as positive Discussion sensitivity (Se) and specificity (Sp) of two serum ELISAs and one fecal qPCR, depending on different scenarios Dataset Mean (95% PCI) Se Sp Doubtful ELISA results as negative, fecal qPCR cut-off Ct ≤42 ELISA A 17.3 (11.0; 24.8) 95.3 (93.7; 96.7) ELISA B 14.7 (8.5; 22.4) 95.6 (94.0; 97.0) Fecal qPCR 50.9 (31.3; 73.3) 99.2 (98.2; 99.8) Doubtful ELISA results as positive, fecal qPCR cut-off Ct ≤40 ELISA A 21.0 (12.8; 30.8) 94.8 (93.1; 96.3) ELISA B 20.0 (11.9; 30.2) 94.0 (92.2; 95.6) Fecal qPCR 40.7 (23.5; 63.2) 99.0 (98.0; 99.7) Doubtful ELISA results as negative, fecal qPCR cut-off Ct ≤40 ELISA A 21.2 (12.9; 31.0) 95.3 (93.7; 96.7) ELISA B 17.4 (9.5; 27.5) 95.5 (94.0; 96.9) Fecal qPCR 45.8 (26.2; 69.3) 99.2 (98.2; 99.8) Estimates were obtained with model 1 We used a Bayesian latent class approach to estimate the diagnostic accuracy of two serum ELISAs and one fecal qPCR for the detection of 2- to 3-year-old sub- clinically infected sheep. This evaluation follows the standards for the reporting of diagnosis accuracy for paratuberculosis [1] that were recently extended to Bayesian latent class models [2]. y Latent class models are highly sensitive to assumptions made regarding the conditional dependence between tests [10]. We found that treating all three tests as conditionally independent (model 3) led to biased results, with strongly overestimated sensitivities for both ELISAs. This finding is supported by the high DIC value obtained for this model and was already emphasized by simulation studies [20]. In the same way, we found that the assumption of condi- tional independence between fecal qPCR and ELISAs (model 2) was not supported by the data. Although the conditional independence between fecal culture and ELISA may hold [16, 17], to our knowledge, there is no available study evaluating the conditional dependence be- tween fecal qPCR and ELISA. Indeed, the a priori assump- tion of conditional independence is made in most cases but not formally tested [12, 15]. In our study, covariance terms between fecal qPCR and ELISAs were only signifi- cant in the specificity part of the model and were consid- erably less than the one found between the two ELISAs. However, based on DIC values, models that accounted for this dependence were unambiguously favored and led to estimates that were moderately lower than those obtained under the conditional independence assumption. Discussion These findings may or may not apply to evaluations of other commercial ELISAs and PCRs, depending on the antigens used and gene targets, respectively. In some instances, moreover, the dependence between tests may be of minimal importance, especially if the individual estimates (i.e., specificity) are close to one [2]. However, our results suggest that, when possible, models accounting for all dependence of sensitivities and specificities should be evaluated first and possibly simplified based on a rigorous selection process. Complete saturated models may, how- ever, not be identifiable (i.e., with the number of parame- ters greater than the degrees of freedom permitted by the data), allowing only restrained covariance structures to be evaluated [44]. Estimates were obtained with model 1 (40.7%) and slightly higher estimated sensitivity for ELISA A (21.0%) and ELISA B (20.0%). In any case, the estimated specificity of the three tests remained mostly unchanged. Estimated accuracy of diagnostic tests Similarly, changing the positive cut-off for fecal qPCR from Ct ≤42 to Ct ≤40 yielded a slightly lower estimated sensitivity for fecal qPCR Table 3 Mean and 95% posterior credible intervals (PCI) for the sensitivity (Se) and specificity (Sp) of two serum ELISAs and on fecal qPCR and true prevalence (Ps) of MAP in sub-populations 1 to 4 Mean (95% PCI) Model 1a Model 2b Model 3c DIC 120.4 134.2 227.9 ELISA A Se 17.9 (11.4; 25.6) 19.7 (13.1; 27.3) 70.0 (56.0; 85.6) Sp 94.8 (93.1; 96.3) 95.0 (93.4; 96.5) 98.7 (97.4; 99.9) ELISA B Se 17.4 (10.6; 25.9) 23.2 (15.8; 31.5) 80.0 (63.7; 95.3) Sp 94.0 (92.2; 95.7) 94.8 (93.2; 96.4) 98.9 (97.5; 99.9) Fecal qPCR Se 47.5 (29.3; 69.9) 56.3 (36.9; 77.3) 31.3 (21.8; 41.9) Sp 99.0 (97.9; 99.7) 99.4 (98.4; 99.9) 93.2 (91.6; 94.7) Sub-pop. 1 P1 0.01 (0.0; 0.03) 1.2 (0.1; 3.8) 6.6 (3.1; 11.0) Sub-pop. 2 P2 5.4 (1.6; 11.5) 6.2 (2.5; 11.9) 8.1 (4.7; 12.3) Sub-pop. 3 P3 21.7 (13.1; 34.5) 19.2 (12.1; 29.4) 8.4 (5.2; 12.3) Sub-pop. 4 P4 57.3 (35.4; 88.8) 47.2 (31.0; 70.4) 15.5 (9.7; 22.6) ΔELISA A – ELISA B Se 0.5 (−5.6; 5.9) −3.5 (−9.9; 2.5) −10.0 (−28.8; 9.3) Sp 0.8 (−0.8; 2.4) 0.2 (−1.2; 1.5) −0.2 (−1.9; 1.5) ΔFecal PCR – ELISA A Se 29.6 (13.7; 50.9) 36.7 (18.6; 57.9) −38.8 (−56.4; −22.4) Sp 4.2 (2.5; 6.0) 4.4 (2.6; 6.2) −5.5 (−7.4; −3.7) Δ Fecal PCR – ELISA B Se 30.1 (14.5; 50.8) 33.1 (16.0; 53.4) −48.8 (−66.4; −30.8) Sp 5.0 (3.4; 6.7) 4.6 (2.8; 6.4) −5.7 (−7.7; −3.8) Doubtful ELISA and fecal qPCR Ct ≤42 results were treated as positive aModels with pairwise dependence between ELISA A and ELISA B for sensitivity and pairwise dependence between the three tests for specificity b Page 7 of 11 Page 7 of 11 Mathevon et al. BMC Veterinary Research (2017) 13:230 Serial and parallel testing Serial and parallel testing were evaluated for model 1 (Table 5). For both ELISA and fecal qPCR combinations, serial testing was associated with a slight increase in specificity but a strong drop in sensitivity to below 9%. The use of ELISA and fecal qPCR in parallel testing led to an increased estimated sensitivity compared to fecal qPCR alone, though at the price of a loss of specificity. Sensitivity analysis and model assumption checking Rather, this might reflect the potential of pass through of orally ingested organisms by uninfected animals [30, 31] or the small yet existent possibility of cross-contamination of samples during collection or laboratory processing. The multi-copy presence of the IS900 target in the MAP genome (14-18 copies) might conversely provide higher analytical sensitivity compared to some specific alternative targets (f57, ISMAP02, hspx) that are only present in six or fewer copies [52, 53]. Moreover, 10.0 g of feces were processed for the qPCR detection, lowering the possibility of missing MAP aggregates [54]. Nevertheless, as stated in our results, the epidemiological sensitivity of fecal qPCR, even based on the IS900 target, remains low in 2- to 3-year-old sub- clinically infected sheep (40-50%). This might reflect the low number of infected animals that shed MAP in their feces within this age cohort, or that intermittent shed- ding prevented their detection at the time of sampling, or both. difficult to achieve in practice. In large herds/flocks where only partial sampling is often applied due to cost constraints, focusing on specific age cohorts may also allow for an easier and more robust comparison of prevalence estimates between herds/flocks. Finally, at the herd/flock level, a narrow age range may facilitate year-over-year comparison of results. The drawback of such an approach is that our results may be strongly linked to our study population and should be carefully extrapolated to other situations. As both ELISA and fecal qPCR provide a continuous range of result values, the classification of samples as positive or negative results in a loss of information [45] and in inconclusive test results (in our case, doubtful ELISA results and characteristic amplification curves with Ct > 40 for fecal qPCR according to the manufac- turer’s recommended positive threshold). As they may have a strong influence on accuracy estimates [46], in- conclusive results were classified either as positive or negative in separate analyses following standards for reporting of diagnosis accuracy studies. However, be- cause there were only a few, doubtful ELISA results did not cause any considerable differences in the summaries of test performances. In the same way, choosing a Ct ≤42 rather than Ct ≤40 threshold for the fecal qPCR did not lead to a dramatic change in sensitivity estimates. These changes were of the order of magnitude as those ob- served between statistical models 1 and 2. Sensitivity analysis and model assumption checking The use of poorly informative prior distributions for specificities and resampling subpopulations or flocks did not yield any substantial change of the parameter estimates. This suggests a very weak influence of prior distributions on estimation and that the assumption of constant sensitiv- ities and specificities was not unreasonable. Furthermore, model selection based on DIC remained unchanged, strengthening our findings regarding the conditional de- pendence between test results. Table 5 Mean and 95% posterior credible intervals (PCI) for the sensitivity (Se) and specificity (Sp) of serial and parallel testing using one serum ELISA and the fecal PCR Table 5 Mean and 95% posterior credible intervals (PCI) for the sensitivity (Se) and specificity (Sp) of serial and parallel testing using one serum ELISA and the fecal PCR Mean (95% PCI) ELISA A – Fecal qPCR ELISA B – Fecal qPCR Serial testing Seser 8.7 (3.8; 15.8) 8.5 (3.5; 16.0) Spser 99.8 (99.3 – 100.0) 99.3 (98.5; 99.9) Parallel testing Separ 56.6 (38.8;76.1) 55.8 (37.4; 75.9) Sppar 93.8 (91.8; 95.6) 93.1 (90.7; 95.1) Estimates were obtained with model 1, with doubtful ELISA results and fecal qPCR Ct ≤42 treated as positive One other assumption underlying latent class models is that the various infection stages among the different populations are homogeneously distributed [2]. Our study was based on animals belonging to a narrow age range (2 to 3 years), which, to our point of view, offers several advantages. First, it might have lessened the se- lection biases related to a non-homogenous sampling across the different infection stages among populations, since an age representative sample of animals might be Mathevon et al. BMC Veterinary Research (2017) 13:230 Page 8 of 11 Page 8 of 11 without improvement of specificity estimates (results not shown). Although the specificity of the IS900 target for the detection of MAP is of concern, as other mycobac- teria with IS900-like sequences have been described [48], considerable improvements have been made in PCR probe and primer designs in recent years [49, 50], and this hypothesis is currently unlikely. However, other tar- gets exclusive to MAP, such as the hspx gene [51], have shown non-perfect specificity for the detection of infec- tious animals when evaluated in Bayesian latent class models [14]. Sensitivity analysis and model assumption checking Point estimates of both ELISA sensitivities obtained in our study (14 - 21%) are similar to or slightly lower than those obtained in other studies for the detection of sub- clinically infected sheep reviewed in Nielsen and Toft (2008) [29]. ELISA B was recently applied on serum and milk in Greek dairy sheep and yielded higher sensitivity estimates (46-49%) [47]. The reasons for these discrep- ancies are not known but could be related to the age structure of study samples, breed differences or possibly regional MAP strain variations. Conversely, our specifi- city estimates (94-96%) were in concordance with those found in already mentioned studies in sheep [16, 35–37] and support the idea that ELISA is far from being perfectly specific. Our specificity estimates for ELISAs and qPCR are based on data collected in flocks suspected or known to be infected by MAP. Therefore, they may not reflect those that would have been obtained in truly paratuberculosis- free flocks, in which they could be expected to be higher [55]. However, the large-scale application of an imperfectly specific test (even with specificity as high as 99.5%) is questionable for detection purposes, as it would lead to numerous false positive results in paratuberculosis-free flocks that would require further investigation. Con- versely, this lack of specificity may have fewer adverse im- pacts on infected flock monitoring programs, as the positive predictive value of tests will be higher, and no confirmatory testing will generally be requested [56]. Fecal qPCR has the potential to be a rapid and sensi- tive method of MAP diagnosis, especially in sheep in which fecal cultures performed poorly. We found that fecal qPCR had higher diagnostic accuracy than ELISA, with sensitivity estimates close to those obtained by Baumann et al. [14] in sheep when using the Ct ≤40 cut-off for positive results. Moving the cut-off up to Ct ≤42 was associated with slightly enhanced sensitivity estimates with almost no change in specificity estimates. While the specificity of fecal qPCR was very high, it was not absolute at the Ct ≤42 or Ct ≤40 cut-off. An even more conservative value (i.e., Ct ≤38) was also evaluated Finally, the estimated sensitivity of fecal qPCR had wide credible intervals. Sensitivity analysis and model assumption checking In latent class model analysis, reasons responsible for such findings are low true values of diag- nostic test accuracy, low true prevalence, small sample size, small difference in prevalence between sub-populations, lack of global identifiability of the model, or parameter esti- mates close to 0.5 [20, 44, 57]. Although a large number of sheep were sampled, the estimated true prevalence was rather low in two out of four sub-populations (0.8% and 5.4%, respectively), and therefore, the sensitivity estimates were based on a limited number of positive results. This is Mathevon et al. BMC Veterinary Research (2017) 13:230 Page 9 of 11 also illustrated by the very narrow intervals for fecal qPCR sensitivity estimates provided by multiplying the original data by ten (11,970 animals) (results not shown). Additional file 2: Aggregated data set. Aggregated diagnostic test results for the 4 sub-populations, given whether doubtful ELISA results were handled as positive or negative and based on two thresholds for fecal qPCR. (DOCX 15 kb) y The serial use of fecal qPCR for the confirmation of ELISA-positive individuals allows for an almost perfect specificity, especially for ELISA A (99.8%). Serial testing was, however, associated with a very low global sensitivity, meaning that the true infectious status of an ELISA- positive individual that would be subsequently tested as qPCR-negative in feces would remain uncertain. The interferon-gamma release assay provides a positive re- sponse earlier in the course of the disease than fecal cul- ture [58] and would therefore be advised in such cases. However, this assay also requires careful interpretation, as it cannot distinguish between infected and exposed ani- mals [59]. As shown in Table 5, the diagnostic accuracy at the individual level could be enhanced by the use of serum ELISA and fecal qPCR in parallel testing. This reflects the fact that fecal shedding of MAP and the humoral response are poorly correlated and that parallel testing might target different individuals. This is also stated by the non- significant covariance terms for sensitivity between fecal qPCR and serum ELISAs in our Bayesian latent class model and is in accordance with experimental infection results indicating that some persistently shedding sheep may develop clinical disease in the absence of an antibody response [60]. The use of tests in combination, however, substantially adds to the cost of control, which may or may not be acceptable to sheep owners. Competing interests h h d l h p g The authors declare that they have no competing interests. The authors declare that they have no competing interests. Availability of data and materials d d d h Aggregated data are presented within the manuscript (Table 1) and in Additional file 2. Individual data analyzed during the current study are available from the corresponding author on reasonable request. Sensitivity analysis and model assumption checking Moreover, the higher cost of individual fecal qPCR (approximately 35 euros or 39 USD) compared to serum ELISA (approximately 6 euros or 7 USD) limits its use at a large scale in France. Ethics approval and consent to participate Animal studies were compliant with all applicable provisions established by the European Commission Directive 2010/63/UE. All animals used in this study were handled in strict accordance with good clinical practices and all efforts were made to minimize suffering. g All animal owners gave written consent for their animals to be used in this study. All animal owners gave written consent for their animals to be used in this study. Funding This study was funded by the INRA GISA Metaprogram PICSAR and Region Midi-Pyrénées (PAROVLOT program). The lead author received a PhD grant from both funding bodies. The funding bodies did not have any direct role in the study design or sample collection. Acknowledgements The authors thank all farmers, farm technicians and veterinary practitioners and students for their involvement in and dedication to this study. Abbreviations CovSe: covariance term on sensitivity; CovSp: covariance term on specificity; CpD2: second derivative maximum; Ct: cycle threshold; DIC: deviance information criterion; MAP: Mycobacterium avium spp. paratuberculosis; MCMC: Markov Chain Monte Carlo; PCI: Bayesian posterior credible interval; PPD: Bayesian posterior probability of difference; qPCR: quantitative polymerase chain reaction; S/ P: sample-to-positive ratio; Se: sensitivity; Separ: sensitivity of parallel testing; Seser: sensitivity of serial testing; SNIG: Systeme National d’Information Génétique; Sp: specificity; Sppar: specificity of parallel testing; Spser: specificity of serial testing Author details 1 1IHAP, Ecole Nationale Vétérinaire de Toulouse, 23 chemin des Capelles, F-31076 Toulouse Cedex 3, France. 2CAPEL Ovilot, 237 avenue Pierre Semard, 46000 Cahors, France. Received: 14 April 2017 Accepted: 19 July 2017 Received: 14 April 2017 Accepted: 19 July 2017 Authors’ contributions YM, FC and GF developed and designed the study and participated in the animal sampling. YM carried out the laboratory analysis. YM and FC carried out the data analysis. YM, FC and GF drafted the initial manuscript. RF provided additional assistance in coordination of the study and animal sampling. All authors read and approved the final manuscript. Consent for publication Not applicable. Consent for publication Not applicable. An accurate appraisal of diagnostic test accuracy is of critical importance for a better evaluation of paratuberculo- sis control programs. In this study, we showed that the as- sumption of conditional independence between fecal qPCR and serum ELISA was not supported by the data and that accounting for this dependence provided slightly different accuracy estimates. Fecal qPCR demonstrated a higher sen- sitivity and specificity than serum ELISA, but the overall sensitivity of both diagnostic approaches remains low in 2- to 3-year-old sub-clinically infected animals. These findings advocate for more frequent testing of animals in a longitu- dinal follow-up scenario. Studies are in progress to evaluate the consequence of these estimated diagnostic test accuracy for surveillance programs at the flock level. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Gardner IA, Nielsen SS, Whittington RJ, Collins MT, Bakker D, Harris B, et al. Consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis in ruminants. Prev Vet Med. 2011;101:18–34. Additional files Norton S, Johnson WO, Jones G, Heuer C. Evaluation of diagnostic tests for Johne's disease (Mycobacterium Avium subspecies paratuberculosis) in New Zealand dairy cows. J Vet Diagn Investig. 2010;22:341–51. 39. Lunn D, Spiegelhalter D, Thomas A, Best N. The BUGS project: Evolution, critique and future directions. Stat Med. 2009;28:3049–67. 16. Kostoulas P, Leontides L, Enoe C, Billinis C, Florou M, Sofia M. Bayesian estimation of sensitivity and specificity of serum ELISA and faecal culture for diagnosis of paratuberculosis in Greek dairy sheep and goats. Prev Vet Med. 2006;76:56–73. 40. Sturtz S, Ligges U, Gelman A. R2WinBUGS: a package for running WinBUGS from R. J Stat Softw. 2005;12:1–16. 41. Brooks SP, Gelman A. General methods for monitoring convergence of iterative simulations. J Comput Graph Stat. 1998;7:434–55. 17. Weber MF, Verhoeff J, van Schaik G, van Maanen C. Evaluation of Ziehl-Neelsen stained faecal smear and ELISA as tools for surveillance of clinical paratuberculosis in cattle in the Netherlands. Prev Vet Med. 2009;92:256–66. 42. Gelman A, Rubin DB. Inference from iterative simulation using multiple sequences. Stat Sci. 1992:457-72. 18. Bermingham ML, Handel IG, Glass EJ, Woolliams JA, de Clare Bronsvoort BM, McBride SH, et al. Hui and Walter's latent-class model extended to estimate diagnostic test properties from surveillance data: a latent model for latent data. Sci Rep. 2015;5:11861. 43. Plummer M, Best N, Cowles K, Vines K. Coda: convergence diagnosis and output analysis for MCMC. R News. 2006;6(1):7–11. 44. Jones G, Johnson WO, Hanson TE, Christensen R. Identifiability of models for multiple diagnostic testing in the absence of a gold standard. Biometrics. 2010;66:855–63. 19. Nielsen SS, Toft N. Age-specific characteristics of ELISA and fecal culture for purpose-specific testing for paratuberculosis. J Dairy Sci. 2006;89:569–79. 45. Nielsen SS, Toft N, Jorgensen E, Bibby BM. Bayesian mixture models for within-herd prevalence estimates of bovine paratuberculosis based on a continuous ELISA response. Prev Vet Med. 2007;81:290–305. 20. Toft N, Jorgensen E, Hojsgaard S. Diagnosing diagnostic tests: evaluating the assumptions underlying the estimation of sensitivity and specificity in the absence of a gold standard. Prev Vet Med. 2005;68:19–33. 21. Nielsen SS, Gronbaek C, Agger JF, Houe H. Maximum-likelihood estimation of sensitivity and specificity of ELISAs and faecal culture for diagnosis of paratuberculosis. Prev Vet Med. 2002;53:191–204. 46. Shinkins B, Thompson M, Mallett S, Perera R. Diagnostic accuracy studies: how to report and analyse inconclusive test results. BMJ. 2013;346:f2778. 47. Angelidou E, Kostoulas P, Leontides L. Additional files The effect of conditional dependence on the evaluation of diagnostic tests. Biometrics. 1985;41:959–68. 32. Joseph L, Gyorkos TW, Coupal L. Bayesian estimation of disease prevalence and the parameters of diagnostic tests in the absence of a gold standard. Am J Epidemiol. 1995;141:263–72. 10. Gardner IA, Stryhn H, Lind P, Collins MT. Conditional dependence between tests affects the diagnosis and surveillance of animal diseases. Prev Vet Med. 2000;45:107–22. 33. Enoe C, Georgiadis MP, Johnson WO. Estimation of sensitivity and specificity of diagnostic tests and disease prevalence when the true disease state is unknown. Prev Vet Med. 2000;45:61–81. 11. Kostoulas P, Leontides L, Billinis C, Florou M. Application of a semi-dependent latent model in the Bayesian estimation of the sensitivity and specificity of two faecal culture methods for diagnosis of paratuberculosis in sub-clinically infected Greek dairy sheep and goats. Prev Vet Med. 2006;76:121–34. 34. Spiegelhalter DJ, Best NG, Carlin BP, Van Der Linde A. Bayesian measures of model complexity and fit. J R Stat Soc Series B Stat Methodol. 2002;64:583–639. 35. Hope A, Kluver P, Jones S, Condron R. Sensitivity and specificity of two serological tests for the detection of ovine paratuberculosis. Aust Vet J. 2000;78:850–6. 12. Wells SJ, Collins MT, Faaberg KS, Wees C, Tavornpanich S, Petrini KR, et al. Evaluation of a rapid fecal PCR test for detection of Mycobacterium Avium subsp. paratuberculosis in dairy cattle. Clin Vaccine Immunol. 2006;13:1125–30. 13. Angelidou E, Kostoulas P, Leontides L. Bayesian validation of a serum and milk ELISA for antibodies against Mycobacterium Avium subspecies paratuberculosis in Greek dairy goats across lactation. J Dairy Sci. 2014;97:819–28. 36. Sergeant ES, Marshall DJ, Eamens GJ, Kearns C, Whittington RJ. Evaluation of an absorbed ELISA and an agar-gel immuno-diffusion test for ovine paratuberculosis in sheep in Australia. Prev Vet Med. 2003;61:235–48. 14. Bauman CA, Jones-Bitton A, Jansen J, Kelton D, Menzies P. Evaluation of fecal culture and fecal RT-PCR to detect Mycobacterium Avium Ssp. paratuberculosis fecal shedding in dairy goats and dairy sheep using latent class Bayesian modeling. BMC Vet Res. 2016;12:212. 37. Gumber S, Eamens G, Whittington RJ. Evaluation of a Pourquier ELISA kit in relation to agar gel immunodiffusion (AGID) test for assessment of the humoral immune response in sheep and goats with and without mycobacterium paratuberculosis infection. Vet Microbiol. 2006;115:91–101. 38. Stevenson M. EpiR : Tools for the analysis of epidemiological data (Version 0.9-79) [https://cran.r-project.org/web/packages/epiR/index.html]. 2016. Accessed 31 July 2016. 15. Additional files p 2. Kostoulas P, Nielsen SS, Branscum AJ, Johnson WO, Dendukuri N, Dhand NK, et al. STARD-BLCM: standards for the reporting of diagnostic accuracy studies that use Bayesian latent class models. Prev Vet Med. 2017;138:37–47. Additional file 1: R code for Bayesian Latent Class models. Bayesian Latent Class Models (model 1 to 3) that were used in this study. (DOCX 25 kb) Additional file 1: R code for Bayesian Latent Class models. Bayesian Latent Class Models (model 1 to 3) that were used in this study. (DOCX 25 kb) Additional file 1: R code for Bayesian Latent Class models. Bayesian Late Class Models (model 1 to 3) that were used in this study. (DOCX 25 kb) Page 10 of 11 Page 10 of 11 27. Spiess AN. qpcR: Modelling and analysis of real-time PCR data. R package version 1.4-0. [https://cran.r-project.org/web/packages/qpcR/index.html]. 2014. Accessed 31 July 2016. 3. Hui SL, Walter SD. Estimating the error rates of diagnostic tests. Biometrics. 1980;36:167–71. 4. Dendukuri N, Joseph L. Bayesian approaches to modeling the conditional dependence between multiple diagnostic tests. Biometrics. 2001;57:158–67. 28. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria [https://www.R- project.org/]. Accessed 31 Jul 2016. 5. Georgiadis MP, Johnson WO, Gardner IA, Singh R. Correlation-adjusted estimation of sensitivity and specificity of two diagnostic tests. J R Stat Soc Series B Stat Methodol. 2003;52:63–76. 29. Nielsen SS, Toft N. Ante mortem diagnosis of paratuberculosis: a review of accuracies of ELISA, interferon-gamma assay and faecal culture techniques. Vet Microbiol. 2008;129:217–35. 6. Berkvens D, Speybroeck N, Praet N, Adel A, Lesaffre E. Estimating disease prevalence in a Bayesian framework using probabilistic constraints. Epidemiology. 2006;17:145–53. 30. Sweeney RW, Whitlock RH, Hamir AN, Rosenberger AE, Herr SA. Isolation of mycobacterium paratuberculosis after oral inoculation in uninfected cattle. Am J Vet Res. 1992;53:1312–4. 7. Johnson WO, Gastwirth JL, Pearson LM. Screening without a 7. Johnson WO, Gastwirth JL, Pearson LM. Screening without a gold standard : the Hui-Walter paradigm revisited. Am J Epidemiol. 2001;153:921–4. the Hui-Walter paradigm revisited. Am J Epidemiol. 2001;153:921–4. 31. Moloney BJ, Whittington RJ. Cross species transmission of ovine Johne's disease from sheep to cattle: an estimate of prevalence in exposed susceptible cattle. Aust Vet J. 2008;86:117–23. 8. Branscum AJ, Gardner IA, Johnson WO. Estimation of diagnostic-test sensitivity and specificity through Bayesian modeling. Prev Vet Med. 2005;68:145–63. 9. Vacek PM. Mathevon et al. BMC Veterinary Research (2017) 13:230 Additional files Bayesian estimation of sensitivity and specificity of a commercial serum/milk ELISA against the Mycobacterium Avium subsp. Paratuberculosis (MAP) antibody response for each lactation stage in Greek dairy sheep. Prev Vet Med. 2016;124:102–5. 22. Stevenson K. Genetic diversity of Mycobacterium Avium subspecies paratuberculosis and the influence of strain type on infection and pathogenesis: a review. Vet Res. 2015;46:64. 23. Kawaji S, Begg DJ, Plain KM, Whittington RJ. A longitudinal study to evaluate the diagnostic potential of a direct faecal quantitative PCR test for Johne's disease in sheep. Vet Microbiol. 2011;148:35–44. 48. Englund S, Bolske G, Johansson KE. An IS900-like sequence found in a mycobacterium sp. other than Mycobacterium Avium subsp. paratuberculosis. FEMS Microbiol Lett. 2002;209:267–71. 49. Kawaji S, Taylor DL, Mori Y, Whittington RJ. Detection of Mycobacterium Avium subsp. paratuberculosis in ovine faeces by direct quantitative PCR has similar or greater sensitivity compared to radiometric culture. Vet Microbiol. 2007;125:36–48. 24. Laurin EL, Chaffer M, McClure JT, McKenna SL, Keefe GP. The association of detection method, season, and lactation stage on identification of fecal shedding in Mycobacterium Avium Ssp. paratuberculosis infectious dairy cows. J Dairy Sci. 2015;98:211–20. 25. Timms VJ, Mitchell HM, Neilan BA. Optimisation of DNA extraction and validation of PCR assays to detect Mycobacterium Avium subsp. paratuberculosis. J Microbiol Methods. 2015;112:99–103. 50. Plain KM, Marsh IB, Waldron AM, Galea F, Whittington AM, Saunders VF, et al. High-throughput direct fecal PCR assay for detection of Mycobacterium Avium subsp. paratuberculosis in sheep and cattle. J Clin Microbiol. 2014;52:745–57. 26. Fock-Chow-Tho D, Topp E, Ibeagha-Awemu EA, Bissonnette N. Comparison of commercial DNA extraction kits and quantitative PCR systems for better sensitivity in detecting the causative agent of paratuberculosis in dairy cow fecal samples. J Dairy Sci. 2017;100:572–81. 51. Ellingson JL, Bolin CA, Stabel JR. Identification of a gene unique to Mycobacterium Avium subspecies paratuberculosis and application to diagnosis of paratuberculosis. Mol Cell Probes. 1998;12:133–42. Page 11 of 11 Mathevon et al. BMC Veterinary Research (2017) 13:230 52. Li L, Bannantine JP, Zhang Q, Amonsin A, May BJ, Alt D, et al. The complete genome sequence of Mycobacterium Avium subspecies paratuberculosis. Proc Natl Acad Sci U S A. 2005;102:12344–9. 53. Mobius P, Hotzel H, Rassbach A, Kohler H. Comparison of 13 single-round and nested PCR assays targeting IS900, ISMav2, f57 and locus 255 for detection of Mycobacterium Avium subsp. paratuberculosis. Vet Microbiol. 2008;126:324–33. 54. Mathevon et al. BMC Veterinary Research (2017) 13:230 Additional files Christopher-Hennings J, Dammen MA, Weeks SR, Epperson WB, Singh SN, Steinlicht GL, et al. Comparison of two DNA extractions and nested PCR, real-time PCR, a new commercial PCR assay, and bacterial culture for detection of Mycobacterium Avium subsp. paratuberculosis in bovine feces. J Vet Diagn Investig. 2003;15:87–93. 55. Brenner H, Gefeller O. Variation of sensitivity, specificity, likelihood ratios and predictive values with disease prevalence. Stat Med. 1997;16:981–91. 56. Thrusfield M. Veterinary epidemiology. Third ed. USA: Wiley-Blackwell; 2007. p. 305–30. 57. Georgiadis MP, Johnson WO, Gardner IA. Sample size determination for estimation of the accuracy of two conditionally independent tests in the absence of a gold standard. Prev Vet Med. 2005;71:1–10. 58. Huda A, Jungersen G, Lind P. Longitudinal study of interferon-gamma, serum antibody and milk antibody responses in cattle infected with Mycobacterium Avium subsp. paratuberculosis. Vet Microbiol. 2004;104:43–53. 59. Mortier RA, Barkema HW, De Buck J. Susceptibility to and diagnosis of Mycobacterium Avium subspecies paratuberculosis infection in dairy calves: a review. Prev Vet Med. 2015;121:189–98. 60. Stewart DJ, Vaughan JA, Stiles PL, Noske PJ, Tizard ML, Prowse SJ, et al. A long-term study in merino sheep experimentally infected with Mycobacterium Avium subsp. paratuberculosis: clinical disease, faecal culture and immunological studies. Vet Microbiol. 2004;104:165–78. 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https://openalex.org/W1982508950	http://oro.open.ac.uk/36023/1/149-1470-1-PB.pdf	English	null	The added value of implementing the Planet Game scenario with Collage and Gridcole	Journal of interactive media in education	2,008	cc-by	7,463	Policy This document has been downloaded from Open Research Online, The Open University's repository of research publications. This version is being made available in accordance with Open Research Online policies available from Open Research Online (ORO) Policies Open Research Online Citation Hernández Leo, Davinia; Villasclaras Fernandez, Eloy; Asensio Pérez, Juan I.; Dimitriadis, Yannis; Bote Lorenzo, Miguel and Jorrín Abellán, Iván (2008). The added value of implementing the Planet Game scenario with Collage and Gridcole. Journal of Interactive Media in Education, article no. , 22. https://oro.open.ac.uk/36023/ (CC-BY 3.0)Creative Commons: Attribution 3.0 The added value of implementing the Planet Game scenario with Collage and Gridcole 3 University of Valladolid, Faculty of Education Paseo de Belén 1 47011 Valladolid, Spain http://hera.fed.uva.es 2University of Valladolid, School of Telecommunications Engineering, Campus Miguel Delibes 47011 Valladolid, Spain http://gsic.tel.uva.es 1 Universitat Pompeu Fabra Information and Communication Department, Passeig de Circumval.lació 8 08003 Barcelona, Spain http://gti.upf.edu Abstract: This paper discusses the suitability and the added value of Collage and Gridcole when contrasted with other solutions participating in the ICALT 2006 workshop titled “Comparing educational modelling languages on a case study.” In this workshop each proposed solution was challenged to implement a Computer-Supported Collaborative Learning situation (CSCL) posed by the workshop’s organizers. Collage is a pattern-based authoring tool for the creation of CSCL scripts compliant with IMS Learning Design (IMS LD). These IMS LD scripts can be enacted by the Gridcole tailorable CSCL system. The analysis presented in the paper is organized as a case study which considers the data recorded in the workshop discussion as well the information reported in the workshop contributions. The results of this analysis show how Collage and Gridcole succeed in implementing the scenario and also point out some significant advantages in terms of design reusability and generality, user-friendliness, and enactment flexibility. Keywords: IMS Learning Design, Computer-Supported Collaborative Learning, authoring, en Interactive Demonstration: Collage authoring tool is published in SourceForge and can be also downloaded from http://gsic.tel.uva.es/collage Versions If this document is identified as the Author Accepted Manuscript it is the version after peer review but before type setting, copy editing or publisher branding JIME http://jime.open.ac.uk/22 Pre-print 1 Introduction The suitability of IMS Learning Design (IMS LD) (IMS 2003) specification for modelling Computer- Supported Collaborative Learning (CSCL) situations in the form of CSCL scripts (Kollar, Fisher and Hesse 2006) has raised interesting controversy during the recent years (see for instance (Miao 2005) and (Hernández-Leo, Burgos, Tattersall and Koper 2007) and new Educational Modelling Languages (EML) are being proposed (see other articles in this special issue). Nevertheless, any conclusion on that debate is incomplete without considering whether the additional complexity of collaborative learning situations and the associated requirements is properly tackled by existing authoring and enacting computing tools (compliant or not with IMS LD). The ICALT 2006 workshop on “Comparing educational modelling languages on a case study” (Vignollet, David, Ferraris, Martel, and Lejeune 2006) provided an excellent opportunity to carry out such an assessment by comparing not only modelling approaches for CSCL scripts but also key capabilities of associated tools in terms of observation functions, trace/log generation, and re-use/adaptation support. For the aforementioned workshop, the authors of this paper proposed a design-based approach to the “Planet Game” scenario, introduced in the first paper of this special issue, based on the usage of “Collaborative Learning Flow Patterns” (CLFP) (Hernández-Leo, Asensio-Pérez and Dimitriadis 2005). CLFPs are implemented as IMS LD templates which seek to capture the essence of good practices in arranging participants in collaborative learning situations, sequencing types of collaborative learning activities, etc. Examples of good practices captured by CLFPs are Brainstorming and Jigsaw (Aronson and Patnoe 1997; Johnson and Johnson 1997). CLFPs provide a way of JIME http://jime.open.ac.uk/22 Pre-print communicating collaborative learning expertise to other (novice) practitioners: instead of trying to create their own collaborative designs from scratch, practitioners can reuse CLFPs as templates or guides for structuring their own collaborative situations (Hernández-Leo, Harrer, Dodero, Asensio- Pérez and Burgos 2007). Regarding the tool support for the proposed modelling approach based on patterns, the Collage authoring tool (Hernández-Leo, Villasclaras-Fernández, Jorrín-Abellán, Asensio-Pérez, Dimitriadis, Ruiz-Requies and Rubia-Abi 2006) provides a user-friendly way of selecting and completing one or several CLFPs so as to generate an IMS LD Unit of Learning (UoL) that models the targeted collaborative learning situation. Collage enables educators to select CLFPs by indicating the pursued learning objectives (both attitudinal and procedural) as well as by deciding the task type to be carried out in the designed collaborative scenario (see Figure 1). 1 Introduction Collage then recommends those CLFPs whose associated best practices are more suitable to fulfil the educator’s requirements. Once one or several CLFPs have been selected, the educator simply has to customize the flow of collaborative learning activities proposed by the CLFPs by defining the description of the activities, activity completion policy, roles, and group-size limits. Finally, it is necessary to determine and configure the resources to support the activities so that Collage can pack them all into the final IMS LD Unit of Learning. The educator performs all those steps by means of a Graphical User Interface (GUI) that hides the complexity of the XML-based formalism of IMS LD. IMS LD UoLs generated by Collage are then intended to be enacted by the Gridcole tailorable CSCL system (Bote-Lorenzo, Gómez-Sánchez, Vega-Gorgojo, Dimitriadis, Asensio-Pérez, Jorrín-Abellán 2008). In addition to the interpretation of the IMS LD UoL, Gridcole is capable of providing the participants with the set of learning materials and computing tools needed for the completion of each collaborative learning activity. Gridcole differs from other well-known Learning Management Systems (LMS), such as Moodle or .LRN, in that it does not incorporate a limited set of learning computing tools. On the contrary and following the principles of Service-Oriented Computing (Papazoglou, Traverso, Dustdar and Leymann 2007), Gridcole provides a framework in which third-party web-based learning tools are offered to students in an integrated fashion. The only constraint associated to this type of system is the availability of third-party providers offering the needed tools. This way of enhancing and customizing Gridcole functionality is called “tailoring by soft integration” (Morch 1995). LMSs are not tailorable, in the respect that they do not integrate existing tools (beyond those already available in each specific system) in order to support educational scenarios satisfying their specific needs. Having a limited set of tools limits or constrains the teacher when designing the educational scenario. For example, (Bote-Lorenzo et al., 2008) describes a real collaborative learning scenario framed in a “Computer Architecture” course where Gridcole integrates a benchmarking tool. This tool is specific to the learning situations of this subject matter and is not offered by LMS’s. 1 Introduction Figure 1 sketches the authors’ proposed solution for the “Planet Game” scenario pointing out to the role played by Collage and Gridcole in both the design of the “Planet Game” scenario (the creation of the desired IMS LD UoL and its enactment by integrating external tools). In addition to detailing the authors’ proposed modelling and implementation approach to the “Planet Game” scenario, this paper expands on the results of the ICALT 2006 workshop by analysing them as a case study intended to contribute to the validation of the CLFP-based approach enforced by Collage and Gridcole. This case study is of special research interest as it tries to answer the key question on whether the proposed approach is capable of satisfying the requirements of a learning scenario designed by a third-party (the workshop organizers, who are not biased by previous knowledge on the capabilities of the challenged approach). Additionally the case study provides a straightforward means of identifying the added value of the approach, as well as its weaknesses, with respect to other significant research works in the field, thus enabling the identification of adequate improvement efforts and redundancies. JIME http://jime.open.ac.uk/22 Pre-print EDUCATOR Computer- interpretable collaboration script (IMS LD) Patterns (pedagogical approaches) COLLABORATIVE LEARNING “EXPERTS” • Objectives • Activities • Tools • Documents ChatTool THIRD-PARTY TOOLS INTEGRATED BY GRIDCOLE FOR THE CASE STUDY DESIGN OF THE SCENARIO ENACTMENT OF THE SCENARIO GROUPS OF STUDENTS Synchronous discussion Questionnaires Shared repository Figure 1: Global outline of the implementation approach Therefore, the paper presents in section 2 the methodology adopted for analysing the ICALT workshop as a case study. The methodology guides the collection of data and its subsequent analysis for the drawing of comparative conclusions which is undertaken after the implementation of the “Planet Game” scenario, being both aspects (implementation and data analysis) described in section 3. Finally, section 4 concludes the paper by presenting the main research results and the current and future related work. ChatTool Figure 1: Global outline of the implementation approach Therefore, the paper presents in section 2 the methodology adopted for analysing the ICALT workshop as a case study. The methodology guides the collection of data and its subsequent analysis for the drawing of comparative conclusions which is undertaken after the implementation of the “Planet Game” scenario, being both aspects (implementation and data analysis) described in section 3. 1 Introduction Finally, section 4 concludes the paper by presenting the main research results and the current and future related work. Therefore, the paper presents in section 2 the methodology adopted for analysing the ICALT workshop as a case study. The methodology guides the collection of data and its subsequent analysis for the drawing of comparative conclusions which is undertaken after the implementation of the “Planet Game” scenario, being both aspects (implementation and data analysis) described in section 3. Finally, section 4 concludes the paper by presenting the main research results and the current and future related work. 3 Implementing the scenario and data analysis The results of the case study are discussed through this section which is organized consistent with the conceptual structure of the case study. • A video that records the session in which the discussion takes place. The data have been analyzed an interpreted following the “triangulation” method devoted to gaining assurance of the interpretations. Triangulation has to do with redundancy (Guba, 1981). It is the comparative analysis and critical review of evidence proceeding from different data sources and/or from different participants. The data has been aggregated in accordance with the focuses of interest outlined in the case study (the conceptual structure as defined by Stake, 1995). In particular, the topics on which the case study focuses are: testing the application of the authors’ pattern-based design process for the creation of LD scripts, as it is implemented in Collage, to a scenario proposed by a third party, and understanding the pros and cons of the approach compared with the related approaches that participate in the workshop. The concrete information questions that derive from these topics are: • Topic 1: Implementing the scenario • Topic 1: Implementing the scenario o To which extent is it possible to design a script proposed by a third party using Collage? o Can the script created with Collage be enacted by an actual LMS? • Topic 1: Implementing the scenario o To which extent is it possible to design a script proposed by a third party using Collage? C h i d i h C ll b d b l LMS? o To which extent is it possible to design a script proposed by a third party using Collage? o Can the script created with Collage be enacted by an actual LMS? • Topic 2: Comparison of the authors’ proposed solutions with related approaches o What are the pros and cons of the authors’ approach compared to other approaches regarding computational representations? o What are the pros and cons of the authors’ approach compared to other approaches regarding design? o What are the pros and cons of the authors’ approach compared to other approaches regarding enactment? o What are the pros and cons of the authors’ approach compared to other approaches regarding re-use/adaptation aspects? 2 Methodology: the ICALT workshop as a case study As described in the introduction, the case study entails the participation in a workshop integrated in the ICALT 2006 conference (Vignollet et al., 2006). The motivation of the organizers, as indicated in the introductory paper of this special issue, is concerned with whether existing EMLs (including IMS LD) and their associated tooling can be satisfactorily used in the design and enactment of CSCL situations. Nine participants proposing different approaches to solve the scenario contribute in the workshop. The introductory paper of the special issue lists the workshop participants and describes in more detail their proposed solutions, that is to say, the language used to specify the script and the tools employed to author and execute them. In the paper we only consider those participants present in the special issue for readability purposes: Collage and Gridcole (use IMS LD, Hernández-Leo et al., 2006b), Reload LDE and Coppercore (use IMS LD, Tattersall, 2006), MOT+LD (use IMS LD, Paquette and Léonard, 2006), F-logic complemented with the use of Reload LDE and Coppercore (Amorim, Lama and Sánchez, 2006), LAMS (Dalziel, 2006), ModX and LDI (using LDL Martel, Vignollet and Ferraris, 2006), CPM with Objecteering and UML profile (Nodenot and Laforcade, 2006). Extended explanations of each solution can be found in the corresponding paper of this special issue. Due to the particular characteristics of this case study, three types of data sources are used: • Papers written by the participants regarding their general approach and their application to the script proposed by the workshop organizers. • Regarding our approach, the UoL package created with Collage is also available [1]. Consequently, the conclusions and screenshots of resulting from its design and execution are also used as supporting data. • Regarding our approach, the UoL package created with Collage is also available [1]. l h l i d h f l i f i d i d i g g pp , p g g [ ] Consequently, the conclusions and screenshots of resulting from its design and execution are also used as supporting data. JIME http://jime.open.ac.uk/22 Pre-print 3.1.1 The main aspects of the scenario can be designed with Collage It is possible to apply the authors’ pattern-based design process for the creation of LD scripts, as it is implemented in Collage, to designing the Planet Game scenario. Though the script is not rigorously a JIGSAW-based situation (students do not collaborate to jointly solve a problem but they “compete to propose individually the solution”), its learning flow structure is inspired in its essence. That is, the script considers a “Jigsaw group”, which is the whole class, that is divided into two “Expert groups” representing teams A and team B, each of which accesses complementary information. Therefore, the IMS LD template representing JIGSAW CLFP [2] can be selected in Collage and particularized as illustrated in figure 2 and figure 3. JIME http://jime.open.ac.uk/22 Pre-print Figure 2: Authoring the script with Collage Figure 3: Refining the JIGSAW-based template with the description of the activities and the collaborative tool supporting the activities The “Individual” phase of JIGSAW is devoted to present the rules of the Planet Game and clue istribution depending on the team to which each student belongs. In this respect it should be noticed hat although the expert group phase of the JIGSAW is not strictly considered in this scenario, the orresponding expert-group role must exist to differentiate between members of team A and team B. This is needed for providing the right expert interview (through a shared document repository) and the pecific chat room in the discussion activity of the “Jigsaw Group” phase. The particular solution dopted in this script regarding a general way of specifying a group-service (not necessarily dedicated o conferences) is using the conference service element of IMS LD and an external binding document Figure 2: Authoring the script with Collage Figure 2: Authoring the script with Collage g g p g Figure 3: Refining the JIGSAW-based template with the description of the activities and the Figure 3: Refining the JIGSAW-based template with the description of the activities and the collaborative tool supporting the activities The “Individual” phase of JIGSAW is devoted to present the rules of the Planet Game and clue distribution depending on the team to which each student belongs. In this respect it should be noticed that although the expert group phase of the JIGSAW is not strictly considered in this scenario, the corresponding expert-group role must exist to differentiate between members of team A and team B. 3.1.1 The main aspects of the scenario can be designed with Collage questionnaire tool chat expert_interview Table 1: Summary of the UoL (based on JIGSAW CLFP) created using Collage JIGSAW CLFP phase Group/ role Activity Activity description Resources Jigsaw group Individual study At the end of this game you have to be able to classify the planets according to their distance to the Sun (from the nearest one to the most distant). Extract planets’ properties from the assigned expert interview. (Team A members’ interview contains planets’ order and some properties (without names) and team B’s interview informs about planet’s names and some properties.) expert_interview Individual study Teacher Activity control You have privileged access to the expert interviews expert_interview Expert group Subproblem Teacher Empty! (NOT VISIBLE) Global discussion Cooperate with the other team using a forum to exchange information. Each team can use a chat to discuss. forum chat expert_interview Jigsaw group Solution proposal Fill in (individually) a questionnaire about the planet classification. questionnaire tool Global problem Teacher Activity control You have access to the forum, and you can participate to discussions. You can also add new clues in any expert interview. You have to nominate a winner according to the questionnaires. questionnaire tool chat expert_interview Table 1: Summary of the UoL (based on JIGSAW CLFP) created using Collage Table 1: Summary of the UoL (based on JIGSAW CLFP) created using Collage Concluding, the main aspects of the script can be modelled with Collage. There are only two details that cannot be rigorously authored. It is not possible to specify that “the teacher decides when the exchanges are finished” because the IMS LD elements that enable its computational representation (the teacher makes the activity visible setting a property or the act is completed only when the teacher finishes her/his activity) are not included in the JIGSAW-based template. However, it is possible to add the necessary IMS LD constructs to the script using Reload LDE or another non-constrained IMS LD compliant editor (even a plain XML or text editor). Similarly, an additional activity to describe that “the game finishes when a winner is nominated” cannot be added in the current version of Collage. In any event, this can be solved by simply using the forum for the nomination of the winner or by manually modifying the script. 3.1.1 The main aspects of the scenario can be designed with Collage Instead of using a new activity to model this requirement, it is possible to design it as the feedback (viewing a property value) of the solution proposal activity of the last phase of JIGSAW. 3.1.1 The main aspects of the scenario can be designed with Collage This is needed for providing the right expert interview (through a shared document repository) and the specific chat room in the discussion activity of the “Jigsaw Group” phase. The particular solution adopted in this script regarding a general way of specifying a group-service (not necessarily dedicated to conferences) is using the conference service element of IMS LD and an external binding document JIME http://jime.open.ac.uk/22 Pre-print that indicates which groups need a different instance of the service (Bote-Lorenzo et al., 2008). Therefore, a differentiated instance of the chat and the shared repository will be available only to the members of a particular team (each team is an instance of the expert-group role, thanks to the use of the created-new attribute of the IMS LD role element). Each instance of the group-service that models the repository storing the interviews will be also available to the teacher, so that s/he can add new clues. An analogous approach is adopted for the forum, which is available to all the participants, and the questionnaire tool that will be answered in the last activity (solution proposal) of the JIGSAW. Table 1 shows a summary of the resulting UoL as created by Collage (it also generates this table). cates which groups need a different instance of the service (Bote-Lorenzo et al., 2008). y g y g ( g ) JIGSAW CLFP phase Group/ role Activity Activity description Resources Jigsaw group Individual study At the end of this game you have to be able to classify the planets according to their distance to the Sun (from the nearest one to the most distant). Extract planets’ properties from the assigned expert interview. (Team A members’ interview contains planets’ order and some properties (without names) and team B’s interview informs about planet’s names and some properties.) expert_interview Individual study Teacher Activity control You have privileged access to the expert interviews expert_interview Expert group Subproblem Teacher Empty! (NOT VISIBLE) Global discussion Cooperate with the other team using a forum to exchange information. Each team can use a chat to discuss. forum chat expert_interview Jigsaw group Solution proposal Fill in (individually) a questionnaire about the planet classification. questionnaire tool Global problem Teacher Activity control You have access to the forum, and you can participate to discussions. You can also add new clues in any expert interview. You have to nominate a winner according to the questionnaires. 3.1.2 The UoL created with Collage can be enacted using Gridcole Gridcole system, whose prototype includes the CopperCore IMS LD engine (Vogten, Martens, Nadolski, Tattersall, van Rosmalen, Koper, 2006) and other modules in charge of integrating the external tools (Bote-Lorenzo, 2008), is capable of interpreting the UoL created using Collage. Making use of the UoL and an external binding document that indicates which groups need a different instance of a service, Gridcole provides the required service instance to users. Therefore, this system guides users through the flow of collaborative learning activities integrating the tools needed to support them. In this scenario (see table 1) the selected collaborative tools are the GSIC-UVA chat, Synergeia (a shared repository for the interviews and the forum [3]) and the Quest tool (for the final questionnaires, (Gómez, Rubia, Dimitriadis, and Martínez, 2002)). Figure 4 is a screenshot of Gridcole giving access, to a student of team A, to the repository folder that contains the expert interview assigned to her/his team. The top left frame of the interface indicates the sequence of activities that should be performed JIME http://jime.open.ac.uk/22 Pre-print by the user. If the user clicks on the name of the activity, its description is shown in the right frame. Also, in the bottom left frame students can see the documents and tools available for the support of the activity (in this shared repository). When the user selects a web-based tool or a document, the selected resource is provided by the system using the right-hand frame. activity (in this shared repository). When the user selects a web-based tool or a document, the selected resource is provided by the system using the right-hand frame. Figure 4: Enacting the Planet game script created with Collage using Gridcole integrating a shared repository: clue distribution Figure 5 shows how Gridcole makes available the common forum for exchanging information with the other team and the particular chat room that students can use to discuss with their team’s members. In this case the integrated chat is a grid service-based tool which is installed, configured and launched (as a Java client in the user’s machine) by Gridcole. 3.1.2 The UoL created with Collage can be enacted using Gridcole Figure 5: Enacting the Planet game script created with Collage using Gridcole integrating a discussion forum and a chat: cooperative phase Figure 4: Enacting the Planet game script created with Collage using Gridcole integrating a shared repository: clue distribution Figure 5 shows how Gridcole makes available the common forum for exchanging information with the other team and the particular chat room that students can use to discuss with their team’s members. In his case the integrated chat is a grid service-based tool which is installed, configured and launched (as Java client in the user’s machine) by Gridcole. Figure 4: Enacting the Planet game script created with Collage using Gridcole integrating a shared repository: clue distribution Figure 5 shows how Gridcole makes available the common forum for exchanging information with the other team and the particular chat room that students can use to discuss with their team’s members. In this case the integrated chat is a grid service-based tool which is installed, configured and launched (as a Java client in the user’s machine) by Gridcole. Figure 5 shows how Gridcole makes available the common forum for exchanging information with the other team and the particular chat room that students can use to discuss with their team’s members. In this case the integrated chat is a grid service-based tool which is installed, configured and launched (as a Java client in the user’s machine) by Gridcole. Figure 5: Enacting the Planet game script created with Collage using Gridcole integrating a discussion forum and a chat: cooperative phase Figure 5: Enacting the Planet game script created with Collage using Gridcole integrating a discussion forum and a chat: cooperative phase JIME http://jime.open.ac.uk/22 Pre-print Similarly, Gridcole provides direct access to the questionnaire published in Quest in such a way that students can use immediately the tool to indicate the answer of the game (figure 6). Similarly, Gridcole provides direct access to the questionnaire published in Quest in such a way that students can use immediately the tool to indicate the answer of the game (figure 6). y g ( g ) Figure 6: Enacting the Planet game script created with Collage using Gridcole integrating a questionnaire tool: proposing the solution Figure 6: Enacting the Planet game script created with Collage using Gridcole integrating a questionnaire tool: proposing the solution 3.2.2 Design Tattersall uses the basic design procedure recommended in (IMS, 2003) and Reload LDE to create the UoL representing the script (Tattersall, 2006). As he comments (in the discussion session, recorded in the video), “Reload and CopperCore are tools at the notation level […] it is nice to see other approaches here … that makes easier to use LD…” Again, the Collage approach is pioneering in this trend: hiding the concepts of IMS LD by providing a design process that offers templates based on sound educational practice. The authors of LDL (and the associated Learning Design Infrastructure, LDI) also accept the need of this kind of design processes implemented in authoring tools: “The building phase is not completely achieved yet in the current version of LDI. Indeed, a user-friendly scenario editor destined to the teachers is required… (Martel et al., 2006).” The possibilities of MOT+LD (Paquette et al., 2006) to specify knowledge/competency are rich; however the target users of MOT+LD are learning designers familiar with the specification and its capacity to represent CSCL scenarios is limited. CPM provides a rich graphical formalism to designers of PBL situations from the initial requirements phase to the detailed design steps (Nodenot et al., 2006). However, though it is an independent language of IMS LD (and other EMLs), UML-based CPM is also intended for users with advanced technological skills. Collage implements a design process that fosters the reuse of patterns capturing successful CL flow structures. However, other types of reusable elements are possible in general (Hernández-Leo et al., 2007a). Reusable elements can be of different level of granularity - exemplars (ready-to-run) vs. templates (incomplete exemplars, such as the Collage patterns) - and completeness - chunks (portion of exemplar) vs. building blocks (incomplete chunks, such as the LAMS activity tools). Systems providing different types of reusable elements that can be assembled or combined will offer further design functionalities to the user. The very easy way of assembling LAMS activity tools is complementary to the refinable Collage templates. Building blocks similar to LAMS activity tools could be assembled in Collage templates to refine its activities or as new ones that enlarge the learning flow. Adding different types of reusable elements and constrained connecting rules between them to the design process behind Collage would provide more design options without endangering the principles of the reused element. 3.2.1 Computational representations It is straightforward to compare the author’s proposed solution with related approaches defended in the workshop since they are applied to the same example. In this respect the contributions of (Tattersall, 2006; Paquette et al., 2006; Amorim et al., 2006) confirm the statement that IMS LD supports the implementation of this script, with the interoperability advantages that it implies. However, it is worth mentioning that the way of modelling the script using IMS LD notation diverges. This shows the many possibilities of the specification which is flexible enough to describe scripts with the same core design but with different details open to author interpretations, intentions, authoring tool design constraints or features of the available runtime systems. For example, while (Tattersall, 2006; Amorim et al., 2006) and the Collage-based design use two IMS LD acts to model the script, (Paquette et al., 2006) employ four acts. Another example refers to the definition of roles. The Collage and Gridcole solutions do not follow the approach adopted by the other participants using IMS LD that consists of defining a role for each team at design time (Tattersall, 2006). In contrast, the JIGSAW-based template, used as a basis to create the script with Collage, undertakes a challenge that is also pointed out by Tattersall: “One interesting challenge with respect to the approach is to generalize to several teams depending on the cohort size. As the approach stands, the number of roles is fixed, but a solution which allowed any number of teams would clearly require a different approach.” In effect, only one role (expert-group) is defined in the UoL created with Collage and the two occurrences of the role (Team A and Team B) are determined when the script is instantiated. This is a possibility enabled by the attribute “create-new” set to “allowed” of the IMS LD role that incorporates the JIGSAW-based template as implemented in Collage. The determination of the actual number of groups at instantiation time provides flexibility and generality, however a service making available the clues to users depending on their group is necessary. In this respect we include a reference to this service at design time (shared folder with the interview in a repository) generalizing the way of specifying a collaborative tool. Using external tools the Collage and Gridcole approach also takes advantage of their special functionalities (e.g. 3.2.2 Design However, not only do design constraints limit flexibility at design time, the available enacting system can also influence this effect. This idea is also pointed out by Dalziel (in the video), “…quite different approaches of modelling the scenario […] limited to the features of the available tools,” and discussed next. 3.2.1 Computational representations adding comments to the expert interviews) and of JIME http://jime.open.ac.uk/22 Pre-print the familiarity that users may already have with the tools. Only few details cannot be formally expressed using the LD notation itself, such as the automatic random allocation of participants to groups, which on the other hand it is not required by the scenario. In LAMS “the “Grouping” tool – set to divide students randomly into two groups (Dalziel, 2006).” 3.2.3 Enactment There are two different perspectives in the approaches participating in the workshop that include execution environments. LAMS is an integrated system for authoring and execution, while the approaches of (Tattersall, 2006; Amorim et al., 2006; Martel et al., 2006; Hernández-Leo et al., 2006b) employ different systems for design and enactment. Besides, Gridcole and current developments around CopperCore advocate the integration of external tools according to service-oriented technologies. The first conclusion in this respect is that the differences in the enacted scripts of the different approaches are influenced by the available tools. This conclusion supports one of the ideas behind the design of Gridcole: having a limited set of tools constrains the teacher when designing the educational scenario. Teaches may end using some tools because they are available in an LMS, but they may prefer using other tools which satisfy in a better way the needs of their educational situation. (Tattersall, 2006) indicates that service integration (according to a Service-oriented Architecture) into Coppercore- based environments can solve the problem of limited availability of tools. This approach is also JIME http://jime.open.ac.uk/22 Pre-print implemented by Gridcole system with especial emphasis in CSCL requirements (integrating CSCL tools according to the user’s group) and the possibility of using tools requiring super-computing capabilities potentially available in a computational grid. This service-oriented approach is more general (though not very different and probably more ambitious) than the solution of LAMS V2.0 to tackle this problem. The approach of LAMS consists on a new architecture based on a “tool contract” that specifies the requirements for activity tools that can be integrated in LAMS. On the other hand, an aspect that is not satisfactorily covered by current IMS LD tooling is user- friendly administrative facilities needed when instantiating UoLs. As Tattersall (2006), we use the “command line” Clicc functionality of Coppercore to manually associate users to groups (in our case also to create the occurrence of groups). In contrast, LDI includes such facilities so that they can be easily used: “This consists in putting at the teacher’s disposal an interface allowing him to choose the participants, to attribute the roles, and to select the services and contents required by the scenario… (Martel et al., 2006)” On the other hand, further advances regarding enacting UoLs are envisaged. 3.2.3 Enactment They are related to a tighter integration of design and enactment systems to increase flexibility at runtime: “A tighter integration of design-time and runtime perspectives on IMS LD will occur, so that designs can be critiqued and improved on the basis of log data (Tattersall, 2006)”. 3.2.4 Re-use/adaptation Most of the approaches participating in the workshop declare that the resulting script can be easily re- used by changing the content associated to the activities: “Though this UoL is planned for a study of the solar system, it can be reused for other subjects by changing document titles and associating different item locations (Paquette et al., 2006).” The contribution of the authors’ approach is however that Collage already allows the reuse of a general structure (JIGSAW CLFP). Besides, since the JIGSAW-based template implemented in Collage specifies the groups in a general manner (the actual groups need to be created at instantiation time, allowed by the use of the IMS LD “create-new” attribute), the possibilities of re-using the script increase (forming more teams or several (jigsaw) groups, i.e. mixing different members of team A and team B for cooperating in different forums. That would allow, for example, the study of several negotiation strategies. In both cases it is only necessary to create more instances of the corresponding roles). The idea of using the structure of the script as a template is also pointed out in (Tattersall, 2006), “… the Unit of Learning can easily be turned into a template by modifying the resources to address a different topic […] In essence the Unit of Learning could be used for many different areas.” 4 Conclusion and current work Furthermore, the design process implemented in Collage is being extended so that it explicitly considers assessment aspects (Villasclaras-Fernández, Hernández-Leo, Asensio-Pérez, and Dimitriadis, in press). With respect to Gridcole, its architecture has been complemented with the functionality of automatic exchange of learning outcomes from different activities among groups (Palomino-Ramírez, Bote-Lorenzo, Asensio- Pérez, Dimitriadis and de la Fuente-Valentín 2008) and with a semantic searcher of CSCL tools that supports the teacher when looking for services to be integrated by Gridcole (Vega-Gorgojo et al., 2008). All these works are part of an ongoing effort devoted to the development of educator-friendly and tailorable technological settings covering the whole lifecycle of flexible scripted collaborative learning scenarios. Acknowledgements: This work has been partially funded by the Spanish Ministry of Education and Science (TSI-2005-08225-C07-04) and the Autonomous Government of Castilla y León, Spain (project VA00905). DHL also acknowledges a fellowship of the Innovation, University and Enterprise Department from the Generalitat de Catalunya. The authors would also like to thank the rest of GSIC/EMIC Group at the University of Valladolid for their support and ideas. 4 Conclusion and current work This paper has described the authors’ proposed solution for the ICALT 2006 workshop “Comparing educational modelling languages on a case study”. Also, the paper has detailed how the results of the workshop itself have been analyzed under the considerations of an evaluation case study aimed at answering a set of questions regarding the authors’ research. It is noteworthy that the case study showed how the authors’ proposed solution for designing CSCL scripts using the pattern-based approach, enforced by the Collage tool and enacted by the Gridcole system succeeded in implementing a CSCL situation posed by a third-party. Other case studies have been carried out in workshops with teachers where they have used Collage to create their own scripts according to their educational requirements and situations (Hernández-Leo et al., 2007c). Each of these scripts is different and represents challenges drawn from real practice. Additionally, and when compared with other research initiatives presented at the ICALT workshop, the authors’ proposed solution showed to have significant advantages in terms of facilitating the authoring and reuse (thanks to the create-by-reuse approach inherent to the CLFPs (Hernández-Leo et al., 2007a)), as well as enabling the enactment (thanks to the functional flexibility offered by the tailorable nature of Gridcole). The comparison among the different works presented at the ICALT 2006 workshop refers to the status of the proposed solutions as of 2006, including that presented by this author. Improvements to those proposed solutions presented in the other papers of this special issue have not been considered as the JIME http://jime.open.ac.uk/22 Pre-print main goal was to analyze the data derived from presentations and discussions that took place at the workshop itself. Regarding the authors’ proposed solution, several improvements have already been made to the Collage and Gridcole tools. For instance, one complementary tool to Collage (called InstanceCollage or iCollage, Hernández-Gonzalo, Villasclaras-Fernández, Hernández-Leo, Asensio- Pérez and Dimitriadis, 2008) has been developed so as to assist educators in the task of assigning students to groups at instantiation time. It provides an intuitive GUI using similar visualizations to those employed in Collage. The possibility of dynamically changing the assignation of students to groups at runtime is an important research line of future work. Additionally, Collage itself has been upgraded with new CLFPs and new ways of combining them. 5 References Amorim, R., Lama, M., & Sánchez, E. (2006). Modelling and implementation of the astronomy case study with an IMS LD ontology. In Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1166-1167) Kerkrade, the Netherlands: IEEE Computer Society. Amorim, R., Lama, M., & Sánchez, E. (2006). Modelling and implementation of the astronomy case study with an IMS LD ontology. In Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1166-1167) Kerkrade, the Netherlands: IEEE Computer Society. Aronson, E., & Patnoe, S. (1997). The jigsaw classroom: building cooperation in the classroom. (2 ed.). United States: Addison-Wesley Educational Publishers Inc. Bote-Lorenzo, M.L., Gómez-Sánchez, E., Vega-Gorgojo, G., Dimitriadis, Y., Asensio-Pérez, J.I., Jorrín-Abellán, I.M. (2008). Gridcole: a tailorable grid service based system that supports scripted collaborative learning, Computers & Education, 51(1):155-172 Dalziel, J.R. (2006). Modeling a team-based astronomy task using LAMS, Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1152-1153) Kerkrade, the Netherlands: IEEE Computer Society. Gómez, E., Rubia, B., Dimitriadis, Y., & Martínez, A. (2002). Quest, A Telematic Tool for Automatic Management of Student Questionnaires in Educational Research. In Proceedings of 2nd European Conference on Technology, Information, Education Citizenship, Barcelona, Spain. Guba, E.G. (1981). Criteria for assessing the trustworthiness of naturalistic inquiries Educational Communication and Technology: A Journal of Theory, Research, and Development, 29(2), 75-91. Hernández-Gonzalo, J., Villasclaras-Fernández, E., Hernández-Leo, D., Asensio-Pérez, J.I., & Dimitriadis, Y. (2008) InstanceCollage: a graphical tool for the particularization of role/group structures in pattern-based IMS-LD collaborative scripts, Proceedings of the 8th International Conference on Advanced Learning Technologies (pp. 506 - 510) Santander, Spain. JIME http://jime.open.ac.uk/22 Pre-print Hernández-Leo, D., Asensio-Pérez, J.I., & Dimitriadis, Y. (2005). Computational representation of Collaborative Learning Flow Patterns using IMS Learning Design. Educational Technology & Society, 8(3), 75-89. Hernández-Leo, D., Villasclaras-Fernández, E.D., Jorrín-Abellán, I.M., Asensio-Pérez, J.I., Dimitriadis, Y., Ruiz-Requies, I., & Rubia-Avi, B. (2006a). COLLAGE, a collaborative learning design editor based on patterns. Educational Technology and Society, 9(1), 58-71. Hernández-Leo, D., Villasclaras-Fernández, E.D., Asensio-Pérez, J.I., Dimitriadis, Y., Bote-Lorenzo, M.L., & Marcos-García, J.A. (2006b). Tuning IMS LD for implementing a collaborative lifelong learning scenario, Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1160-1161) Kerkrade, the Netherlands: IEEE Computer Society. Hernández-Leo, D., Harrer, A., Dodero, J.M., Asensio-Pérez, J.I., Burgos, D. (2007a). A Framework for the Conceptualization of Approaches to “Create-by-Reuse” of Learning Design Solutions. Journal of Universal Computer Science. 13(7), 991-1001. 5 References Hernández Leo, D., Burgos, D., Tattersall, C., Koper, R. (2007b). Representing Computer-Supported Collaborative Learning macro-scripts using IMS Learning Design Proceedings of the 2nd European Conference on Technology Enhanced Learning (EC-TEL'07) Crete, Greece. Hernández Leo, D., Villasclaras Fernández, E.D., Asensio Pérez, J.I., Dimitriadis, Y. (2007c). Some findings from the evaluation of the Collage authoring tool TENCompetence Open Workshop on Current research on IMS Learning Design and Lifelong Competence Development Infrastructures (pp. 27-32) Barcelona, Spain. IMS (2003). IMS Learning Design specification. Retrieved Accessed online on May 2008 at: http://www.imsglobal.org/learningdesign/ Johnson, D.W., & Johnson, R.T. (1999). Learning together and alone: cooperative, competitive, and individualistic learning. (5 ed.). Needham Heights, MA, USA: Allyn and Bacon. Kollar, I., Fischer, F., & Hesse, F.W. (2003). Cooperation scripts for Computer-Supported Collaborative Learning. In B. Wasson, R. Baggetun, & U. Hoppe (Eds.), Proceedings of Computer Supported Collaborative Learning 2003, Community Events - Communication and Interaction (pp. 59- 61) Bergen, Norway: InterMedia. Martel, C., Vignollet, L., & Ferraris, C. (2006). Modeling the case study with LDL and implementing it with LDI. In Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1158-1159) Kerkrade, the Netherlands: IEEE Computer Society. Miao, Y., Hoeksema, K., Hoppe, H.U., & Harrer, A. (2005). CSCL scripts: Modelling features and potential use. In T. Koschmann, D. Suthers, & T. W. Chan (Eds.), Proceedings of Computer Supported Collaborative Learning 2005 (pp. 423-432) Mahwah, NJ: Lawrence Erlbaum Associates. Morch, A. (1995). Three levels of end-user tailoring: customization, integration and extension. Proceedings of the 3rd Decennial Aarhus Conference. Aarhus, Denmark, 41-45. Nodenot, T., & Laforcade, P. (2006). Learning from a planets game: elements of a didactical transposition described with the CPM language, Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1164-1165) Kerkrade, the Netherlands: IEEE Computer Society. Palomino-Ramírez, L., Bote-Lorenzo, M.L., Asensio-Pérez, J.I., Dimitriadis, Y., de la Fuente- Valentín, L. (2008). The Data Flow Problem in Learning Design: A Case Study, Proceedings of the 6th International Conference on Networked Learning (pp. 285-292), Halkidiki, Greece. Papazoglou, M.P., Traverso, P., Dustdar, S., Leymann, F. (2007) Service-Oriented Computing: State of the Art and Research Challenges. IEEE Computer, 40(11), 38-45. JIME http://jime.open.ac.uk/22 Pre-print Paquette, G., & Léonard, M. (2006). The educational modelling of a collaborative game using MOT+LD, Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1156-1157) Kerkrade, the Netherlands: IEEE Computer Society. ake, R.E. (1995). The art of case study research. 5 References London: Sage Publications. Tattersall, C. (2006). Comparing educational modelling languages on a case study: an approach using IMS Learning Design, Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1154-1155) Kerkrade, the Netherlands: IEEE Computer Society. Vega-Gorgojo, G., Bote-Lorenzo, M.L., Gómez-Sánchez, E., Asensio-Pérez, J.I., Dimitriadis, Y., Jorrín-Abellán, I.M. (2008). Ontoolcole: Supporting Educators in the Semantic Search of CSCL Tools Journal of Universal Computer Science, 14(1), 27-58. Vignollet, L., David, J.P., Ferraris, C., Martel, C., & Lejeune, A. (2006). Comparing educational modelling languages on a case study, Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 1149-1150) Kerkrade, The Netherlands: IEEE Computer Society. Villasclaras-Fernández, E., Hernández-Leo, D., Asensio-Pérez, J.I., Dimitriadis Y., (in press): Incorporating assessment in a pattern-based design process for CSCL scripts, International Journal Computers in Human Behaviour. Vogten, H., Martens, H., Nadolski, R., Tattersall, C., van Rosmalen, P., & Koper, R. (2006). CopperCore Service Integration - Integrating IMS Learning Design and IMS Question and Test Interoperability, Proceedings of the 6th IEEE International Conference on Advanced Learning Technologies (pp. 378-382) Kerkrade, the Netherlands: IEEE Computer Society. 6 Footnotes [1] The ready-to-run IMS LD compliant Unit of Learning: http://gsic.tel.uva.es/~dherleo/icalt06workshop/l3astronomy.zip [2] Jigsaw CLFP: http://gsic.tel.uva.es/~dherleo/clfp/jigsaw-en/ [3] Synergeia Website: http://bscl.fit.fraunhofer.de/ (last visited: May 2008) [1] The ready-to-run IMS LD compliant Unit of Learning: http://gsic.tel.uva.es/~dherleo/icalt06workshop/l3astronomy.zip [2] Jigsaw CLFP: http://gsic.tel.uva.es/~dherleo/clfp/jigsaw-en/ [3] Synergeia Website: http://bscl.fit.fraunhofer.de/ (last visited: May 2008)
https://openalex.org/W4384024344	https://qjae.scholasticahq.com/article/84183.pdf	English	null	Book Review: Criminal (In)Justice: What the Push for Decarceration and Depolicing Gets Wrong and Who It Hurts Most	The quarterly journal of Austrian economics/Quarterly journal of Austrian economics	2,023	cc-by	2,682	Book Review Book Review * Tate Fegley (tatefegley@protonmail.com) is assistant professor and chair of the Business and Economics department at Montreat College. Criminal (In)Justice: What the Push for Decarceration and Depolicing Gets Wrong and Who It Hurts Most Rafael A. Mangual New York, N.Y.: Center Street, 2022. 212 pp. Quart J Austrian Econ (2023) 26.1:XX–XX https://qjae.scholasticahq.com/ doi: 10.35297/qjae.010160. Tate Fegley* R afael A. Mangual’s Criminal (In)Justice is an engaging challenge to the dominant narratives regarding criminal justice reform in post-2020 America. It is very readable and compact enough to serve as a primer on the topic for the interested layman, yet also engages with current literature to such an extent to be useful to researchers. And, as an explicit challenge to dominant narratives, it has the advantage of giving the reader a more complete picture of competing views. It is unfortunately the case that in our current era the purveyors of “The Narrative” feel no need to even acknowledge, let alone address, competing viewpoints, other than to apply the litany of common labels (e.g., “racist,” “sexist,” “xenophobe”) to those who hold them. As such, even if imperfect, Mangual’s work R Creative Commons CC BY 4.0 License Quart J Austrian Econ (2023) 26.1:XX–XX https://qjae.scholasticahq.com/ doi: 10.35297/qjae.010160. 326 Quart J Austrian Econ (2023) 26.1:XX–XX is useful for those who wish to broaden their understanding of the current issues surrounding criminal justice reform, including “defunding” the police, qualified immunity, “decarceration,” cash bail reform, and the so-called progressive prosecutors. In the first chapter, Mangual contextualizes the current Overton window regarding criminal justice reform (in other words, how we got to a point where ideas such as not prosecuting theft under $950 and charging armed carjacking as a misdemeanor have been imple mented as a matter of policy). He begins by noting the observed cyclical pattern in crime and criminal justice policies, where rising violence sparks crime control efforts but once crime decreases, control efforts are relaxed and the conditions that led to the initial upswing in crime return. Violent crime was in a sharp decline in the late 1990s, and therefore calls to relax certain controls became more politically palatable. Class action lawsuits against police departments and prisons became more common and mandatory minimum sentences for certain crimes were repealed. The media became more focused on publicizing police uses of force, aided by the growing prominence of cell phone cameras and social media. After 2014, progressive prosecutors with the explicit goal of reducing racial disparities in incarceration won races in several major metro areas around the country. In 2020, the deaths of Ahmaud Arbery, Breonna Taylor, George Floyd, and Rayshard Brooks broke the camel’s back and led to a “slew of far-reaching policy shifts that . . . 1 I am unsure what the modifier “transaction” adds to “costs” in this context. Tate Fegley* sharply raised the transaction costs of law enforcement while lowering the trans action costs of lawbreaking” and “the single-largest annual spike in homicides in American history” (pp. 20–21).1 Mangual argues against the common explanations for this uptick in crime. The pandemic seems an unlikely explanatory candidate given the lack of correlation between it and crime in similarly impacted countries, as well as the fact that crime in America is highly geographically and demographically concentrated. So concentrated, in fact, that 50 percent of murders in the US occur in just 2 percent of counties, while 54 percent of counties experience no murders in a given year (Crime Prevention Research Center 2017). This fact should give us pause when we are assured the fact that a number of cities are experiencing record numbers of homicides is Book Review: Criminal (In)Justice: What the Push for Decarceration and… 327 no big deal because the national crime rate is going down. Neither can poverty explain changes in criminal behavior. According to Mangual (p. 38), “What drives criminal violence has a lot more to do with the antisocial dispositions of violent criminals and a street culture that elevates violence as both a legitimate means of dispute resolution and a basis for respect. In other words, violent crime is a social problem we can’t simply buy our way out from under.” Chapters 2 and 3 address the goal of mass decarceration (i.e., making the reduction of the incarceration rate a goal in and of itself) on two fronts, respectively: post-conviction incarceration and pretrial detention. Mangual attacks four arguments made in favor of decarceration: 1. It is problematic that the US has a significantly higher incar ceration rate than other industrialized nations. 2. A large portion of those incarcerated are serving time for non-violent, low-level offenses. 3. It is unjustifiable that incarceration levels continued to rise during the decline in crime seen since the mid-1990s.i 4. Any public safety benefits from incarceration are outweighed by its harms; incarceration is criminogenic and breaks families apart. The section attacking this first argument is what I found to be the least satisfactory in the entire the book (and was, perhaps, the one to which I was most looking forward, given my own research on this very question (Fegley, 2015). The comparisons Mangual makes seem questionable. Tate Fegley* For example, to illustrate the point that the US has a higher rate and volume of serious violent crime, he makes the point that in 2018, four neighborhoods in Chicago, two districts in Baltimore, two precincts in Detroit, and 3 neighborhoods in St. Louis “saw more than 10.5 percent of the homicides seen in the whole of England and Wales and Germany that year, despite housing just 0.33 percent of the combined population of those countries” (p. 45). He also states, “as recently as 1992, Germany sentenced a slightly higher percentage of those convicted of willful homicide to lengthy prison sentences than did the US, with 14 percent of those convicted in Germany being sentenced to life versus just 9 percent being sentenced to life here” (pp. 45–46). There is also the fact that the US is very wealthy, and therefore can spend more on incarceration. 328 Quart J Austrian Econ (2023) 26.1:XX–XX Does this sufficiently explain why even US states with the lowest incarceration rates still have rates several times that of comparable industrialized nations? However, Mangual’s case against the decarceration only gets stronger from this initial low point. Mangual gets deep into the nuances of the statistics regarding the charges against those who are convicted and incarcerated, how long they are confined, and the rates at which they reoffend when on probation or parole, disabusing the reader of the common notions that prisons are primarily ware houses of non-violent, low-level offenders. In doing so, he makes a compelling case that, other than for marginal offenders, decarceration will not likely come at a negligible cost to public safety. In the chapter on pretrial detention reform efforts—particularly getting rid of cash bail—Mangual (p. 65). appears to agree with the reformers that cash bail can lead to unjust outcomes in terms of undue burdens on individual liberty, and “makes the question of pretrial release one of means rather than one of risk.” He goes on to state that “a better approach is to structure reforms in such a way that empowers judges to remand dangerous or high-risk offenders to pretrial detention, irrespective of the charges they face” since it is “likely for a high-risk offender to be arrested for what would generally be regarded as a low-level offense” and then commit more serious crimes while awaiting trial (p. 69). Tate Fegley* For Mangual, ideally there would be a better funded system with more prosecutors, investigators, and judges in order to shorten pretrial detention. The following chapter addresses the question of the effect on children of having a parent incarcerated, something both Republican and Democratic politicians increasingly lament. In addition to decrying the separation of illegal immigrant adults and children at the border, demanding that criminal parents not be separated from their children is one of those select cases where progressives loudly claim to care about intact families. Mangual highlights several papers, including Norris et al.’s (2021) paper published in the American Economic Review, measuring the beneficial effects incarceration of criminal family members can have on children. I note this paper in particular, given the journal’s prominence in the field of economics, as well as the negative backlash the authors faced. The fact that they were willing to write it and the editors were Book Review: Criminal (In)Justice: What the Push for Decarceration and… 329 willing to publish it despite its non-PC findings (which may be the largest hurdle to peer-reviewed publication these days), suggests their results are quite convincing. Chapter 5, “Use of Force and the Practical Limits of Popular Police Reforms,” presents strong arguments in favor of the author’s conclusions and that is fine as far as it goes; Mangual does not overpromise in his thesis of the chapter, which is that progressive reforms of policing are unlikely to lead to a large overall reduction in violence. But it can feel as though the issues facing police in America are framed in such a way that serious problems are ignored. For example, Mangual includes many statistics regarding fatal police shootings of suspects,2 uses of force, injuries associated with use of force, and complaints filed against police officers. In percentage terms, one can make these issues seem rather small. For example, Mangual asks, “In the context of almost 700,000 officers making more than 10 million arrests and conducting tens of millions of traffic and pedestrian stops every year, can you honestly say that the data on uses of force establish an institutional police violence problem?” A problem with making this inference is that, like crime, police misconduct is not uniformly distributed across the country. 2 Including the fact that approximately 93 percent of the shootings in the Washington Post’s database were of armed suspects. Many on the political right strangely tend to like to cite this figure, despite the facts that bearing arms is a recognized right in the US Constitution and that being unarmed is not necessarily indicative of being harmless or innocent. Tate Fegley* If Mangual is going to claim that it is misleading to dismiss concen trated upticks in crime over the last two years by pointing to overall crime rates, it seems incongruent to dismiss problems of police misconduct and violence by pointing to overall rates. Likewise, those who are convinced that policing is a dangerous job are unlikely to have their minds changed by someone citing the number of violently injured or killed police officers and then being told to contextualize those numbers in terms of a country with 700,000 officers conducting tens of millions of stops each year. Another problem is that Mangual’s argument does not acknowledge or address the fact that officers engaging in reprehensible behavior frequently escape any meaningful punishment. Even if misconduct is not being detected at a rate that Mangual thinks is indicative of 330 Quart J Austrian Econ (2023) 26.1:XX–XX an institutional problem, the impunity with which officers engage in it is certainly an institutional problem. However, given that police violence can be considered an insig nificant problem from a statistical standpoint, any effort to further reduce it is unlikely to lead to a massive reduction. Mangual argues against defunding the police, citing the literature regarding the relationship between more police and less crime (and, relatedly, even if more police lead to more police violence, it will be dwarfed by the reduction in deaths caused by non-police). Mangual (p. 103) also argues that police militarization is not driving police use of force, given that “SWAT officers took suspects under fire in just 342 of the tens of thousands of operations they undertook” [emphasis added]. Again, while Mangual’s argument— that SWAT teams account for a very small percentage of the docu mented uses of force3—is fine as far as it goes, it seems to ignore a bigger question: if violence is so infrequently used, why are SWAT teams used so frequently? It won’t do to say that it is the presence of the SWAT teams themselves that deter violence, given the fact that so many of their deployments are in the form of no-knock raids. Mangual rounds out his chapter on police reform addressing the calls to get rid of qualified immunity and to send out mental health professionals, rather than police officers, to certain calls for help. 3 Though the force used to break down people’s doors in the middle of the night is not counted as a “use of force.” Tate Fegley* Regarding the former, the effect on police behavior is likely to be small for the simple fact that police officers are almost always indemnified even when qualified immunity does not apply (Schwartz 2014). Though there are some efforts to increase the involvement of civilian crisis intervention teams, such as the Crisis Assistance Helping Out on the Streets (CAHOOTS) program in Eugene, Oregon, they respond to a relatively small proportion of emergency calls (and most of those to which they do respond being welfare checks, providing transportation, or assisting police already on the scene). The idea that mental health professionals can significantly replace police officers in responding to calls is unrealistic given the size of the labor pool with the skills to fill such positions. 331 Book Review: Criminal (In)Justice: What the Push for Decarceration and… Chapter 6, “The Other Side of the ‘False-Positive Problem,’” addresses the issue of racial profiling in the context of stop-and-frisk. Mangual discusses his own experience, growing up as a Latino in New York City, with the “Code of the Street” in which individuals intend to look like they are capable of and willing to use violence as a form of self-protection. Unfortunately, this behavior can also draw the attention of police. Mangual acknowledges that the false- positive problem is a difficult issue, but just as racial disparities in incarceration are largely a function of differential crime rates, so is the larger police presence in minority communities (which leads to differential rates of stops). The penultimate chapter before the conclusion is “Race: The Elephant in the Room.” Mangual rightfully attacks the anti-concept of “systemic racism,” which defines a system as racist if it results in statistical disparities, even if no racial animus is present. Mangual presents the side of the argument in defense of the criminal justice system as not systemically racist from a standpoint of individual animus. He also emphasizes the other side of the ledger: the increase in public safety due to increases in incarceration rates and enforcement. Critics focus exclusively on the on racial disparities in enforcement outcomes—arrests, incarceration rates, sentence lengths—but neglect the massive declines in crime over the last three decades, which disproportionately benefit racial minorities. Criminal (In)Justice is recommended for anyone interested in learning about current criminal justice policy debates, especially from a conservative, yet thoughtful, perspective. Tate Fegley* While it is limited in mainly being a challenge to dominant progressive narratives on criminal justice rather than proposing a detailed positive program of reform, it does its job effectively. So, although a reader may leave the book with many questions on how to improve criminal justice in the United States, he will at least be assured that it could be much worse. REFERENCES Crime Prevention Research Center. 2017. Murders in US Very Concen trated: 54% of US Counties in 2014 had Zero Murders, 2% of Counties Have 51% of the Murders. Crime Prevention Research Center. April 25. Available at https://crimeresearch.org/2017/04/number-murders- county-54-us-counties-2014-zero-murders-69-1-murder/ 332 Quart J Austrian Econ (2023) 26.1:XX–XX Fegley, Tate. 2015. “Land Of The Free, Home Of The Imprisoned: A Comparison Of Incarceration Rates Among The US And Other Industrialized Nations.” Dialogi Polityczne 19: 21–32. https://dx.doi. org/10.12775/DP.2015.014 Mangual, Rafael A. 2022. Criminal (In)Justice: What the Push for Decarceration and Depolicing Gets Wrong and Who It Hurts Most. New York: Center Street. Norris, Samuel, Matthew Pecenco, and Jeffrey Weaver. 2021. “The Effects of Parental and Sibling Incarceration: Evidence from Ohio.” American Economic Review 111, no. 9 (September): 2926–63. https://doi. org/10.1257/aer.20190415 Schwartz, Joanna C. 2014. “Police Indemnification.” NYU Law Review 89, no. 3 (June): 885–1005.
https://openalex.org/W2991362318	https://hal.archives-ouvertes.fr/hal-02472560/file/000086.pdf	English	null	FOLDED CQT RCNN FOR REAL-TIME RECOGNITION OF INSTRUMENT PLAYING TECHNIQUES	null	2,019	cc-by	6,091	HAL Id: hal-02472560 https://hal.science/hal-02472560v1 Submitted on 10 Feb 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. FOLDED CQT RCNN FOR REAL-TIME RECOGNITION OF INSTRUMENT PLAYING TECHNIQUES IRCAM (UMR9912 STMS) esling@ircam.fr Philippe Esling CICM – MUSIDANSE – Université Paris 8/ IRCAM (UMR9912 STMS) ducher@ircam.fr Jean-François Ducher Jean-François Ducher CICM – MUSIDANSE – Université Paris 8/ IRCAM (UMR9912 STMS) ducher@ircam.fr Jean François Ducher One major cause of this gap in research is the lack of IPT sound banks. Lostanlen [17] has recently addressed the question of IPT recognition but limited his experiment to samples from isolated notes in a unique sound bank. ABSTRACT In the past years, deep learning has produced state-of-the- art performance in timbre and instrument classification. However, only a few models currently deal with the recognition of advanced Instrument Playing Techniques (IPT). None of them have a real-time approach of this problem. Furthermore, most studies rely on a single sound bank for training and testing. Their methodology provides no assurance as to the generalization of their results to other sounds. In this article, we extend state-of- the-art convolutional neural networks to the classification of IPTs. We build the first IPT corpus from independent sound banks, annotate it with the JAMS standard and make it freely available. Our models yield consistently high accuracies on a homogeneous subset of this corpus. However, only a proper taxonomy of IPTs and specifically defined input transforms offer proper resilience when addressing the “minus-1db” methodology, which assesses the ability of the models to generalize. In particular, we introduce a novel Folded Constant Q-Transform adjusted to the requirements of IPT classification. Finally we discuss the use of our classifier in real-time. Here, our aim is to build a real-time classifier of IPT from solo recordings. Our system should be reactive to possibly rapid changes in the technique. Therefore, the preprocessing of the audio has to maintain temporal coherence and induce as little latency as possible. For instance, any segmentation of the audio (such as proposed by [21]) in order to subsume our task into a problem of classification of isolated notes would be irrelevant. Our study focuses on the cello but the methodological issues we raise are similar for other instruments and the process we use to build and train the classifier could be generalized as long as the IPTs of these instruments are included in a sufficient number of sound banks. We show that trying to categorize cello IPTs in a unidimensional way produces weak results. The classifier performs well on homogeneous sets of data but generalizes poorly. Therefore we introduce a taxonomy of the playing techniques of the cello along 4 axes ( n a m e d exciter/vibrator, left-hand, waveform, and interaction position). We aim to build a single network which classifies audio sequences in a multi-task [4] manner according to these axes. Then, we implement a rule-based system on top of this network, in order to simplify the model and yield a classification along the 18 main IPT categories. © Jean-François Ducher, Philippe Esling. Licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). Attribution: Jean-François Ducher, Philippe Esling. “Folded CQT RCNN for Real-time Recognition of Instrument Playing Techniques”. 20th International Society for Music Information Retrieval Conference, Delft, The Netherlands, 2019. © Jean-François Ducher, Philippe Esling. Licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). Attribution: Jean-François Ducher, Philippe Esling. “Folded CQT RCNN for Real-time Recognition of Instrument Playing Techniques”. 20th International Society for Music Information Retrieval Conference, Delft, The Netherlands, 2019. 1. INTRODUCTION Throughout modern history, western composers have diversified and refined Instrument Playing Techniques (IPTs) in order to foster innovation in the timbre space [14]. In folklore and oral traditions, IPTs sometimes stand out as a distinctive feature of the musical style [16, 22]. Therefore, their identification could contribute to the more general task of style recognition in the process of browsing in music databases. Moreover, interactive computer music systems (for instance in the field of improvisation or score-following) could hugely benefit from the development of real-time IPT classifiers [24]. In order to train our classifier, we produce a large corpus of labeled synthetic data with 5 IPT sound banks and their proprietary samplers. This corpus is annotated using the JAMS standard [11]. We make it available to the MIR community. We adapt state-of-the-art models successfully used for instrument classification [11,16] to the multi-task and low latency requirements of IPT recognition. Front-end classifiers along the 4 IPT dimensions are built as fully- connected (FC) or recurrent layers on top of deep convolutional neural networks (CNNs). All tested system configurations achieve high accuracies on homogeneous subsets of our annotated corpus. This alone provides no indication of their ability to generalize to other databases or actual solo recordings. Therefore, we adapt the minus- 1db methodology presented by Livshin [15] to the needs of our system. When subject to this methodology, we In the last two decades, the MIR community has produced a lot of research in the field of timbre recognition, but there has been little effort in IPT classification, often considered as its last frontier [17]. © Jean-François Ducher, Philippe Esling. Licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0). Attribution: Jean-François Ducher, Philippe Esling. “Folded CQT RCNN for Real-time Recognition of Instrument Playing Techniques”. 20th International Society for Music Information Retrieval Conference, Delft, The Netherlands, 2019. 708 Proceedings of the 20th ISMIR Conference, Delft, Netherlands, November 4-8, 2019 proprietary database of soli, which were first segmented using a harmonicity-based method. With the leave-1-CD- out methodology, accuracy still reached 88.1%. show that RNN front-ends generalize better than FC. Our adapted Folded Constant-Q Transform (FCQT) also yields more stable performance than Log-Mel- Spectrograms. Finally, we assess the reactivity of our models for each of the 4 IPT dimensions. Lostanlen and Cella [18] used two separate solo instrument databases to train and test a deep CNN to classify 8 instruments. 1 Pooling reduces the size of the output of the convolution (called a feature map) by downsampling ; generally, the maximum value of a local neighborhood is taken (max-pooling) 2.1 Deep Learning and MIR Following their success in the field of computer vision [10], deep learning techniques have been quickly adopted by the MIR community. Instead of using sets of hand-designed audio descriptors [1,7,15], these techniques rely on basic representations of the audio signal and let the algorithm learn suitable features for a given task. Convolutional (CNN), recurrent (RNN) neural networks and their combinations have been among the most popular architectures used in MIR. Convolutional layers seek local correlations within their input by training sets of convolutional kernels. CNNs are built by stacking such layers with pooling layers1 at increasingly bigger scales. Therefore, they can detect large and complex patterns while being computationally efficient. RNNs have been developed in order to forecast or classify temporal sequences. As their hidden units have connections from one time step to the next, they can carry information through various temporal states. Long Short- Term Memory (LSTM) units, where gates enable to control this flow of information, have been proficiently used in MIR [6]. Regarding predominant instrument classification in polyphonic textures, Han and al. [11] achieved state-of- the-art F1-scores with a deep CNN applied to log-mel- spectrograms. This study was performed with two independent subsets of the IRMAS database for training and testing. 1. INTRODUCTION Their network relied on the CQT of the audio signal. Through proper optimization of their convolution strategy, their system reaches average accuracies of 74%, against 61.4% for a decision tree forest applied to a large set of audio descriptors. 2.2 Instrumental Timbre Classification Early research in instrument classification relied on samples of isolated notes played with ordinary techniques. In most studies, experiments were performed with a single sound bank. This practice overlooked variability related to the instrument model, player or recording environment. A detailed review of generalization issues by Livshin [15] shows that the performance of classifiers trained and tested with a single sound bank gives no hint on their accuracy when confronted to new sounds. He suggests to use several independent sound banks, pick one for testing while training the classifier on the rest joined together. Then, the experiment has to be repeated with all the possible test banks. This methodology named minus-1db provides more reliable indications on the ability of the classifier to generalize. In the case of instrument classification from solo recordings, it translates into a leave-1-CD-out policy. 2.3 IPT Classification IPT classification studies have been carried out on the clarinet [19], the snare drum [27], and the electric guitar [5]. The first two studies pose methodological issues since they perform training and evaluation with a single database. Chen and al. [5] focus on the detection in electric guitar solos of five techniques which all have an impact on the melodic contour. This feature is key to the design of their classifier. Therefore, their research can hardly be generalized to other IPTs. Lostanlen and al. [17] tackle the issue of IPT recognition in a transversal manner. They work with samples from isolated notes belonging to 143 IPTs from 16 instruments. Their query-by-example system relies on a variant of the k-nearest neighbors algorithm where the metric used is subject to a training process. Applied to Mel-Frequency Cepstral Coefficients enriched by second- order scattering coefficients, it reaches rank-5 accuracy of 61%. Yet, again, they train and test their system on a single sound bank. 3. IPT TAXONOMY As we will show in Section 5., trying to classify IPTs without taking into account their multi-dimensional structure results in a poor ability to generalize. This motivates our newly introduced taxonomy. 2 The resonator is assumed to remain unchanged (body). 3 For instance, a pizzicato will never be classified along the 3rd (waveform) axis. But it still could be classified along the 2nd axis (e.g. glissando) or the 4th (e.g. harmonics). 4 All notes above G2 can be played on several strings. String change is regarded as a component of intra-class variability. 5 Available upon request 6 e.g. UVI Workstation for ISI, Vienna Instruments for VSL. 7 https://drive.google.com/open? id=1HYqHxxd2ZDkU2TL_1EXa6WNv9lY37hU9 Proceedings of the 20th ISMIR Conference, Delft, Netherlands, November 4-8, 2019 Proceedings of the 20th ISMIR Conference, Delft, Netherlands, November 4-8, 2019 Proceedings of the 20th ISMIR Conference, Delft, Netherlands, November 4-8, 2019 – excitation movements should be characterized by their position (e.g. sul tasto or ponticello), length (e.g. staccato), eventual periodicity (e.g. jettato, tremolo), and the amount of speed and pressure involved (e.g. flautando vs. pressured). Some IPTs which would require a separate axis (should we aim at an exhaustive taxonomy for musicological purposes) were forced onto an existing dimension. Pressured bowing was included in axis 1 as it results in strong inharmonicity and noisiness. Harmonics, both natural and artificial, were included in axis 4, as they are most commonly played sul ponticello, but imply specific spectral envelopes. Finally, string classification4 was excluded from the taxonomy, as well as the use of mutes. – excitation movements should be characterized by their position (e.g. sul tasto or ponticello), length (e.g. staccato), eventual periodicity (e.g. jettato, tremolo), and the amount of speed and pressure involved (e.g. flautando vs. pressured). 3.3 Proposed Taxonomy We match the list of IPTs in our sound banks with the theoretical approach in section 3.1. Bearing in mind our real-time constraint, we want to prevent an inflation of the number of classifiers and parameters in our model. In the remainder of this article, we will use the term database X to designate the sequences which were generated with this process applied to the sound bank X. Therefore, we retain only 4 dimensions in our taxonomy (see Table 1). The first axis refers to the exciter/vibrator couple, which has a strong impact on the harmonicity and noisiness of the resulting sound. The second axis refers to how the left hand shapes the pitch contour. The third axis, called waveform, classifies IPTs depending on the nature and length of the bow/string interaction. The last axis refers to the position of the interaction with the string, which induces different spectral envelopes in the sound. 4.1.1 Sequence Generation Principles Bearing in mind our goal to generalize to actual solo recordings, we have generated series of audio sequences which simulate such recordings. This simulation tool was developed as a series of Max/Msp patches5 controlling the proprietary samplers6 of our 5 sound banks. The generated sequences include randomly generated notes and chords of all available IPTs. The chords are bounded by the playability of the instrument (as presented in [28]) and the specific ranges of the sound banks. 4.1.2 Data Augmentation We augment the data to increase the robustness of the classifier to variations in the recording environment and tuning of the instrument. Therefore, we generate sequences with a randomly detuned reference A4 in a 20Hz range around 440Hz (through transposition of the original samples). We also use various levels and types of reverb in the samplers. Finally, we average the stereo channels provided by the sampler to get the final signal. After augmentation, the sequences represent 13.5 hours of music and over 4 Gb of AIF files sampled at 44100Hz and encoded at 16 bits PCM. Axis 1 Axis 2 Axis 3 Axis 4 Exciter/Vibrator Left-hand Waveform Int. Position NONE NONE NONE vibrato sustained ordinario staccato sul tasto glissando sul ponticello pressured bow/string trill harmonics hand or knuckle hit on body tremolo bow hair/string (ordinario) bow wood/string (con legno) finger/string (pizzicato) non vibrato finger/string+body (pizz. Bartok) spiccato, battuto marcato, sfz 3.2 Availability of Data Proper definitions of all these IPTs should then be provided in order to annotate a recorded corpus of audio in a consistent manner. On each dimension, each class of the taxonomy had to be represented at least in two sound banks for the minus 1-Db methodology to be implemented. However, the cost of such a study would be prohibitive. Therefore, we decided to rely upon available sound banks and their IPT definitions. We identified 5 IPT sound banks which had different players and recording setups: EastWest Quantum Leap (EWQL), Vienna String Library (VSL), IRCAM Solo Instruments (ISI), Virtual Orchestra (VO) and ConTimbre (CONT). These banks suffer from two drawbacks. First, as expected, the absence of standardized definitions causes gaps in the realization of given IPTs between them, even for such a basic feature as the vibrato of an ordinario class. Second, each of the sound banks includes only a fraction of all the technical possibilities mentioned above. Several IPT combinations, albeit perfectly playable, are not available (e.g. harmonic trills). 3.1 Theoretical Background A proper IPT taxonomy requires identifying what exciter, vibrator and resonator are selected (Schaeffer [26]), and what modification and excitation movements are undertaken (Cadoz [3]). Taking the example of the cello and following Feron [8] : To our knowledge, very few studies follow the methodological principles of Livshin, and, as such, can be regarded as being state-of-the-art in this matter. – among the possible exciters are the bow hair, bow wood, as well as various parts of the hand (finger, nail, knuckle). The natural vibrators are the four strings, but the body can be involved2; Patil and al. [21] proposed a classifier built upon a support vector machine applied to spectro-temporal receptive fields. Trained on isolated notes of the RWC database to classify 6 instruments, this model reached 98.7% accuracy. Its resilience was assessed on a – modification movements are mainly the ornaments and other IPTs realized by the left hand (e.g. vibrato, glissandi, harmonics); 2 The resonator is assumed to remain unchanged (body). 709 709 4.2.1 Preprocessing As shown in Figure 1, we have tested several possibilities8 for the spectral transform. Log-mel-spectrograms (LMS): following [11], we first downsample the signal to 22,050Hz, then compute LMS on 128 bins, with FFT windows of 2048 samples and hopsize of 512 samples (~23ms). Figure 2. F-CQT example for a C4 note: (i) each CQT frame is reshuffled on an octave-per-octave basis, folded (ii) using 2-octave wide half-overlapping bands, in order to avoid side effects. A kernel (iii) of size 12 captures 8 out of the first 12 harmonics including the fundamental. Adapted low-res CQT: following [18], we compute a 12 bin-per-octave CQT from C3(~130Hz)9 t o B10(~15.8kHz). Hop size is 1024 samples (~23ms). Only the logarithm of the amplitude of the CQT is retained. In order to preserve the temporal coherence of the preprocessed signal, we halve the Q-factor of the bins in the C3-B3 octave. In our adapted CQT, the size of the analysis windows are limited to 2850 samples (~64ms). Adapted high-res CQT: to better account for IPTs such as glissandi and vibrato, we also experiment with a doubled bin-per-octave resolution above C5. The total number of bins of the CQT increases from 84 to 144. Since analysis windows are bounded, we cannot extend this resolution to the lower two octaves without deteriorating further the corresponding Q-factors. Figure 2. F-CQT example for a C4 note: (i) each CQT frame is reshuffled on an octave-per-octave basis, folded (ii) using 2-octave wide half-overlapping bands, in order to avoid side effects. A kernel (iii) of size 12 captures 8 out of the first 12 harmonics including the fundamental. In all configurations, we filter out low-energy frames (with average LMS or CQT < -79dB), normalize the data and cut it into fixed-length sequences of 60 frames (~1.4 s). Short sequences are less likely to include the attack of long notes, which is critical information for the network. A sequence length of 60 frames is an empirical compromise between this loss of information and the computing cost of longer sequences. As shown in Figure 2, bins related to the harmonics f2i+3j= f1 2i 3j of a given fundamental f1 remain in its close neighborhood. Therefore, a small convolutional kernel of 3 fifths x 4 octaves10 will capture 8 out of the first 12 harmonics. This is achieved without resorting to a computationally expensive harmonic-CQT [2]. 10 Tradeoff between the number of harmonics captured and the size of the kernel. A higher number of octaves results in blurring the picture with several harmonics on the same bin. 8 We use the Librosa [20] implementation for LMS and CQT. 9 It has been assumed that the lower octave of the cello would be analyzed with enough detail without the fundamental frequency being reported. 4.1.3 JAMS Standard Annotation Our simulation tool exports both the annotation files with the JAMS format [13] and the corresponding audio. IPT classification along the 4 axes is provided with the tag_open namespace. Onset times and note/chord pitches were added when available, using onset (resp. pitch_contour) namespaces. Both audio and annotation files are made available to the MIR community7. Table 1. IPT taxonomy (axes, classes) proposed in this study. This taxonomy is partly hierarchical in the sense that classification along Axes 2-4 is optional and dependent upon classification on Axis 1. When no classification is desirable, the NONE class is used in the training process3. 710 Proceedings of the 20th ISMIR Conference, Delft, Netherlands, November 4-8, 2019 Figure 1. System architecture (incremental configurations A to E). A is directly inspired by [11]. B introduces CQT along with adapted filtering in the convolutional process. The resolution of CQT is increased in C. Recurrent layers process the output of the CNN in D. Finally, in configuration E, we experiment with the joint classification of onsets. Figure 1. System architecture (incremental configurations A to E). A is directly inspired by [11]. B introduces CQT along with adapted filtering in the convolutional process. The resolution of CQT is increased in C. Recurrent layers process the output of the CNN in D. Finally, in configuration E, we experiment with the joint classification of onsets. Figure 1. System architecture (incremental configurations A to E). A is directly inspired by [11]. B introduces CQT along with adapted filtering in the convolutional process. The resolution of CQT is increased in C. Recurrent layers process the output of the CNN in D. Finally, in configuration E, we experiment with the joint classification of onsets. 4.2.2 Folded Constant Q-Transform (F-CQT) 4.2.2 Folded Constant Q-Transform (F-CQT) We introduce F-CQT as a generalization of the pitch spiral method [18] in order to capture efficiently the spectral envelope of a signal. It is obtained by first changing the pitch order of the CQT chromatic bins to match the cycle of fifths. The reshuffled CQT is then folded in 2 dimensions so as to put successive octaves on adjacent lines, in the same manner as the pitch spiral. We introduce F-CQT as a generalization of the pitch spiral method [18] in order to capture efficiently the spectral envelope of a signal. It is obtained by first changing the pitch order of the CQT chromatic bins to match the cycle of fifths. The reshuffled CQT is then folded in 2 dimensions so as to put successive octaves on adjacent lines, in the same manner as the pitch spiral. 11 2D filters are noted: N time frames x M frequency bins. 12 Detailed architecture available here:https://drive.google.com/open? id=1GvS6VQ3iJP6e9MBajEPL0VOXva-iuUmS 5.1 Direct classification (18 classes) 18-class accuracy of configuration A18 with a single 18-class IPT classifier front-end. Results are given for 5 different training/testing subsets and averaged on 3 alternative learning schedules. 4.2.1 Preprocessing Convolution with such a kernel can be seen as a 1D frequency-wise convolution of the usual CQT with a disjoint filter: the F-CQT filter. To capture the same 711 Proceedings of the 20th ISMIR Conference, Delft, Netherlands, November 4-8, 2019 harmonics with a regular 1D-kernel would require a much bigger kernel size (43 parameters instead of 12). harmonics with a regular 1D-kernel would require a much bigger kernel size (43 parameters instead of 12). 4.2.3 CNN Back-end We assume that the nature of our task is somewhat similar to the recognition of instruments in a challenging environment such as polyphonic textures. Therefore, we follow the main characteristics of the CNN architecture presented in [11], while adapting its capacity to our data. 4.2.4 IPT Classifiers (Front-end) In configurations A to C, IPT classifiers are built with a fully-connected (FC) layer of 32 neurons with ReLU activation, followed by another FC layer with softmax activation. The latter comprises as many neurons as there are classes in the corresponding axis [10]. 13 Detailed training parameters available at the same URL as 12. 4.3. Training Configuration The system is trained by minimizing the sum of the cross- entropy loss function of the classifiers. Mini-batch gradient descent is performed with ADAM optimization [10] and exponential decay of the learning rate13. 4.2.3 CNN Back-end 4.2.3 CNN Back-end 5.1 Direct classification (18 classes) We first attempted to build directly a classifier among 18 cello IPTs, chosen simply because they were the most represented IPTs in our data. Our network architecture (called A18) was similar to configuration A, but with a single 18-class classifier front-end. This effort resulted in excellent accuracies when the classifier was tested on homogeneous subsets of our corpus (see Table 2). However, these results collapsed when the minus-1db methodology was implemented. Not only average accuracies dropped below 50% but they were very irregular from one test base to the other. This indicated a poor ability to generalize. The proposed CNN is made of 3 modules which operate at increasing scales. In each module we stack two identical convolutional layers, with batch normalization and Rectified Linear Unit (ReLU) activation. A max- pooling layer and dropout at 0.25 probability are implemented at the output of the module. Following the architecture design of [11], we use as baseline square (3x3) filters11 for all layers. However it has been suggested in recent research [23] that domain-specific filtering could improve CNN performance, especially in the deeper layers. Therefore, we evaluate the use of three separate filters, namely our F-CQT, the baseline (3x3) and a (6x2) filter; the latter is designed to capture longer patterns such as vibrato or trills. The concatenation of 8 feature maps for each filter is used as input of the second module. Max-pooling layers are in charge of downsampling the features while the number of feature maps increases12. The output of the CNN is a 10 step long sequence of 64 maps with a single feature (one step equals six frames ~125ms). Table 2. 18-class accuracy of configuration A18 with a single 18-class IPT classifier front-end. Results are given for 5 different training/testing subsets and averaged on 3 alternative learning schedules. 18-class accuracy CONT 92,9% 49,9% EWQL 94,0% 30,3% ISI 95,2% 51,1% VSL 97,3% 44,0% VO 93,4% 32,4% 94,6% 41,5% Database excluded from training Homogeneous corpus Heterogeneous corpus (minus-1db) Average A18 18-class accuracy CONT 92,9% 49,9% EWQL 94,0% 30,3% ISI 95,2% 51,1% VSL 97,3% 44,0% VO 93,4% 32,4% 94,6% 41,5% Database excluded from training Homogeneous corpus Heterogeneous corpus (minus-1db) Average A18 Table 2. 18-class accuracy of configuration A18 with a single 18-class IPT classifier front-end. Results are given for 5 different training/testing subsets and averaged on 3 alternative learning schedules. Table 2. 5.2 Introduction of our taxonomy Trained and tested on 5 homogeneous subsets of our data, our models still yield in average 18-class accuracies over 90% in all the configurations (see Table 3). In the framework of the minus-1db methodology, they now exhibit much greater resilience. Their 18-class accuracies, averaged on all test bases, are above 50% in most configurations. These accuracies are also less sensitive to the choice of the test base, as shown by Figure 3 in the case of configuration D. In configuration D (resp. E), we replace the first FC layer with a double (resp. single) layer of 32 unidirectional LSTM cells with an input of 64 features per temporal step. In configuration E, following [12], an additional classifier with the same design is jointly trained to locate the attack of the last note of the sequence. Its eleven classes coincide with the 10 steps of the sequence plus one: this additional class is used to categorize sequences of long notes where the attack occurs prior to the beginning of the sequence. The prediction of this onset classifier is concatenated with the original features and used as input to the 4 IPT classifiers. Table 3. Parameter count and 18-class accuracy of configurations A-E, averaged on 5 different homogeneous or heterogeneous training/testing subsets and 3 alternative learning schedules. Configuration 18-class accuracy A (LMS) 184K 93,5% 49,6% B (Low-res CQT) 180K 90,1% 52,2% C (High-res CQT) 181K 91,2% 54,3% D (2 layers LSTM) 150K 91,3% 57,6% E (Joint onset class.) 142K 91,7% 57,6% Parameter count Homogeneous corpus Heterogeneous corpus (minus-1db) A rule-based system computes an 18-class linear classification from the network predictions along the 4 IPT axes. The same rules are applied to the ground truth. An 18-class accuracy is provided as an additional assessment of the performance of the system. On all axes but the interaction position, whatever the resolution, CQT with adapted filtering performs better than 128 bins log-mel-spectrograms (see Table 4). For 712 Proceedings of the 20th ISMIR Conference, Delft, Netherlands, November 4-8, 2019 instance, configuration C achieves better average and 18- class accuracy than A with roughly the same parameter count (Student t-test resp. p=0.046 and 0.004). reactivity, we select sequences in our test database where a change of IPT just occurred on the last note (or chord) of the sequence. We compute the average accuracy of the system as each time frame goes by. 5.2 Introduction of our taxonomy As Figure 4 exhibits, exciter is the axis where the classifier is most reactive, achieving >70% accuracy within 70ms of the attack. Unsurprisingly, it takes much longer for the network to categorize left-hand activity (e.g. vibrato, trills) or discriminate between waveforms, which often requires the note to be released (e.g. staccato). Finally, not only the bow-position axis yields poorer overall accuracy, but it is also the least reactive. Table 4. Minus-1db framework : per-axis and average accuracies in each configuration, for all 5 test databases and 3 alternative learning schedules. Accuracy / Configuration A (LMS) 72,6% 84,2% 66,6% 77,1% 62,7% B (Low-res CQT) 73,6% 85,6% 71,1% 78,1% 59,7% C (High-res CQT) 74,7% 86,0% 72,6% 78,6% 61,7% D (2 layers LSTM) 76,0% 87,0% 73,4% 79,9% 63,5% E (Joint onset class.) 75,6% 86,7% 73,8% 79,5% 62,2% Average on all axes Exciter/ vibrator Left- hand Wave- form Int. Position Table 4. Minus-1db framework : per-axis and average accuracies in each configuration, for all 5 test databases and 3 alternative learning schedules. Table 4. Minus-1db framework : per-axis and average accuracies in each configuration, for all 5 test databases and 3 alternative learning schedules. Table 4. Minus-1db framework : per-axis and average accuracies in each configuration, for all 5 test databases and 3 alternative learning schedules. When the attack of the note gets out of the 60 frame- wide analysis window (see Figure 4, right side), the system has to categorize IPTs without information about the attack. However, its performance is not harmed as one could expect. Indeed, the actual length of the note provides information about the technique used. Longer notes tend, for instance, to be produced with the bow and to be vibrated or trilled. We hypothesized that increasing the resolution of the CQT beyond the tempered scale would improve system accuracy for such IPTs as vibrato or glissando. Our experiment confirms that configuration C yields better average accuracies than configuration B on the left-hand axis (p=0.023). This results in better 18-class accuracy (p=0.045). Figure 4. Average accuracy for sequences with an IPT change vs. time lapsed between change and prediction (10 frames~0,23s). Test base: ISI, configuration D. Configuration D with a 2-layer LSTM front-end achieves better average and 18-class accuracy than C with a fully-connected front-end (resp. p=0.039 and 0.02). 6. CONCLUSIONS In this article, we have extended state-of-the-art methods regarding instrument recognition to the real-time classification of IPTs from cello solo recordings. First experiments in the framework of the minus-1db methodology show a good resilience of models which are based on a meaningful taxonomy and process an adapted CQT through the proper combination of deep CNN and LSTM layers. Our methodology, from the realization of the databases to the architecture and training of the networks, can be extended with little effort to other string instruments. Other orchestral instruments first require an adaptation of the IPT taxonomy, which could be grounded on the same principles as ours [3,8,26]. Figure 3. Accuracy per base and axis (configuration D). In all tested configurations, the best accuracies are observed on the exciter/vibrator axis, while the worst- performing axis is interaction position. This statement is valid across most test databases, as seen in Figure 3 for configuration D. Bow position classification is likely to be a very difficult task even for a human expert. In the medium register, the choice of the string has a strong impact on the timbre, which makes the bow position harder to guess. In the higher register, sul tasto (with stronger emphasis or lower rank harmonics) and ordinario may be hard to distinguish. Finally, this is the axis where variability due to the instrument model is likely to be most perceivable. To further assess the ability of our models to generalize, a database of contemporary cello solo recordings has been built, some of which will be annotated with the JAMS standard and used for testing. Finally, several unsupervised adaptation techniques, such as Maximum Classifier Discrepancy [25] or back- propagation through Gradient Reversal Layer [9], will also be tested in this environment. 5.2 Introduction of our taxonomy Configuration E with joint onset classification but single-layer LSTMs also achieves better 18-class accuracy than C (p=0.016) with even lower parameter count. In both configurations E and D, all axes exhibit average improved performance compared to C but detailed results show discrepancies from one test base to the other. Figure 3. Accuracy per base and axis (configuration D). Figure 4. Average accuracy for sequences with an IPT change vs. time lapsed between change and prediction (10 frames~0,23s). Test base: ISI, configuration D. 7. REFERENCES [1] D.G. Bhalke, C.B. Rama Rao, and D.S. Bormane: “Automatic Musical Instrument Classification using Fractional Fourier Transform based-MFCC Features and Counter Propagation Neural Network”, Journal Int. Inform. Syst., Vol. 46.3, pp. 425–446, 2016. [15] A. Livshin: “Automatic Musical Instrument Recognition and Related Topics. Acoustics”, PhD Thesis, Université Pierre et Marie Curie, Paris VI, 2007. [2] R.M. Bittner, B. McFee, J. Salamon, P. Li, J.P. Bello: “Deep Salience Representations for f0 Estimation in Polyphonic Music”, Proc. International Society for Music Information Retrieval (ISMIR), 2017. [16] D.M. Lloyd: “A Classical Clarinetists Guide to Klezmer Music”, PhD Thesis, Ohio State University, pp. 37-43, 2017. [17] V. Lostanlen, J. Andén, and M. Lagrange: “Extended Playing Techniques: the Next Milestone in Musical Instrument Recognition”. 5th International Workshop on Digital Libraries for Musicology, Paris, France, 2018. [3] C. Cadoz: “Instrumental gesture and musical composition”, Proc. Int. Computer Music Conf., 1988. [4] R. Caruana: “Multitask Learning”. Machine Learning, Vol. 28, 1997. [18] V. Lostanlen, C.-E. Cella: “Deep Convolutional Networks on the Pitch Spiral for Music Instrument Recognition”, Proc. ISMIR, 2016. [5] Y.-P. Chen, L. Su, and Y.-H. Yang: “Electric Guitar Playing Technique Detection in Real-World Recording Based on F0 Sequence Pattern Recognition”, Proc. ISMIR, 2015. [19] M.A. Loureiro, H. Bastos de Paula and H.C. Yehia: “Timbre Classification Of A Single Musical Instrument”. Proc. ISMIR, 2004. [6] K. Choi, Fazekas G., K. Cho and M. Sandler: “A Tutorial on Deep Learning for Music Information Retrieval”, arXiv:1709.04396v2, 2018. [20] B. McFee, C. Raffel, D. Liang, D.P.W. Ellis, M. McVicar, E. Battenberg, and O. Nieto: “Librosa: Audio and Music Signal Analysis in Python”, Proc. of the 14th Python in Science Conf. (SCIPY), 2015. [7] S. Essid: “Classification Automatique des Signaux Audio-fréquences: Reconnaissance des Instruments de Musique”, PhD Thesis, Université Pierre et Marie Curie, Paris, 2005. [21] K. Patil, M. Elhilali, “Biomimetic Spectro-Temporal Features for Music Instrument Recognition in Isolated Notes and Solo phrases”, EURASIP J. Audio Speech Music Process, 2015. [8] F.-X. Féron, B. Carat: “Catégorisation des Actions Instrumentales dans Pression pour un(e) Violoncelliste de Lachenmann”, Manifeste 2014, Conférence “Composer (avec) le geste”, https://medias.ircam.fr/x23f46d, 2014. [22] C. Perret: “Une Rencontre entre Musique Savante et Jazz, Musique de Tradition Orale et les œuvres aux Accents Jazzistiques d’Érik Satie, Darius Milhaud, Igor Stravinsky et Maurice Ravel”, Volume !, Vol. 2:1, pp. 43-67, 2003. [9] Y. Ganin, E. Ustinova, H. Ajakan, P. Germain, H. Larochelle, F. Laviolette, M. Marchand and V. 5.3 Reactivity study Our real-time classifier has to be as reactive as possible to sudden changes in the play of the cellist. To assess that 713 Proceedings of the 20th ISMIR Conference, Delft, Netherlands, November 4-8, 2019 [14] S. Kostka: Materials and Techniques of Post Tonal Music, Taylor&Francis, pp. 216-223, 2016. [14] S. Kostka: Materials and Techniques of Post Tonal Music, Taylor&Francis, pp. 216-223, 2016. 7. REFERENCES Lempitsky: “Domain-Adversarial Training of Neural Networks”, Journal of Machine Learning Research, Vol. 17, p. 1-35, 2016. [23] J. Pons, O. Slizovskaia, R. Gong, E. Gómez and X. Serra, “Timbre Analysis of Music Audio Signals with Convolutional Neural Networks”, 25th European Signal Processing Conference (EUSIPCO), 2017. [10] I. Goodfellow, Y. Bengio, and A. Courville: Deep Learning, MIT Press, Cambridge MA, USA, 2017. [24] R. Rowe: Interactive Music Systems: Machine Listening and Composing, Chapter 5, MIT Press, Cambridge MA, USA, 1993. [11] Y. Han, J. Kim, and K. Lee: “Deep Convolutional Neural Networks for Predominant Instrument Recognition in Polyphonic Music”. IEEE/ACM Transactions on Audio, Speech, and Language Processing, Vol. 25, no. 1, pp. 208–221, 2017. [25] K. Saito, K. Watanabe, Y. Ushiku, T. Harada: “Maximum Classifier Discrepancy for Unsupervised Domain Adaptation”, arXiv:1712.02560, 2018. [26] P. Schaeffer: Traité des objets musicaux, Editions du Seuil, pp. 52-53, 1966. [12] C. Hawthorne, E. Elsen, J. Song, A. Roberts, I. Simon, C. Raffel, J. Engel, S. Oore, D. Eck: “Onsets and Frames: Dual-Objective Piano Transcription”, Proc. ISMIR, 2018. [27] A.R. Tindale, A. Kapur, G. Tzanetakis, and I. Fujinaga: “Retrieval of Percussion Gestures using Timbre Classification Techniques”, Proc. ISMIR, 2004. [13] E.J. Humphrey, J. Salamon, O. Nieto, J. Forsyth, R.M. Bittner, J.P. Bello: “JAMS : a JSON Annotated Music Specification for Reproducible MIR Research”, Proc. ISMIR, 2014. [28] J. Wiederker: The contemporary Cello, pp. 59-61, Ed. L'Oiseau d'or, Sainte-Geneviève-des-Bois, France. 714
https://openalex.org/W2019384036	https://zenodo.org/records/2368317/files/article.pdf	German	null	Über die Emission von Elektronen und positiven Ionen an glühenden Drähten	Annalen der Physik	1,917	public-domain	12,275	1) Tag der miindlichen Priifung 30. Juli 1914. Die Promotion uncl damit die Veriiffentlichung der Arbeit wurde durch den Heeresdienst dea Verfasaera so lange hhausgeachoben. 2) J. J. Thomson, Phil. Mag. (5) 48. p. 54'7. 1899. 3. 8ber die Em.lse%on uon EZektjomwn und poe4t.lven Ionm an ylWbenden Dr U k t m ; von Walter Hattemann. [Auszug nus der Berliner Dissertation. ')I [Auszug nus der Berliner Dissertation. ')I Die vorliegende Arbeit sol1 die bisherigen Untersuchungen uber die Emission von Elektronen (I. Teil) und positiven Ionen (11: Teil) nach zwei Riohtungen hin erweitern. In dem emten Teil werden Beobachtungen wiedergegeben uber die Aussendung von Elektronen gluhender Metalldriihte bei hiiheren Tempe- raturen, und der zweite Teil der -4rbeit untersucht die che- rnische Natur der positiven Ionen, die von verschiedenen DrlShten ausgesandt werden. Es werden hierbei durch Be- stimmung des Verhaltnisses e/m, der Ladung zur Masse, ver- schiedene Arten von positiven Ionen nachgewiesen, wahrend bei den bisherigen Beobachtungen immer nur eine Gattung gefunden wurde. 81 6 81 6 1) Tag der miindlichen Priifung 30. Juli 1914. Die Promotion uncl damit die Veriiffentlichung der Arbeit wurde durch den Heeresdienst dea Verfasaera so lange hhausgeachoben. g g 2) J. J. Thomson, Phil. Mag. (5) 48. p. 54'7. 1899. 1) 0. W. Riaherdson, Phil. Trans. 901. p. 616. 1903. 2) R, die Gaskonstaute fiir ein Elektron, ist identiech mit der Konstenten x des Plenckeohen StmhlungageeetEeS, d. h. sie ist gleich dem Produkt au8 w . R, wo R die absolute Gaakonstmta, tmd w das Verhiiltnis der Moleahl zur Eiektronentahl ist (Planak, Vorler. iiber Wiirmesfnrhlung. p. 147. 1906). I. Teil. Zu Beginn des ersten Teiles sollen nun kurz die Tab sachen rekapituliert werden, auf die sich diese ganzen Unter- suchungen s tu tzen . Gliiht man ein Metall, dem man am besten die Foriii eines Drahtes gibt, durch den elektrischen Strom im Innern eines Metallzylinders und legt zwischen Draht und Zylinder ein elektrisches Feld, indem man den Zylinder positir auf- ladet, so tritt ein elektrischer Strom auf von solcher Rich- tung, als ob der gluhende Draht negative Ladungen aus- sendet. Thomson2) hat gefunden, daB diese negativen La- dungen aus Elektronen bestehen. Mit wachsender Potential- differem nimmt auch der Elektronenstrom zu und erreicht Die Emission von Ekktronen und positiven h e n usto. 817 schlieSlich einen Maximalwert, den Sattigungsstrom, u-elcher dem Punkte entsprioht, bei dem in der Sekunde ebensoviel Elektronen duroh den Strom befordert als in der gleiohen Zeit neu gebildet werden. 1st s der SIttigungastrom, N die Anzahl abgegebener Elektronen pro Oberfliicheneinheit und E deren Ladung, so ist also S = & . N . S = & . N . (1) Inwiefern nun N von der Temperatur abhangt, hat Richard- son1) in einer von ihm aufgestellten Theorie gezeigt. Mit der Annahme, daS die Elektronen im Innern eines Korpers sioh wie die Molekiile eines Gases verhalten und dem Boltz- ma nn- Max w e llaohen Oesohwindigkeitsvertgesetze ge- horohen, fand er die Ab-keit der Zahl N von der Tem- peratur schlieSlich dergestellt durch die Oleichung (2) (2) (2) Hierin bedeut'eii : N = die Aneahl der pro Oberfliicheneinheit abgagebenen Elek- tronen, N = die Aneahl der pro Oberfliicheneinheit abgagebenen Elek- n = die Aneehl der Elektronen in der Volumeneinheit, R = die Gaakomtmte fiir ein Elektron'), T = die absolute Temperatur, m = die &se einea Elektmns, e = die Basis der natiirliahen Logarithmen, und 0 = die Arbeit, die ein Elektron beim D u r c w durch die tronen, Oberfbhe leisten mu& n = die Aneehl der Elektronen in der Volumeneinheit, , R = die Gaakomtmte fiir ein Elektron'), T = die absolute Temperatur, p m = die &se einea Elektmns, , e = die Basis der natiirliahen Logarithmen, und 0 = die Arbeit, die ein Elektron beim D u r c w durch die Oberfbhe leisten mu& Die Gleichung (9) liefert dam zusammen mit der Gleichung (1) den Stittigungestrom der Elektronen fiir die Oberfliichen- einheit 13) 13) Die Beobachtungen, die bis jetzt gemaoht worden sind, habep nun stets ergeben, daS diese Formel innerhalb der Fehler- 818 W. Hiittemann. grenzen den Sattigungsstrom mit befriedigender Genauigkeit wiedergibt. Demnach kann die Gleichung tiir den Sattigungs- strom auch dargestellt werden durch x = ~ f F . e T , B - _ (4) x = ~ f F . e T , B - _ (4) wo A und B zwei fiir den betreffenden Korper charakte- ristische Konstanten sind, die im Vergleich mit (3) die Werte ha ben Urn nun festzustellen, ob tatsachlich beobachtete Werte des Siittigungsstromes bei verschiedenen Temperatwen mit der Gleichung fiir den Siittigungsstrom fibereinstimen, hat Ri- chardson die Gleichung logarithmiert und sie dadurch in eine lineare Form gebracht. 1 B logs=lOgA+glogZ'- --.loge. T Es sei (6) a = logA und b = B-loge. (6) Dann erhiilt man die Gleichung Y = u - ~ . x , d. h. also die Gleichung einer geraden Linie. S = & . N . Nach Richard- sons Theorie miissen also die am Temperatur und Slittigungs- strom berechneten Werte von z und y auf einer geraden Linie liegen, wenn man sie in ein rechtwinkliges Koordinaten- system eintragt, wobei die z zu Abszissen und die y zu Ordi- naten zu wahlen sind. Aus der Neigung der geraden Linie gegen die Abszissenachse, also aus dem Werte von b, kann man sofort feststellen, welche Arbeit ein Elektron beim Durchdringen der Oberflhhe eines besthunten gegliihten Korpers zu leisten hat, und damit zugleich findet man den Potentialsprung 6 in Volt an der Oberfhche des Korpers, da beide GroSen durch die Gleichung verbunden sind (6) @ = e . d p l . Konstruiert man fiir versohiedene Metalle diese gersden Linien, so sieht man sofort, welchem Draht der groSere Potential- Die Einission von E l e k t r m und positiven Ionen WE. 819 sprung entspricht. Ist der Potentialsprung 0, so ist die Ge- rade eine Parallele zur Abezisaenachse, ist er unendlich p S , 80 lPuft die Gerade der Ordinotenachse perallel. p Um nun ziu sehen, wie sich Metalle bei hiiheren Tem- pepaturen als den bis jetzt untenuchten in h u g auf die Theorie von Richardson verbalten, habe ioh Wolfram und Tsntal, die ja einen eehr hohen S,chmelzpunkt haben, auf den Effekt der Ebktronenemiseion bis ziu den h6ehstzuliissigen Temperaturen hin untersncht und uber die Frage entechieden, wie sie sich zur Richardsonschen Theorie verhalten. Vo..llrah~nerdnanp. Zu den Messungen wurde das in der Fig. 1 angegebene GlaRrohr R verwendet. Im Innern desselben befand sich Fig. 1. der zu untersuchende Draht, weloher bei Wolfram 7,3 cm lang war und einen Dnrohmesser von 0,s mm hat&, wa- rend bei Tantal die LHnge 7,6 cm und der Durohmesser 0,a mm betragen. Der Vemchsdraht, war an swei starken Nickelglenmen Kl und KI befmtigt, die ihrerseits wieder an starken Nickelstiiben angeschraubt waren. Unten trugen diese starke Plstindrghte, die direkt in die Glaaansiltae des Fig. 1. der zu untersuchende Draht, weloher bei Wolfram 7,3 cm lang war und einen Dnrohmesser von 0,s mm hat&, wa- rend bei Tantal die LHnge 7,6 cm und der Durohmesser 0,a mm betragen. Der Vemchsdraht, war an swei starken Nickelglenmen Kl und KI befmtigt, die ihrerseits wieder an starken Nickelstiiben angeschraubt waren. Unten trugen diese starke Plstindrghte, die direkt in die Glaaansiltae des der zu untersuchende Draht, weloher bei Wolfram 7,3 cm lang war und einen Dnrohmesser von 0,s mm hat&, wa- rend bei Tantal die LHnge 7,6 cm und der Durohmesser 0,a mm betragen. Der Vemchsdraht, war an swei starken Nickelglenmen Kl und KI befmtigt, die ihrerseits wieder an starken Nickelstiiben angeschraubt waren. Unten trugen diese starke Plstindrghte, die direkt in die Glaaansiltae des W. Hiittemann. 820 Schliffes S eingeschmolzen waren. Den Draht umgab ein Nickelzylinder, der an der Seite geschlitzt war, um erstens den Draht mit seinen Zuleitungen bequem in den Zylinder hineinzubringen und zweitens den Draht gut beobachten zu konnen. Auch der Zylinder war seinerseits mit seinem Nickel- ansatz, der wiederum am Ende in Platin auslief, in das eine seitliche Ansatzrohr des Versuchsapparates eingeschmolzen. Das Rohr wurde evakuiert durch eine Gaedepumpe, die es er- moglichte, den Druck in dem Versuchsrohr bis zu unmeJ3bar kleinen Werten herabzusetzen. Der Druck wurde gemessen mit einem MacLeodmanometer , das bis auf l/lm Millimeter geeicht war. Der Draht wurde geglat durch eine Batterie von sechs groBen Akkumulatoren H, die durch einen Regulier- widerstand R direkt an die Zuleitungen des Versuchsdrahtes geschlossen waren. Der negative Pol des Heizdrahtes lag stets an Erde. Zwischen Draht und Zylinder lag eine Spannung, die durch NebenschlhB von der Akkumulatorenbatterie A gewonnen wurde; in demselben Stromkreis lag das Spiegel- galvanometer G, das zur Messung der Siittigungsstrome be- stimmt war. Die Batterie A bestand aus 45 kleinen Akku- mnlatoren, die durch einen grol3en Widerstand von 700 Ohm kurzgeschlossen waren. Vo..llrah~nerdnanp. Von diesem Widerstande konnte eine beliebige Potentialdifferens abgeschaltet und zwischen Draht und Zylinder gelegt werden. Durch Regdierung der Potential- differenz von 5 zu 5 Volt konnten verschiedene Punkte der Siittigungsstromkurven bestimmt werden. Schliffes S eingeschmolzen waren. Den Draht umgab ein Nickelzylinder, der an der Seite geschlitzt war, um erstens den Draht mit seinen Zuleitungen bequem in den Zylinder hineinzubringen und zweitens den Draht gut beobachten zu konnen. Auch der Zylinder war seinerseits mit seinem Nickel- ansatz, der wiederum am Ende in Platin auslief, in das eine seitliche Ansatzrohr des Versuchsapparates eingeschmolzen. Das Rohr wurde evakuiert durch eine Gaedepumpe, die es er- moglichte, den Druck in dem Versuchsrohr bis zu unmeJ3bar kleinen Werten herabzusetzen. Der Druck wurde gemessen mit einem MacLeodmanometer , das bis auf l/lm Millimeter geeicht war. Der Draht wurde geglat durch eine Batterie von sechs groBen Akkumulatoren H, die durch einen Regulier- widerstand R direkt an die Zuleitungen des Versuchsdrahtes geschlossen waren. Der negative Pol des Heizdrahtes lag stets an Erde. Zwischen Draht und Zylinder lag eine Spannung, die durch NebenschlhB von der Akkumulatorenbatterie A gewonnen wurde; in demselben Stromkreis lag das Spiegel- galvanometer G, das zur Messung der Siittigungsstrome be- stimmt war. Die Batterie A bestand aus 45 kleinen Akku- mnlatoren, die durch einen grol3en Widerstand von 700 Ohm kurzgeschlossen waren. Von diesem Widerstande konnte eine beliebige Potentialdifferens abgeschaltet und zwischen Draht und Zylinder gelegt werden. Durch Regdierung der Potential- differenz von 5 zu 5 Volt konnten verschiedene Punkte der Siittigungsstromkurven bestimmt werden. 1) Dr. R. Hase, Hannover, Beschreibung des Wannerpyrorneters. Idearungen. Um konstante Verhgltnisse zu bekommen, wurde der Versuchsdraht erst mehrere Stunden lmg im Vakuum aus- gegliiht. Dann ging nach einiger Zeit die Wolfram- bzw. Tantaloxydschicht von dem Draht herunter und er bekam einen vollkommen metallischen Glanz. Jetzt wurde er noch einige Stunden gegliiht und war schlieJ3lich so weit entgast., daS mit den Messungen begonnen werden konnte. Zur Temperaturbestimmung des Drahtes wurde das op- tische Pyrometer von Wanner l) benutzt, welches gestattete, Temperaturen von 1000-20000 C bequem zu messen. Die Emission vmt Elelctranen und positiven Ione?t mu'. 82 821 Der Gang einer Messung ging folgendermaBen vor sich. Nachdem der Draht genugend ausgegliiht war und auch von den anderen im Innern des Versuchsrohres befindlichen Nickel- teilen kein Gas mehr abgegeben wurde, d. h. wenn das Mac Leodmanometer keinen meBbaren Druck mehr anzeigte, wurde zunachst die Temperatur des Drahtes bestimmt. Dann wurde zwischen Draht und Zylinder ein elektrisches Feld gelegt, wobei der Draht negativ war, und zwar wurde die Potentialdifferenz von 5 zu 5 Volt oder auch von 10 zu 10 Volt bis zu 90 Volt gesteigert und die Sattigungsstromkurven auf- gnommen. Bei einer Spannung von SO Volt war stets Siittigung vorhanden, und bis zu Temperaturen von uber 2000 O C konnten sehr gute Sattigungsstromkurven aufgenommen werden. Wurden d a m jedoch die Temperaturen noch hoher, so trat Zerstiiubung des Drahtes ein. Die Oberfliiche und die Dicke des Drahtes iinderten sich also, so daS weder konstante Strome erreicht wurden, noch die Temperatur bestimmt werden konnte. Nach- dem bei einer Temperatur der Siittigungastrom gemessen worden war, wurde nochmals die Temperatur des Drahtes gemessen und aus beiden Messungen dann das Mittel genommen. Die Empfindlichkeit des benutzten Spiegelgalvanometers zur Messung der Stromkurven betrug pro Zentimeter Ausschlag des Gal- vanometers 5,5 . 10-0 Amp. Durch Parallelschalten eines ge- eichten Stopselrheostaten von 10000 Ohm zum Galvanometer konnte durch passende Stopselung die Empfindlichkeit herab- gesetzt werden. Um gute Siittigungsstromkurven zu erhalten, waren siimtliehe Apparate, die ich benutzte, durch Paraffin sehr gut isoliert und die Drahtverbindungen waren kurz und dick. Ergebniase der Measungen. Die ausgefuhrten Versuche ergaben Siit tigungsstrom- messungen bei Wolfram bis 2069O C und bei Tantal bis 2097O C, d. h. also den Zusammenhang zwischen der Poten- tialdifferenz V zwischen Draht und Zylinder und der Strom- stkke J und gleichzeitig die Abhangigkeit des Sattigungs- stromes s, mithin auch der Amah1 N der abgegebenen Elek- tronen, von der Temperatur t. Die Sattigungsstromkurven =den in der vorher erwiihnten Weise aufgenommen. Bei einer Potentialdifferem von SO Volt misohen Draht und Zylinder wurde regelmiiBig Siittigung des Stromes er- W. H4ttema.m. W. H4ttema.m. a22 reicht, und zwar hielt diese Stittigung an, wenn ich die Potentialdifferenz bis zu der von mir bei den Versuchen er- reichbaren Potentialdifferenz von 90 Volt steigerte. Nur bei B sehr hohen Temperaturen setzte bei einer Potentialdifferenz von ca. 50 Volt aufwlirts eine erhebliahe Steigerung des Stromes ein, die wohl sohon mit anfangender Zerstliubung des Drahtes sehr hohen Temperaturen setzte bei einer Potentialdifferenz von ca. 50 Volt aufwlirts eine erhebliahe Steigerung des Stromes ein, die wohl sohon mit anfangender Zerstliubung des Drahtes Die Emission ~xm Ekktrvnen usld positiven Ionen ww. Die Emission ~xm Ekktrvnen usld positiven Ionen ww. 098 098 und StoSionisotion ~usammenhing, da infolge der mhr grobn Erwiirmnng des Versnohsapparates die &wsbgabe in seinem b e m groSer war ale bei geringeren Temperaturen. Figg. 2 Erwiirmnng des Versnohsapparates die &wsbgabe in seinem b e m groSer war ale bei geringeren Temperaturen. Figg. 2 e! gi 6 5 0 8 E d 9 8 8 d I-! !-I OoOz 0081 0091 OW1 00.3 0001oo(i00800L 009004'00~00t'OOz OOT 0 =dmv UI =wW=@nss U 3 a 3 5 s 5 U E 3 0 0 0 2 0002 0081 0091 ow1 0021 ooo1oo8oo8oo~oo~m~moozoor o und 3 geben die graphkche Darstellung der aufgenommenen Stittigmgsstromkurven. Zwiachen l l O O o C und 210Oo C wurden bei Wolfram und Tantal noch eine game Reihe von anderen Punkten gewlihlt -0lm- e! gi 6 5 0 8 E d 9 8 8 d I-! !-I OoOz 0081 0091 OW1 00.3 0001oo(i00800L 009004'00~00t'OOz OOT 0 =dmv UI =wW=@nss 8 d I-! !-I OoOz 0081 0091 OW1 00.3 0001oo(i00800L 009004'00~00t'OOz OOT 0 =dmv UI =wW=@nss U 3 a 3 5 s 5 U E 3 0 0 0 2 0002 0081 0091 ow1 0021 ooo1oo8oo8oo~oo~m~moozoor o -0lm- und 3 geben die graphkche Darstellung der aufgenommenen Stittigmgsstromkurven. g g Zwiachen l l O O o C und 210Oo C wurden bei Wolfram und Tantal noch eine game Reihe von anderen Punkten gewlihlt W. Hiittemann. t(24 und der Siittigungsstrom und die Temperatur bestimmt. Fiir diese Punkte jedoch nahm ich nicht mehr die ganzen Strom- kurven auf, sondern beobachtete immer nur drei Punkte bei 30, 40 und 70 Volt Potentialdifferem. hderte sich fiir diese drei verschiedenen Punkte der Ausschlag des Galvanometers y = logJ - +log T y = l o g J - ~ l o g T t T 1 T -+ x:..-.104 1 -+ = - .lo4 Hg. 6. W. H4ttema.m. T 1 T -+ x:..-.104 1 -+ = - .lo4 Hg. 6. T nicht, so war eben Sattigung des Stromes vorhanden. Die graphische Darstellung der Figg. 4 und 6 seigt die Abhiingig- keit des Siittigungsstromes von der Temperatur, wo als Ab- ssissen die Temperaturen in Celsiusgraden aufgetragen sind und als Ordinaten die Stromstarken in Ampere. Hg. 6. nicht, so war eben Sattigung des Stromes vorhanden. Die graphische Darstellung der Figg. 4 und 6 seigt die Abhiingig- keit des Siittigungsstromes von der Temperatur, wo als Ab- ssissen die Temperaturen in Celsiusgraden aufgetragen sind und als Ordinaten die Stromstarken in Ampere. nicht, so war eben Sattigung des Stromes vorhanden. Die graphische Darstellung der Figg. 4 und 6 seigt die Abhiingig- keit des Siittigungsstromes von der Temperatur, wo als Ab- ssissen die Temperaturen in Celsiusgraden aufgetragen sind und als Ordinaten die Stromstarken in Ampere. Die Emission 2-'m Elektronen urul positiceti Ionen usw. 8.26 8.26 Die Kurven geben ein Bild von der Zunahme der ab- gegebenen Elektronen von tieferer zu hoherer Temperatur. Man sieht, daI3 bei Vergleichung von mei identischen Tem- peraturen die Anzahl der abgegebenen Elektronen bei Wolf- ram weit hoher ist als bei Tantal. Sollen nun diem Mes- sungen die Richardsonsche Theorie bestiitigen, so miissen die sich ergebenden Werte von x und y, die aus der Tempe- ratur und dem SBttigungsstrom berechnet werden, so be- schaften sein, daI3 sie, als Abszissen und Ordinaten in ein rechtwinkliges Koordinatensystem eingetragen, eine gerade Linie ergeben. Fig. 6 zeigt das Resultat der aus den Beob- achtungen gef undenen Werte. g g Wie man aus der Fig. 6 ersieht, liegen die zugehorigen Werte von z und y also tiitsachlich auf einer geraden Linie; die Formel von Richardson ist somit auch bis zu sehr hohen Temperaturen von uber 2000O C fiir Wolfram und Tantal bestiitigt. Aus der Neigung der geraden Linie kann man nun den Potentialsprung d q , und die Arbeit 0, die ein Elektron beim Durchdringen der Oberflache leistet, berechnen, wobei @ und 8 ip durch die Beziehung der Gleichung (6) miteinander vrrbunden sind. Es ergibt sich nach meinen Messuhgen fiir den Potentialsprung und 6 rp wofiun = 5,O Volt 6 ~ T ~ t . 1 = 4,3 Volt. Diese Zshlen, in elektrostatischen Einheiten ausgedriickt und nit dem Elementarquantum e, welches zu 4,69. W. H4ttema.m. 10-lo ge- nommen ist, multipliziert, ergeben die Arbeiten 0 in Erg Diese Zshlen, in elektrostatischen Einheiten ausgedriickt und nit dem Elementarquantum e, welches zu 4,69. 10-lo ge- nommen ist, multipliziert, ergeben die Arbeiten 0 in Erg @W,,l- = 7,82 lWla Erg, 0~~~t.1 = 6,72 lo-" Erg. Trotzdem nun die Potentialspriinge von Wolfram und Tantal unter sich eine nicht sehr groI3e Verschiedenheit aufweisen, so gehen doch die Anzahl der abgegebenen Elektronen der beiden Metalle bei iiquivalenten Temperaturen sehr stark auseinander. Die Anzahl der abgegebenen Elektronen ist bei Wolfram weit hoher als bei Tantal. dnnalen der Phyeik. 1V. Folge. 62. Resultate. 1. Wolfram und Tantal zeigen im gegliihten Zustande Elektronenemissionen, und zwar bestiitigt sich hinsichtlich der Abgabe der Snzahl der Elektronen die Richardson- ache Formel in allen ihren Teilen bis zu den untersuchten 1. Wolfram und Tantal zeigen im gegliihten Zustande Elektronenemissionen, und zwar bestiitigt sich hinsichtlich der Abgabe der Snzahl der Elektronen die Richardson- ache Formel in allen ihren Teilen bis zu den untersuchten 53 53 dnnalen der Phyeik. 1V. Folge. 62. 826 W. Hiittemann. Temperaturen von 2069O C bei Wolfram und 2097O C bei Tantal. 2. Die Untersuchungen bei noch hoheren Temperaturen, eventuell bis zum Schmelzpunkt, auf Elektronenemission hin, sind bei den genannten Metallen unmoglich, da regelmliBig bei ca. 2100O C eine derartige Zerstaubung der Drlihte ein- tritt, daB die Oberfllichen derselben sich fortwahrend ver- iindern und dann von konstanten Verhliltnissen in dem Ver- suchsrohr nicht mehr die Rede sein kann. 2. Die Untersuchungen bei noch hoheren Temperaturen, eventuell bis zum Schmelzpunkt, auf Elektronenemission hin, sind bei den genannten Metallen unmoglich, da regelmliBig bei ca. 2100O C eine derartige Zerstaubung der Drlihte ein- tritt, daB die Oberfllichen derselben sich fortwahrend ver- iindern und dann von konstanten Verhliltnissen in dem Ver- suchsrohr nicht mehr die Rede sein kann. 3. Die Zahl der abgegebenen Elektronen erweist sich bei iiquivalenten Temperaturen durchweg bei Wolfram groBer als bei Tantal. 4. Die Potentialspriinge werden fiir Woliram zu 5,O und fiir Tantal zu 4,s Volt gefunden, und die Arbeit, die ein Elektron beim Entweichen leisten muB, zu 7,82. 10-l2 hzw. 6,72. 10-l2 Erg. , g 5. Als sehr brauchbares Metal1 fiir die Stromzuleitungen und fiir den Auffangzylinder erweist sich bei derartig hohen Tpnperaturen Nickel. II. Teil. Bei den bisherigen Versuchen war stets der Draht im Verhaltnis zu dem Zylinder negativ aufgeladen, und beim Gliihen der untersuchten Metalle trat ein Elektronenstrom auf, der vom Draht zum Zylinder ging. Lade ich jetzt den Versuchsdraht positiv auf im Verhliltnis zum Auffangzylinder und glihe ihn, so tritt ebenfalls ein Strom auf, der vom Draht zum Zylinder geht, der aber aus positiv geladenen Ionen be- steht. Diese positiven Ionen, welche kleinste geladene Massen- teilchen sind, die infolge des Gldhem von dem Drahte ab- geschleudert werden, haben eiue vie1 kleinere mittlere Ge- schwindigkeit als die Elektronen. Beim Anlegen eines Feldes verlassen die Ionen den Draht und wandern mit bestimmter Geschwindigkeit zum Auffangzylinder, so den positiven Ionen- strom bildend. Was eigentlich aus dem Draht beim Gluhen herauskommt - und das sol1 der zweite Teil der Arbeit be- handeln - kann man daher. feststellen, wenn sich bestimme~ laat, welchen Elementen die Massenteilchen des positiven Ionenstromes angehoren. Dies kann erreicht werden, wenn man den Strom der positiven Ionen durch ein Magnetfeld Die Emission von Elektronerr und positivm Ionen usw. Die Emission von Elektronerr und positivm Ionen usw. 827 ablenkt; dann werden am stiirksten die Elemente mit kleinem Atomgewicht und am schwiichsten diejenigen mit groSem Atomgewicht abgelenkt, d. h. jeder Ionenart entspricht bei &em gewissen Magnetfeld ein ganz bestimmter Kriimmungs- radius, wenn die Ionen auI3erdem alle ein und dasselbe elek- trische Feld durchlaufen haben. Aus dem Kriimmungsradius, dem angelegten Magnetfeld und dem elektrischen Feld zwischen Draht und Zylinder 1aBt sich der Wert von elm, d. i. das Verhaltnis von Ladung zur Masse, bestimmen, der fiir jedes Element einen charakteristischen Wert hat. Nach allen bis- herigen Venuchen konnte jedoch noch nicht gesagt werden, was fiir Atome denn nun den positiven Ionenstrom wirklich ausmachen; es wluden manchmal fur elm Wasserstoffatome gefunden, manchmal Metallatome, dann wieder Kohlenoxyd und such sonst Zahlen fiir das Verhaltnis e/m, die sich nicht auf eine bekannte Molekul- oder Atomgettung zuruckfiihren liehn. Da wurde ich voranlaSt, ausgehend von einer Ar- beit von Richardson') im Jahre 1908 uber Bestimmungen von elm fiir positive Ionen, nach einer von A. Wehnelt erdachten Methode die Bestimmung von elm fiir positive Ionen auszufiihren, und zwar ist dies die Methode der Dreh- zylinder. Ich ging von der Uberlegung aus, daS bei einem gliihenden Drahte - Platin z. B. II. Teil. - Wasserstoff entweicht und hiernache ein entsprechender Wert fiir elm gefunden werden muBte,.und daS ich nicht nur einen einzigen Wert von elm fiir einen bestimmten Draht bekommen mute, son- dern mehrere, wobei ich annahm, daS der positive Ionenstrom aus verschiedenen geladenen Atomen oder Molekiilen von Elementen bestand. Diem ffberlegungen haben sich nach den Messungen mit der Methode der Drehzylinder vollauf bestatigt. b h habe fiinf Korper - Platin, Tantal, Wolfram, Aluminium- phosphat und Calciumoxyd - untersucht und uberall bei der Bestimmung von elm Werte gefunden, die meine An- nahme vollkommen bestatigten. 1) 0. W. Richardson, Phil. Mag. (VI) 16. p. 740. 1908. 55 Beeohreibung der Apparata. Im wesentlichen besteht der Versuchsapparat (Fig. 7) am zwei ineinander drehbaren Zylindern CI und C,,, einer Auffangplatte A und dem Versuchsdraht D mit seinen Zu- 55. W. Hiitternaim. 828 leitungen. Die beiden Zylinder waren aus Messing hergestellt und hatten eine Lhge von 11 cm. Der Durchmesser des inneren Zylinders betrug 3,5 cm und der des iiuSeren 7 cm. Oben und unten waren sie bis auf die in der Figur ange- deuteten offnungen d&ch ’ Messingdeckel verschlossen. Oben und unten waren sie bis auf die in der Figur ange- Fig. 7. deuteten offnungen d&ch Die Zylinder trugen an der Seite je einen Schlita von gleicher Lgnge, jedoch von verschiedener Breite, und zwar war der des inneren Zylinders 0,5 mm breit und der des iiuSeren genau doppelt so breit, 1 111111. Das Magnetfeld wurde erzeugt durch ein langes Solenoid So von bekannter Windungszahl, das sich in der Langsrichtung des Versuchs- apparates erstreckte und so weit uber die Enden desselben hinausragte, daS das Feld an der Stelle des Gliihdrahtes als vollsthdig homogen angesehen werden konnte. Die Richtung des Feldes liegt also dem Gluh- drahte parallel. Gerade gegen- uber dem Schlita von C, war eine Auffangplatte A ange- bracht, die von dem Zylinder vollkommen isoliert war. Diese Auffangplatte war vollkommen umgebon von der Schutz- kappe K. Der iiuBere Zylinder ’ Messingdeckel verschlossen. mit der Auffangplatte und der Schutzkappe saS n& voll- kommen fest auf der ringformigen Platte R, die ihrerseits mit drei Platinansiitaen P, von denen in der Figur nur einer geaeichnet ist., in den Glasayparat eingeschmolzen war und so auch gleichzeitig die Zuleitung nach aul3en bildete. Die Schutakappe K war an ihrer unteren Seito durchbohrt, um hier die Zuleitung nach auSen zu der Auffangplatte zu er- Oben und unten waren sie bis auf die in der Figur ange Fig. 7. deuteten offnungen d&ch Die Zylinder trugen an der Seite je einen Schlita von gleicher Lgnge, jedoch von verschiedener Breite, und zwar war der des inneren Zylinders 0,5 mm breit und der des iiuSeren genau doppelt so breit, 1 111111. Das Magnetfeld wurde erzeugt durch ein langes Solenoid So von bekannter Windungszahl, das sich in der Langsrichtung des Versuchs- apparates erstreckte und so weit uber die Enden desselben hinausragte, daS das Feld an der Stelle des Gliihdrahtes als vollsthdig homogen angesehen werden konnte. Die Richtung des Feldes liegt also dem Gluh- drahte parallel. Beeohreibung der Apparata. Dieses Glasrohr ragte in den Messingansatz M des inneren Zylinders hinein und beide wurden an den Stellen a durch Platineinschmelzungen, kleinen Befestigungen aus Mes- singringen und Kittungen BUS weibm Siegellack SO starr miteinander verbunden, daS bei jeder noch so minimalen Drehung des Schliffes Sch, sich der Zylinder C, in dem gleichen MaSe drehte. In der oberen Glaskappe des Schliffes Sch, waren mehrere kleine Platindriihte eingeschmolzen, die die Strom- znleitung einerseits nachauf3en hin ermoglichten. An diese Platin- drahte schlossen sich hart angelotet die Messingstabe m an, dann kam der Versuchsdraht D, dann wieder ein kleiner Messing stab m, dann eine Spiralfeder aus Messing und dann nochmals kleine Messingstabe m, deren unterster PlatindrWe trug, die in die Kappe des Schliffes Schg eingeschmolzen waren und hier die zweite Stromzufuhrung nach aden hin bildeten. Der Versuchsdraht D lief3 sich durch starke Klemmen in die Messingstabe einschrauben und diese wiedemm waren durch mehrere Verschraubungen unterbrochen, die es erlaubten, durch Drehen der Schliffe Sch, und Sch, diese ganze Ver- bindung auseinander zu nehmen. Die Messingspirale war dam da, den Versuchsdraht, der sich beim Gliihen ausdehnte, durch ihren Zug immer wieder straff zu spannen und ihn dadurch stets in eind symmetrische Lage zu den Zylindern zu bringen.' Da aber die Feder bei ihrer dauernden Erwbnung durch den Strom schlief3lich ganz unelastisch wurde, und auch die Strom- stiirke durch das stete Verhdern des spedisohen Leitungs- widerstandes von Messing zu sehr variierte, so konstruierte ich einen kleinen NebenschluS N, der es ermoglichte, diese Storungen zu beseitigen. An den Stellen, wo diese Zuleitungen zum Versuchsdraht durch den inneren Zylinder gingen, waren sie an den Uffnungen desselben noch von Glasrohren la um moglichen. Diese Zuleitung war vollkommen d w h Bern- stein B isoliert und fiihrte durch den Glaaassatz a naoh auhn. In dem auSeren Zylinder befand sich drehbar der innere Zylinder C,, der an seiner oberen VerschlnSplatte kleine off- nungen a und b trug, die es ermoglichten, den Versuohadraht D wahrend der Messungen stets zu beobachten. Die Drehung des Zylinders wurde ermoglioht durch den hhliff Sch,. In der an dem Schliff befindlichen Glaskappe war von innen ein Glasrohr G eingeschmolzen von der Form, wie es die Figur zeigt. Beeohreibung der Apparata. Gerade gegen- uber dem Schlita von C, war eine Auffangplatte A ange- bracht, die von dem Zylinder vollkommen isoliert war. Diese Auffangplatte war vollkommen umgebon von der Schutz- kappe K. Der iiuBere Zylinder ’ Messingdeckel verschlossen. mit der Auffangplatte und der Schutzkappe saS n& voll- kommen fest auf der ringformigen Platte R, die ihrerseits mit drei Platinansiitaen P, von denen in der Figur nur einer geaeichnet ist., in den Glasayparat eingeschmolzen war und so auch gleichzeitig die Zuleitung nach aul3en bildete. Die Schutakappe K war an ihrer unteren Seito durchbohrt, um hier die Zuleitung nach auSen zu der Auffangplatte zu er- Fig. 7. mit der Auffangplatte und d Fig. 7. Die Zylinder trugen an der Seite je einen Schlita von gleicher Lgnge, jedoch von verschiedener Breite, und zwar war der des inneren Zylinders 0,5 mm breit und der des iiuSeren genau doppelt so breit, 1 111111. Das Magnetfeld wurde erzeugt durch ein langes Solenoid So von bekannter Windungszahl, das sich in der Langsrichtung des Versuchs- apparates erstreckte und so weit uber die Enden desselben hinausragte, daS das Feld an der Stelle des Gliihdrahtes als vollsthdig homogen angesehen werden konnte. Die Richtung des Feldes liegt also dem Gluh- drahte parallel. Gerade gegen- uber dem Schlita von C, war eine Auffangplatte A ange- bracht, die von dem Zylinder vollkommen isoliert war. Diese Auffangplatte war vollkommen umgebon von der Schutz- kappe K. Der iiuBere Zylinder Messingdeckel verschlossen. er Schutzkappe saS n& voll- migen Platte R, die ihrerseits denen in der Figur nur einer parat eingeschmolzen war und ung nach aul3en bildete. Die unteren Seito durchbohrt, um n zu der Auffangplatte zu er- Fig. 7. Fig. 7. Die Emission vim Elektrcmm und positiven I- usw. 829 moglichen. Diese Zuleitung war vollkommen d w h Bern- stein B isoliert und fiihrte durch den Glaaassatz a naoh auhn. In dem auSeren Zylinder befand sich drehbar der innere Zylinder C,, der an seiner oberen VerschlnSplatte kleine off- nungen a und b trug, die es ermoglichten, den Versuohadraht D wahrend der Messungen stets zu beobachten. Die Drehung des Zylinders wurde ermoglioht durch den hhliff Sch,. In der an dem Schliff befindlichen Glaskappe war von innen ein Glasrohr G eingeschmolzen von der Form, wie es die Figur zeigt. Beeohreibung der Apparata. Dieses Glasrohr ragte in den Messingansatz M des inneren Zylinders hinein und beide wurden an den Stellen a durch Platineinschmelzungen, kleinen Befestigungen aus Mes- singringen und Kittungen BUS weibm Siegellack SO starr miteinander verbunden, daS bei jeder noch so minimalen Drehung des Schliffes Sch, sich der Zylinder C, in dem gleichen MaSe drehte. In der oberen Glaskappe des Schliffes Sch, waren mehrere kleine Platindriihte eingeschmolzen, die die Strom- znleitung einerseits nachauf3en hin ermoglichten. An diese Platin- drahte schlossen sich hart angelotet die Messingstabe m an, dann kam der Versuchsdraht D, dann wieder ein kleiner Messing stab m, dann eine Spiralfeder aus Messing und dann nochmals kleine Messingstabe m, deren unterster PlatindrWe trug, die in die Kappe des Schliffes Schg eingeschmolzen waren und hier die zweite Stromzufuhrung nach aden hin bildeten. Der Versuchsdraht D lief3 sich durch starke Klemmen in die Messingstabe einschrauben und diese wiedemm waren durch mehrere Verschraubungen unterbrochen, die es erlaubten, durch Drehen der Schliffe Sch, und Sch, diese ganze Ver- bindung auseinander zu nehmen. Die Messingspirale war dam da, den Versuchsdraht, der sich beim Gliihen ausdehnte, durch ihren Zug immer wieder straff zu spannen und ihn dadurch stets in eind symmetrische Lage zu den Zylindern zu bringen.' Da aber die Feder bei ihrer dauernden Erwbnung durch den Strom schlief3lich ganz unelastisch wurde, und auch die Strom- stiirke durch das stete Verhdern des spedisohen Leitungs- widerstandes von Messing zu sehr variierte, so konstruierte ich einen kleinen NebenschluS N, der es ermoglichte, diese Storungen zu beseitigen. An den Stellen, wo diese Zuleitungen zum Versuchsdraht durch den inneren Zylinder gingen, waren sie an den Uffnungen desselben noch von Glasrohren la um- W. Hiittemann. 830 geben, die an dem Zylinder befestigt waren. Um die Zylinder uberhaupt in den Glasapparat hineinbringen zu konnen, befand sich anihm ein groDer Rezipientenschliff Sclt,. Oben an der Glas- kappe des Schliffes Sch, war an einem kleinen Messinggestell ein Spiegel vollkommen symmetrisch zum Versuchsapparat angebracht, der von einer Nernstlampe beleuchtet wurde und den Stift derselben auf einer Skala abbildete. Diese Skala war im Kreise angeordnet, in dessen Mittelpunkt sich genau der Spiegel befaad. Verauchaanordnung. Fig. 8 zeigt diese. Der Draht wurde gegliiht mit Hilfe einer Batterie von sechs groBen Akkumulatoren H, deren Fig. 8. Strom durch den Widerstand R reguliert wurde. Zur Messung desselben diente das Ampere- meter A. Das Voltmeter 8, melches an dem Versuchsdraht lag, diente dazu, den Spannungs- abfall liings des Drahtes festzu- stellen. Die positive Aufladung erhielt der Draht von der Akkumulatorenbatterie a , die durch einen groBen Ruhstrat- widerstand von 700 Ohm kurz- geschlossen war und durch Ab- schaltung eine Spannung bis zu 90 Volt liefern konnte. Die Zylinder hatten im Verhaltnis zum Draht eine negative Span- nung. Von der Auffangplatte fuhrte ein Draht zu einem Quecksilberniipfchen a, welches sich mit noch drei anderen - b, c, d - quadratisch angeordnet auf einem Paraffinklotz befand. Von c aus fuhrte ein Draht zu einem in hetero- statischer Anordnung aufgestellten Dolezalekelektrometer. Von dem liul3eren Zylinder C,, fuhrte ein Draht zu dem Nlipfchen b, und von dem Niipfchen d wurde er dann weiter zu einem Glimmerkondensator von der Kapazitiit 5.10- Mikrofarad Fig. 8 zeigt diese. Der Draht wurde gegliiht mit Hilfe einer Batterie von sechs groBen Akkumulatoren H, deren Fig. 8. Strom durch den Widerstand R reguliert wurde. Zur Messung desselben diente das Ampere- meter A. Das Voltmeter 8, melches an dem Versuchsdraht lag, diente dazu, den Spannungs- abfall liings des Drahtes festzu- stellen. Die positive Aufladung erhielt der Draht von der Akkumulatorenbatterie a , die durch einen groBen Ruhstrat- widerstand von 700 Ohm kurz- geschlossen war und durch Ab- schaltung eine Spannung bis zu 90 Volt liefern konnte. Die Zylinder hatten im Verhaltnis zum Draht eine negative Span- nung. Von der Auffangplatte fuhrte ein Draht zu einem Quecksilberniipfchen a, welches sich mit noch drei anderen - b, c, d - quadratisch angeordnet auf einem Paraffinklotz befand. Von c aus fuhrte ein Draht zu einem in hetero- statischer Anordnung aufgestellten Dolezalekelektrometer. Von dem liul3eren Zylinder C,, fuhrte ein Draht zu dem Nlipfchen b, und von dem Niipfchen d wurde er dann weiter zu einem Glimmerkondensator von der Kapazitiit 5.10-* Mikrofarad Fig. 8 zeigt diese. Der einer Batterie von sechs gr Fig. 8. Fig. 8. 831 Die Emission von Elektronen und positiven Ionen usw. geleitet. Die andere Platte des Kondensators war mit der Erde verbunden. Sbtliche Zuleitungen zur Auffangplatte sowohl als auch zum Zylinder und zum Kondensator waren durch Metallrohren, die zur Erde abgeleitet waren, elektro- statisch geschutzt. Verauchaanordnung. Liegen die Schlitze nicht mit dem Draht in einer Ebene, so treffen die Ionen nur teil- weise oder gar nicht die Auffangplatte ; die Aufladung dauert dann langer oder findet uberhaupt nicht statt. Dam wird auch der Kondensator hoher oder ganz anfgeladen und dem- nach bei seinem Entladen durch das Elektrometer der Aus- schlag groBer. Alle diese Beobachtungen bei verschiedenen Stellungen der Schlitze ergeben Punkte, die auf einer Kurve liegen, deren Minimum beim Felde 0 den Nullpunkt fiir die Messungen angibt. Zwischen samtliohen einzelnen Messungen wurden die Schlussel S, und S, immer geschlossen, um da- durch stets eine Erdung des Elektrometers, des Kondensators, der beiden Zylinder und der Auffangplatte herbeizufuhren. Uber den Glasapparat wurde nach Feststellung des Null- punktes das erwahnte lange Solenoid So gezogen, durch welches ein elektrischer Strom von bestimmter Amphrezahl geschickt wurde. Dann entstand im Innern desselben ein magnetisches Feld, welches die Ionen ablenkte. Die Dimen- sionen dea Solenoids maren so gewahlt, daB man an der Stekle, wo sich der Draht und die Zylinder befanden, das Feld als homogen ansehen konnte. Die Zylinder mufiten dann so weit gegeneinander gedreht werden, bis bei der Entladung des Kondensators durch das Elektrometer sich wieder ein oder mehrere Minima des Ausschlages einstellten. Diese Punkte ergaben zusammen mit dem elektrischen und magnetischen Feld und der Nullstellung die Werte fur elm und daraus lies sich feststellen, was es ffir Substanzen waren, die von dem Drahte ausgingen. gewendet. Stehen die Schlitze parallel und sind in einer Ebene mit dem Draht beim Felde 0, so treffen alle Ionen, die durch den Schlitz des inneren Zylinders kommen, auf die Auffangplatte, die Sufladung des Elektrometers vollzieht sich am schnellsten; d a m ist die Aufladung des Kondensators aber am kleinsten, also auch der Ausschlag des Elektrometers beim Entladen des Kondensators. Liegen die Schlitze nicht mit dem Draht in einer Ebene, so treffen die Ionen nur teil- weise oder gar nicht die Auffangplatte ; die Aufladung dauert dann langer oder findet uberhaupt nicht statt. Dam wird auch der Kondensator hoher oder ganz anfgeladen und dem- nach bei seinem Entladen durch das Elektrometer der Aus- schlag groBer. Alle diese Beobachtungen bei verschiedenen Stellungen der Schlitze ergeben Punkte, die auf einer Kurve liegen, deren Minimum beim Felde 0 den Nullpunkt fiir die Messungen angibt. Verauchaanordnung. Zwischen samtliohen einzelnen Messungen wurden die Schlussel S, und S, immer geschlossen, um da- durch stets eine Erdung des Elektrometers, des Kondensators, der beiden Zylinder und der Auffangplatte herbeizufuhren. Uber den Glasapparat wurde nach Feststellung des Null- punktes das erwahnte lange Solenoid So gezogen, durch welches ein elektrischer Strom von bestimmter Amphrezahl geschickt wurde. Dann entstand im Innern desselben ein magnetisches Feld, welches die Ionen ablenkte. Die Dimen- sionen dea Solenoids maren so gewahlt, daB man an der Stekle, wo sich der Draht und die Zylinder befanden, das Feld als homogen ansehen konnte. Die Zylinder mufiten dann so weit gegeneinander gedreht werden, bis bei der Entladung des Kondensators durch das Elektrometer sich wieder ein oder mehrere Minima des Ausschlages einstellten. Diese Punkte ergaben zusammen mit dem elektrischen und magnetischen Feld und der Nullstellung die Werte fur elm und daraus lies sich feststellen, was es ffir Substanzen waren, die von dem Drahte ausgingen. Verauchaanordnung. Auch die Bernsteinisolationen waren aus dem gleichen Grunde uberall von zur Erde abgeleitetem Metal1 umgeben. Der Glasapparat befand sich vollstiindig in ge- erdetem Stanniol, so daS irgendwelche Strome oder hie- chende Ladungen von a d e n absolut keinen Einflul3 auf die Messungen hatten. Wurde nun der Draht gegluht, so kamen die Ionen in bestimmten Zeiten nicht gleichmiiSig aus ihm heraus, und aus diesem Grunde hatte ich eben die Anord- nung mit dem Kondensator gewahlt. Die Ionen trafen bei der Nullstellung der Schlitze, das ist die Stellung, bei der der Versuchsdraht und die beiden Schlitze in einer Ebene liegen, ohne Anlegen des Magnetfeldes zuniichst den inneren Zylinder und brachten ihm eine gewisse Ladung in bestimmter Zeit bei, welche sofort auch auf den &uBeren Zylinder iiber- tragen wurde, da beide Zylinder sich beruhrten. An der Stelle, wo sich der Schlitz von Ci befand, gingen die Ionen glatt hindurch, kamen dann m dem Schlitz des aderen Zylinders und gingen bei der Nullstellung der Schlitze hier siimtlich hindurch, da der Schlitz von C, gerade doppelt so groS ist als der von Ct, und ebenfalls die Durchmesser der Zylinder in demselben Verhiiltnis stehen. Diese durchgehenden Ionen prallten dam auf die Auffangplatte und diese wurde auf- geladen. Waren nun die Quecksilberniipfchen durch den Schliissel S, geschlossen, d. h. war a mit c und b mit d verT bunden, so lud sich das Elektrometer durch den Ionenstrom, der die Auffangplatte traf, auf und der Kondensator durch die ubrigen &us dem Draht herauskommenden positiven Ionen. Zeigte das Elektrometer eine bestimmte Aufladung auf der Skala an, so wurde der Schliissel S, schnell herausgenommen und der Schlussel S, geschlossen, d. h. die Nlipfchen c und d miteinander verbunden, der Kondensator also durch das Elektrometer entladen. Dieser Ausschlag war dann immer fiir die Messungen maSgebend. War das Feld 0 und wollte ich feststellen, warm die Schlitze mit dern Draht in einer Ebene lagen, also die Nullstellung suchen, so wurde die beschriebcne MeSmethode fiir verschiedene Stellungen der Zylinder an- 85 2 W . H iitt emann . gewendet. Stehen die Schlitze parallel und sind in einer Ebene mit dem Draht beim Felde 0, so treffen alle Ionen, die durch den Schlitz des inneren Zylinders kommen, auf die Auffangplatte, die Sufladung des Elektrometers vollzieht sich am schnellsten; d a m ist die Aufladung des Kondensators aber am kleinsten, also auch der Ausschlag des Elektrometers beim Entladen des Kondensators. Theorie der Methode. Der aus dem Versuchsdraht austretende Strahl der posi- tiven Ionen gelangt in ein homogenes magnetisches Feld von der Feldstarke Q, welches senkrecht zu seiner Bahn gerichtet ist. Das Feld wird erzeugt durch ein langes Solenoid. Dann wird in jedem Augenblick auf ein positives Ion, welches mit Die Emission von Elektronen und positiven Ionen usto. 833 833 der Geschwindigkeit v bewegt wird und mit der Elektrizitats- menge e geladen ist, ah0 eine Stromstlirke i = v . e darstellt, eine Kraft ausgeiibt, welche senkrecht zu seiner Bewegungs- richtung gerichtet ist. Diese Kraft wird gegeben durch S = Q . i = @ . v . e , d. h., da Q und die konstante Geschwindigkeit v des Ions aufeinander senkrecht stehen, muS das Ion einen Kreis be- schreiben. Die Geschwindigkeit v bei meinen Versuchen konnte als konstant angesehen werden, weil das gesamte elektrische Feld gana in der Niihe des Drahtes liegt und daher die Ionen keinen Geschwindigkeitszuwachs mehr erfahren, sobala sie sich aus der allernachsten N&he des Drahtes entfernt haben. Bewegt sich aber eine Masse m auf einer Kreisbahn, so ist die nach dem Mittelpunkt des Kreises gerichtete Zentnpetalkraft ge- geben durch die Gleichung 8 = ys wo e der Kriimmungsradius des Kreises ist. Diese Gleichung mit der vorigen zusammeu ergibt die neue Gleichuug wo e der Kriimmungsradius des Kreises ist. Diese Gleichung mit der vorigen zusammeu ergibt die neue Gleichuug (7) (7) 1st nun V das Potential der Anode, so besitzt ein positives Ion mit der Laduug e an der Oberfliiche des Drahtes die potentielle Energie e . V, welche bei geniigend niedrigem Druck im Rohre vollkommen in kinetische Energie des Ions umgewandelt wird. Es besteht demnach die Gleichung 1st nun V das Potential der Anode, so besitzt ein positives Ion mit der Laduug e an der Oberfliiche des Drahtes die potentielle Energie e . V, welche bei geniigend niedrigem Druck im Rohre vollkommen in kinetische Energie des Ions umgewandelt wird. Es besteht demnach die Gleichung c . F = - m a s , 1 2 Diese Gleichung ergibt zusammen mit (9) das Verhaltnis der Ladung zur Masse, schlieBlich dargestellt durch Diese Gleichung ergibt zusammen mit (9) das Verhaltnis der Ladung zur Masse, schlieBlich dargestellt durch Ich hatte nun vorausgesetzt, daI3 der Kriimmungsradius der eines Krehes ist, und das whre auch der Fall, wenn der Poten- tialabfall vom Draht zum Zylinder vollkommen am Draht liegen wiirde, was jedoch von vornherein bei meiner Methode der Drehzylinder nicht angenommen werden kann. Der Be- weis dafiir, daB der Kriimmungsradius doch ein Kreis ist, stiitzt sich auf einige Angaben von Greinacherl) und ist in meiner Dissertation ausfiihrlich behandelt. Ich hatte nun vorausgesetzt, daI3 der Kriimmungsradius der eines Krehes ist, und das whre auch der Fall, wenn der Poten- tialabfall vom Draht zum Zylinder vollkommen am Draht liegen wiirde, was jedoch von vornherein bei meiner Methode der Drehzylinder nicht angenommen werden kann. Der Be- weis dafiir, daB der Kriimmungsradius doch ein Kreis ist, stiitzt sich auf einige Angaben von Greinacherl) und ist in meiner Dissertation ausfiihrlich behandelt. 1) H. Greinacher, Verh. d. D. Physik. Ges. 18. p. 866. 1912. Diese Gleichung liefert zusammen mit (7) Diese Gleichung liefert zusammen mit (7) Diese Gleichung liefert zusammen mit (7) (9) 2 T' 9 - . 6 - m @' 4' (9) Das ist aus den die Gleichung fiir elm, nach der die Berechnungen Ergebnissen der Messungen gemacht werden. Das ist aus den die Gleichung fiir elm, nach der die Berechnungen Ergebnissen der Messungen gemacht werden. 54 W Hiit a54 W. Hiittemann. W. Hiittemann. Der Kriimmungsradius ist bestimmt aus drei Punkten, und mar. werden diese dargestellt durch den Draht und die beiden Schlitze der Zylinder. Das magnetische Feld biegt die Strahlen der positiven Ionen ab, und es gehort eine be- stimmte Drehung des inneren Zylinders dazu, diese Strahlen wieder alle auf die Auffangplatte gelangen zu lassen. gp g g Sind r und R = 2 r die Radien der Zylinder und a der Drehungswinkel des inneren gegen den iiuI3eren Zylinder, so ergibt sich der Kriimmungsradius e schlieSlich zu: v R 3 + rs - tz R r COB a n = F 2 sin a v R 3 + rs - tz R r COB a n = F 2 sin a Die Meseungen. Wiihlte ich jedoch ein minimales Zeit- intervall, in welchem ich bei einer bestimmten, vorher be- rechneten Stelle, an der die Zylinderschlitze bei einem be- stimmten elm stehen muSten, einige Messungen ausfuhrte, so konnte ich annehmen, daB die lonen dann regelmaig wiih- rend dieser Zeit den Draht verlassen, und die kiirzeste Zeit, die verstreicht, bis sich das Elektrometer auf ein bestimmtes Potential geladen hat, und die mit einer Stoppuhr gemessen wird, gibt mir charakteristische Punkte fiir elm. Ich habe nun im ganzen flinf Stoffe auf positive Ionisation hin unter- sucht, und zwar sind dies Plath, Wolfram, Tantul, Platm mit Aluminiumphosphat iiberzogen und Pldinc mil Calciunzosyd iiberzogen. Die Meseungen. Die Messungen wurden so ausgefuhrt, mie es schon in der Versuchsanordnung angedeutet war. Es wurde durch die Messungen der Ausschlage der Auffangplatte und des Kon- densators erzielt, daI3 sich immer das Verhaltnis beider Aus- schliige ergab, wodurch das unregelmiiBige Austreten der positiven Ionen aus dem Versuchsdraht weiter nicht storte. Da die Ausfiihrung dieser Messungsmethode ziemlich lange dauerte, so wurde, um eventuell noch Spuren von abgegebenen yositiven Ionen nachweisen zu konnen, noch eine andere un- genauere Methode gewiihlt, und zwar die mit der Stoppuhr. Hierbei war der Kondensator ausgeschaltet, und die Zylinder lagen mit der Schutzkappe direkt an Erde. Die elektro- statische Adladung der Auffangplatte fie1 hier vollkommen Die Envission von E l e k t r m und positiven Ionen usw. 836 Die Envission von E l e k t r m und positiven Ionen usw. weg. Wenn die Schlitze beim Felde 0 nicht mit dem Draht in einer Ebene lagen oder beim Anlegen eines magnetisohen Feldes nicht geeignet zueinander gedreht waren, so prallten die positiven Ionen, die durch den Schlitz des ersten Zylinders kamen, auf den zweiten Zylinder auf und wurden, da der Zylinder geerdet war, direkt nach der Erde abgeleitet. Das Elektrometer blieb dann vollkommen in der Ruhelage. So- bald aber die Schlitze geeignet standen, zeigte das Elektro- meter Aufladung; es kamen also Ionen a d die Auffangplatte, d. h. auch durch den Schlitz des &uBeren Zylinders. Mit Hilfe einer Stoppuhr konnte festgestellt werden, bei welcher Stel- lung der Zylinder zueinander die kiirzeste Zeit verstrich, um auf der Auffangplatte eine bestimmte Aufladung zu haben. Aus verschiedenen Stellungen der Schlitze zueinander eigaben sich verschiedene Zeiten, die mit den Drehungswinkeln der Zylinder zusammen in ein rechtwinkliges Koordinatensystem eingetragen, Kurven ergaben, deren Minima bestimmend fiir die Berechnung von elm waren. Diese Methode hat den Vor- zug, daB sie sehr schnell auszufuhren ist, jedoch den Nach- teil, daB sie von den Ionen annimmt, in bestimmten Zeiten regelmiiI3ig den Draht zu verlassen, was jedoch praktisch nicht der Fall ist. 1) Dasselbe glaubte schon J. J. Thomson (Phil. Meg. (VI) 18. p. 661. 1907) in den Kandstrahlen gefunden zu haben. Platin. Es wurde ein Platindraht in den Versuchsapparat ge- bracht, ein paar Ma1 ganz kurze Zeit gegliiht und dann so- fort mit den Messungen begonnen. Erst n d e der Null- punkt nach der Kondensatormethode festgestellt, und dann durch das Solenoid ein Magnetfeld um den Draht erzeugt. W. Hiitternam. 836 Dadurch wurden die Strahlen der positiven Ionen abgelenkt und die Zylinder mdten dann so weit gedreht werden, bis der Elektrometerausschlag beim Entladen des Kondensators durch das Elektrometer ein Minimum wurde. Nach dieser Methode stellte ich drei Minima fest, die sich bei der Be- rechnung von elm nach Gleichung (11) als Punkte ergaben, an denen positive Ionen von m/H = SO, m/H = 2 und ml H = 1 vorhanden waren. Daraus geht hervor, da6 Wasser- stoffatome und Wasserstoffmolehle am dem Draht heraus- kommenl) und auSerdem noch eine Substanz, die die Bande rn/H = SO hat. Platinionen wurden bei dieser Art der Messung nicht gefunden. Da verfuhr ich nun folgender- maSen: Ich fiihrte zuniichst wieder bei einer Temperatur von ca. 950-100O0 C die Messungen mit dem Kondensator an dem Platindraht aus. Dann wandte ich die zweite Me- thode, die mit der Stoppuhr, an. Vorher berechnete ich genau, wie die Stellung der Schlitze zueinander sein m a t e , wenn Ionen von Platin vorhanden sein sollten. Erhitzte ich jetzt plotslich den Draht bis ca. 1350O C, drehte den Schlitz auf die berechnete Stelle, wohin die Platinionen abgelenkt werden mdten, so bekam ich eine langsame Aufladung des Elektrometers, melche vie1 schwacher wurde, wem ich das Magnetfeld ausschaltete. Um auch sicher zu sein, dab das nicht schon ein Aufladen des Elektrometers durch Ionen von m/H =ca.SO mar, untersuchte ich mit Hilfe der Stoppuhr moglichst schnell die Schlitzstellungen neben diesem Punkt und fand, da13 tatsiichlich bei Drehung des Spiegels Sp um 1 cm von der Nullstellung ein Minimum lag. Im ganzen dauerte die Abgabe von Platinionen bei meiner Elektrometer- empfindlichkeit und bei der Temperatur von ca. 1S5Oo C 10 Minuten lang. Dann ging ich zu hoheren Temperaturen uber und fand dieselbe Erscheinung. Besonders stark waren im Verhiiltnis zu den ersten Messungen positive Ionen von Platin kurz vor dem Schmelzpunkt des Metalles vorhanden. Die Tabb. I u. Platin. I1 geben die Abhiingigkeit des Ausschlages des Elektro- meters beim Entladen des Kondensators durch das Elektro- meter von deru Winkel, um den die beiden Schlitze zuein- ander gedreht sind, wieder, einmal bei dem Felde +314 Gauss Dadurch wurden die Strahlen der positiven Ionen abgelenkt und die Zylinder mdten dann so weit gedreht werden, bis der Elektrometerausschlag beim Entladen des Kondensators durch das Elektrometer ein Minimum wurde. Nach dieser Methode stellte ich drei Minima fest, die sich bei der Be- rechnung von elm nach Gleichung (11) als Punkte ergaben, an denen positive Ionen von m/H = SO, m/H = 2 und ml H = 1 vorhanden waren. Daraus geht hervor, da6 Wasser- stoffatome und Wasserstoffmolehle am dem Draht heraus- kommenl) und auSerdem noch eine Substanz, die die Bande rn/H = SO hat. Platinionen wurden bei dieser Art der Messung nicht gefunden. Da verfuhr ich nun folgender- maSen: Ich fiihrte zuniichst wieder bei einer Temperatur von ca. 950-100O0 C die Messungen mit dem Kondensator an dem Platindraht aus. Dann wandte ich die zweite Me- thode, die mit der Stoppuhr, an. Vorher berechnete ich genau, wie die Stellung der Schlitze zueinander sein m a t e , wenn Ionen von Platin vorhanden sein sollten. Erhitzte ich jetzt plotslich den Draht bis ca. 1350O C, drehte den Schlitz auf die berechnete Stelle, wohin die Platinionen abgelenkt werden mdten, so bekam ich eine langsame Aufladung des Elektrometers, melche vie1 schwacher wurde, wem ich das Magnetfeld ausschaltete. Um auch sicher zu sein, dab das nicht schon ein Aufladen des Elektrometers durch Ionen von m/H =ca.SO mar, untersuchte ich mit Hilfe der Stoppuhr moglichst schnell die Schlitzstellungen neben diesem Punkt und fand, da13 tatsiichlich bei Drehung des Spiegels Sp um 1 cm von der Nullstellung ein Minimum lag. Im ganzen dauerte die Abgabe von Platinionen bei meiner Elektrometer- empfindlichkeit und bei der Temperatur von ca. 1S5Oo C 10 Minuten lang. Dann ging ich zu hoheren Temperaturen uber und fand dieselbe Erscheinung. Besonders stark waren im Verhiiltnis zu den ersten Messungen positive Ionen von Platin kurz vor dem Schmelzpunkt des Metalles vorhanden. Die Tabb. I u. I1 geben die Abhiingigkeit des Ausschlages des Elektro- meters beim Entladen des Kondensators durch das Elektro- meter von deru Winkel, um den die beiden Schlitze zuein- ander gedreht sind, wieder, einmal bei dem Felde +314 Gauss Die Ernisswn con Elelctrmm und positivm Ionm uaw. 837 und einmal beim entgegengesetsten Felde -314 Gauss. Platin. Hier- bei ist der Elektrometereusschlag in Zenthetern angegeben, wobei die Empfindlichkeit des Elektromefers bei den Ver- suchen an den Platindrlihten 160 Teilstriche pro Volt betrug. Ebenfalls ist der Drehungswinkel der Zylinder in Zentimetern angegeben, wobei sich der Spiegel genau im Mittelpunkte eines Kreises drehte, dessen Umfang 188 cm betrug. Der Drehung des reflektierten Strahles um den ganzen Umfang des Kreises entspricht naturlich eine wirkliche Drehung des Spiegels um 180O. Tab. I11 ergibt einige Messungen nach der Stoppuhrmethode bei verschiedenen Temperaturen. Die darauffolgende Fig. 9 gibt die graphische Darstellung der Werte der Tabb. I, I1 und I11 und zeigt deutlich, an wel- chen Stellen die Minima liegen. Die am den Kondensator- messungen gefundene Kurve ist ausgezogen gezeichnet, wkh- rend die nach der Methode der St,oppuhr gefundene gestrichelt geseichnet ist. und einmal beim entgegengesetsten Felde -314 Gauss. Hier- bei ist der Elektrometereusschlag in Zenthetern angegeben, wobei die Empfindlichkeit des Elektromefers bei den Ver- suchen an den Platindrlihten 160 Teilstriche pro Volt betrug. Ebenfalls ist der Drehungswinkel der Zylinder in Zentimetern angegeben, wobei sich der Spiegel genau im Mittelpunkte eines Kreises drehte, dessen Umfang 188 cm betrug. Der Drehung des reflektierten Strahles um den ganzen Umfang des Kreises entspricht naturlich eine wirkliche Drehung des Spiegels um 180O. Tab. I11 ergibt einige Messungen nach der Stoppuhrmethode bei verschiedenen Temperaturen. Die darauffolgende Fig. 9 gibt die graphische Darstellung der Werte der Tabb. I, I1 und I11 und zeigt deutlich, an wel- chen Stellen die Minima liegen. Die am den Kondensator- messungen gefundene Kurve ist ausgezogen gezeichnet, wkh- rend die nach der Methode der St,oppuhr gefundene gestrichelt geseichnet ist. Tabelle I. v = 60 Volt. H = + 314 GLW. Elelrtrometer- 1 Drehungswinkel Elektrometer- ausaohhg in cm in cm euaschleg in cm 13.2 123 12,o 11.5 11.4 11;e 10.7 8,4 6.5 2.2 7,2 10,6 13.0 14.8 16,9 16.1 15,9 4.0 3.1 -~~ ~~ 16.0 15,s 14,7 13.2 9,6 6.1 6.0 8.1 14,4 15.5 15,7 15,3 146 13,2 11,8 12.6 15.1 15,8 Drehungawinkel in cm ~~ ~ ~ ~ _ _ 6 8 9 0 68,s 89,4 70,l 70,9 71.3 71.5 71.7 72.4 73,O 74.0 74.9 753 76,l 76,4 76.7 77;l 77.5 Tabelle I. H = + 314 GLW. 838 W. Hut temann. Tabelle 11. V = 60 Volt. If = - 31 4 Gauss. Platin. Elektrometer- ausschleg in cm 14,l 13,2 12,7 12,o 7J3 593 391 399 999 15-7 16;s 17,O 17,l 15,7 12,6 9,2 57;7 53;5 52,5 I/ Elektrometer- ausschlag in cm 8 8 8.8 ___ __ - 12;o 16,3 17,O 17,2 16,9 16,6 16,O 14,9 14,2 15.3 5213 16;3 16,6 :$ 11 16,8 50,2 Drehungswinkel in cm 5 0 9 0 49,8 49,5 49.0 48,O 47,5 46,l 45,6 45,3 45,O 44,8 4495 4493 4490 43,l ~- __ __ Tabelle 11. 61,5 61,6 61,7 61,8 Tabelle 11. V = 60 Volt. If = - 31 4 Gauss. 992 12,8 61,6 ~ 7,2 16,4 I 61,7 13,4 21,8 61,8 I 18,O 992 61,5 Elektrometer- ausschleg in cm 14,l 13,2 12,7 12,o 7J3 593 391 399 999 15-7 ____ __ __ 61,2 61,3 61,4 01,5 61,7 61,8 61,6 16;s 17,O 17,l 15,7 12,6 9,2 - 14,O 12,2 10,6 7,s 11.8 17,4 7,o 57;7 53;5 52,5 I/ Elektrometer- ausschlag in cm 8 8 8.8 ___ __ - 12;o 16,3 17,O 17,2 16,9 16,6 16,O 14,9 14,2 15.3 5213 16;3 16,6 :$ 11 16,8 50,2 Drehungswinkel in cm 5 0 9 0 49,8 49,5 49.0 48,O 47,5 46,l 45,6 45,3 45,O 44,8 4495 4493 4490 43,l ~- __ __ Tabelle 111. I in sec Drehungswinkel in cm V = 60 Volt. If = - 31 4 Gauss. Tabelle 111. 61,5 61,6 61,7 61,8 992 12,8 61,6 ~ 7,2 16,4 I 61,7 13,4 21,8 61,8 I 18,O 992 61,5 ____ __ __ 61,2 61,3 61,4 01,5 61,7 61,8 61,6 - 14,O 12,2 10,6 7,s 11.8 17,4 7,o Tabelle 111. I in sec Drehungswinkel in cm Die Entission von Elektronen und positiven Ionen usw. 839 839 Die Entission von Elektronen und positiven Ionen usw. 83 Aus den Tabellen und der Figur 1iiBt sich sofort ersehen, an welchen Stellen die einzelnen Minima im Verhiiltnis zum a"" H = - 314 Gauss 4 i w 0 H=+314 Gauss 40 42 44 46 48 50 52 54 56 58 80 62 64 66 88 70 72 74 76 78 80 __c Winkel 1: Fig. 9. Platin. Fig. 9. Platin. Nullpunkte liegen fiir beide Felder + 814 Gauss und - 314 Gauss. Die Drehungswinkel betragen : Nullpunkte liegen fiir beide Felder + 814 Gauss und - 314 Gauss. Platin. Di D h i k l b H = +314 Gauss H = -314 Gauss a) 1 am = 0' 57'38" a) 1,l cm = 10 3' 0" b) 2.7 ,, = 2'36'24" b) 2,s ,. = 2O40'48" C) 10.9 ,, = lOO26'24" C ) 16,6 ,, = 10' 3' 0" d) 16,9 ,, = 15' 13'12" d) 16,7 ,, = 16O 1'48" Aus diesen Drehungswinkeln ergeben sich dann nach der Gleichung (11) fur elm bestimmte Werte und gleichmitig die dazugehorigen Werte von mJH, welche im folgenden beiderseits angegeben sind. H = +314 G ~ U M €I = -314 Gauss a) elm = 44,0; mlH = 216,O a) elm = 53,4; mlH = 181,O b) elm = 324,O; mlH = 29,8 b) elm = M,O; mlH = 27,9 a) elm = 4896,O; mlH = 2,O c) elm = 4660,O; mlH = 2,O d) elm = 9653,O; mlH = 1,0 d) elm = 9404,O; m/H = 1,0 Zur Kontrolle aller dieser Wert.e wurden die Versuche an einem zweiten Platindraht wiederholt. Es ergeben sich hier- bei folgende Werte : 840 I.V. Hiittemann. H = +314 Gauss H = -314 Gauss a) elm = 53,4; mlH = 181,O a) elm = 45; mlH = 216,O b) elm = 299,O; m/H = 32,3 b) elm = 346; mlH = 27,9 c) elm = 4809,O; mlH = 2,O c) elm = 4327; mlH = 2,O d) elm = 9922,O; mlH = 1,0 a) elm = 9200; m/H = 1,O Die erste Bande aller a) stellt also positive Ionen von Platin dar, die dntte Bande c) positive Ionen von Wasserstoff- molekiilen und die vierte d) solche von Wasserstoffatomen. Die Bande b) ld3t sich nicht so leicht diskutieren, weil ge- rade ein Atomgewicht in der Nahe von 30 sehr viele Stoffe haben. Es konnten z. B. Molekule von 0, vorhanden sein, die das Atomgewicht 52 haben, dann solche von N, und auch solche der Verbindung CO. Richardson'), der bei den Ver- suchen mit Platin nur diese eine Bande gefunden hatte, war der Meinung, daS es wohl Ionen von CO seh konnten, schloI3 aber dabei 0, und N, nicht aus. Platin. Ich bin nun der Ansicht, dafj der immer in dem Platin befindliche Sauerstoff zuerst beim Gliihen des Drahtes den Hauptbestandteil der positiven Ionen der b-Bande ausmacht, jedoch nach einiger Zeit wieder verschwindet und dam in dieser Bande nur noch die von An- fang an vorhandenen Ionen von Kohlenoxyd vorhanden sind. 1) 0. W. Richardson, Phil. Mag. (VI) 16. p. 766. 1908. Annden dcr Phplk. IV. F-. 62. 54 Vorstehende Tab. IV ergibt Werte von Ausschlggen des Elektro- meters mit den zugehorigen Drehnngswinkeln. Dabei ist wieder Wolfram. Ganz in der gleichen Weise wie bei Platin wurde bei Wolfram mit den Messungen verfahren. Gliihte ich den Draht bis zur Rotglut und begann sofort mit den Beobach- tungen, so ergab sich als erste Bande ein Wert, der den Wolframatomen entsprach, und zwar wurde diese Bande schon mit der Kondensatormethode gefunden. Die Ionen- abgabe war bei Wolfram sehr vie1 grofjer als bei Platin; hatte ich bei Wolfram ungefghr die gleiche Temperatur wie bei Platin, so muBte ich die Hilfsspannung der Elektrometer- nadel so weit herabsetzen, daS die Empfindlichkeit des Elektro- meters nur 40 Skalenteile pro Volt, im Verhiiltnis zu 160 Skalen- teilen pro Volt bei den Versuchen mit Platin, betrug, um un- gefahr dieselben Ausschliige mie bei den Versuchen mit Platin zu erzielen. Als zweite Bande fand ich wieder Punkte, die un- 1) 0. W. Richardson, Phil. Mag. (VI) 16. p. 766. 1908. Ilie E.nllission von Elektronen und positaven Ionen usw. 84 841 gefahr der Zahl m/H = 30 entsprachen, als dritte Bande Wasserst,offmolehle und als letzte Bande Wasserstoffatome. Wasserst,offmolehle und als letzte Bande Wasserstoffatome. N H=-314Gauss H - +314 Gauss "/Hca 30 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 - Winkd Fig. 10. Wolfram. Tabelle 1V. V = 60 Volt. a - + 314 (hues. -~ Elektrometer - aueachlag in cm 20,o 18,6 17,6 16,5 15.3 15,4 16,9 17.8 18,3 17,9 17,2 15,O 12,2 14,5 16,4 l8,l 19.9 __ I1 Drehungswinkel ; I Elektrometer- in cm 'I ausschlag in cm 60;4 6095 60,7 6039 61,l 61,3 61,5 61,7 61,9 62.1 I1 59,5 24.1 24.1 23,8 608 60.2 22.9 I 2117 2092 19,0 19.8 21,5 23,O 2492 2494 23.9 62;4 23;4 62.6 I 22,4 63.0 21,7 63.6 22.2 Drehungswinkel in cm 6698 67.8 8895 69,4 70,0 70,3 70,5 70.8 71,0 71,2 72,0 73,0 74,1 74,7 76,0 75.2 75,4 75,6 76.0 .- _ ~ _ ~ Vorstehende Tab. IV ergibt Werte von Ausschlggen des Elektro- meters mit den zugehorigen Drehnngswinkeln. Dabei ist wieder Annden dcr Phplk. IV. F-. 62. 54 N H=-314Gauss H - +314 Gauss "/Hca 30 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70 72 74 76 78 80 - Winkd i Tabelle 1V. V = 60 Volt. a - + 314 (hues. Wolfram. -~ Elektrometer - aueachlag in cm 20,o 18,6 17,6 16,5 15.3 15,4 16,9 17.8 18,3 17,9 17,2 15,O 12,2 14,5 16,4 l8,l 19.9 __ I1 Drehungswinkel ; I Elektrometer- in cm 'I ausschlag in cm 60;4 6095 60,7 6039 61,l 61,3 61,5 61,7 61,9 62.1 I1 59,5 24.1 24.1 23,8 608 60.2 22.9 I 2117 2092 19,0 19.8 21,5 23,O 2492 2494 23.9 62;4 23;4 62.6 I 22,4 63.0 21,7 63.6 22.2 Drehungswinkel in cm 6698 67.8 8895 69,4 70,0 70,3 70,5 70.8 71,0 71,2 72,0 73,0 74,1 74,7 76,0 75.2 75,4 75,6 76.0 .- _ ~ _ ~ Vorstehende Tab. IV ergibt Werte von Ausschlggen des Elektro- meters mit den zugehorigen Drehnngswinkeln. Dabei ist wieder Annden dcr Phplk. IV. F-. 62. 54 Vorstehende Tab. IV ergibt Werte von Ausschlggen des Elektro- meters mit den zugehorigen Drehnngswinkeln. Dabei ist wieder Annden dcr Phplk. IV. F-. 62. 54 W. Hiittemann. 842 der Elektrometerausschlag in Zentimetern angegeben und eben- falls der Drehungswinkel auf der Kreisskala. Die vorstehende Fig. 10 gibt die graphische Darstellung der gefundenen Werte wieder. Diesmal lag der Nullpunkt bei den Messungen auf dem Skalenteilstrich 593 der Kreisskala. Das magnetische Feld betrug wieder 314 Gauss und die Messungen wurden bei positivem und negativem Feld ausgefuhrt. Nach den Messungen sind vier Minima vorhanden, die folgende Drehungswinkel haben: H = + 314 Gauss H = - 314 Gauss a) 1,l cm = 10 3’ a) 1,2 cm = 10 9‘ b) 2,6 ,, = 2O29’24” b) 2,5 ,, = 2O23’24“ c) 11,l ,, = 10037‘48” C) 10,3 ,, = 9051’36’’ d) 15,8 ,, = 15O 7’48“ d) 15,l ,, = 14O27‘36” Am diesen Drehuiigswinkeln ergeben sich nach der Gleichung fur die Ladung zur Masse bestimmte Werte fiir elm und fur m l H , die sich zu den nachstehenden Werten berechnen: H = +314 Gauss H = -314 Gauss a) elm = 53,4; mJH = 181.0 a) elin = 64; m/H = 151,O b) elm = 299,O; mlH = 32,3 b) elm 283; m/H = 34,l c ) elm = 4999,O; mJH = 2,O c) elin = 4401; mlH = 2,O d) elm = 9512,O; mlH = 1,0 d) elm = 8797; m/H = 1,0 Die a-Bande ergibt deutlich Wolframionen. Wolfram. Der eine Wert m / H = 151 erscheint im ersten Moment sehr von dem Atom- gewicht von Wolfram abzuweichen; es ist jedoch kein Wert, der ein anderes Atomgewicht vermuten la&, weil er vollkommen in der Grenze der Versuchsfehler liegt, da eine Drehung des Spiegels um 1 mm auf der Kreisskala gleich den Wert m / H = 181 ergibt. Die zweite Bande ergibt beide Male eine Zahl m / H = ca. 32, von der ich behaupte, daI3 sie vorzugs- weise Sauerstoffmolekule neben Kohlenoxyd darstellt, weil der Wert fiir m / H doch vorzugsweise nach den Zahlen 32-34 hiniiberneigt und nicht nach den Zahlen 28-30; wenn auch hier die Fehlergrenzen eine vollstlindig genaue Bestimmung des Atomgewichtes nicht gestatten, so ergab doch die Mes- sung mit einem zweiten Wolframdraht dasselbe Resultat, wie lhe Ehtissim CO)L Elelctrolten und positiven Ionen usw. 843 es bei dem ersten whalten wurde. D a m wurden Wasserstoff- molekule der dritten Bande entsprechend gefunden und schlieB lich Wasserstoffatome. ditrstellrii. Allr vier Banden murden nach der Kondensator- met hodr gefunden. Tantsl. -4quivalent den Messungen an Wolframdrlihten ergaben sich die Beobachtungen an Tantal, einem Metall, welches dem Wolfram verwandt ist. Der Nullpunkt lag bei diesen Ver- suchen auf dem Teilstrich 69 der Kreisskala und Fig. 11 gibt die graphische Darstellung der gefundenen Werte. Die Verhiiltnisse elm und mJH berechnen sich zu den Wrrten : H = +314 G ~ U R S a) elm = 53,4; m H = 181,O h) elm = 299; mfH = 32,3 c) elm = 4560,O; nilH = 2,O d) el& = 10129; ni/H = 1,0 H = +314 G ~ U R S a) elm = 53,4; m H = 181,O h) elm = 299; mfH = 32,3 c) elm = 4560,O; nilH = 2,O d) el& = 10129; ni/H = 1,0 13s rrgeben sic11 vier Banden, clir Ionen von Tantal, von ??z /H = 80, von Wasserstoffmolekiil~n und Wasserstoffatonien 13s rrgeben sic11 vier Banden, clir Ionen von Tantal, von ??z /H = 80, von Wasserstoffmolekiil~n und Wasserstoffatonien 58 ti0 62 64 66 68 70 72 74 76 78 80 - Wlnkd Fig. 11. Tantnl. 58 ti0 62 64 66 68 70 72 74 76 78 80 - Wlnkd Fig. 11. Tantnl. ditrstellrii. Allr vier Banden murden nach der Kondensator- met hodr gefunden. 54 ditrstellrii. Allr vier Banden murden nach der Kondensator- met hodr gefunden. ditrstellrii. Allr vier Banden murden nach der Kondensator- met hodr gefunden. 54 54 844 1) J. J. Thomson, Cembr. Phil. SOC. Proc. 14. p. 105. 1907. Aluminiumphosphat. Die Messungen wurden bei 'einer Temperatur ausgefuhrt, wo das Phosphat gerade anfing, an seiner Oberflache zu gluhen. Bei den Beobachtungen stellte es sich nun zuerst entgegen der Angabe von Thomson heraus, daB Aluminium- phosphat nicht so vide Ionen abgab als frische Drahte von Wolfram, Tantal und auch von Platin. Wartete ich jedoch ca. 1 Stunde und beobachtete dann die Ausschlage des Elektro- meters, so wurde jetzt von Aluminiumphosphst eine weit H- -314 Gauss Fig. 12. Aluminiumphosphat. Fig. 12. Aluminiumphosphat. groBere Anzahl von Ionen abgegeben als von den Metallen, wenn sie die gleiche Zeit gegluht hatten. Daraus geht hervor, daB das Phosphat Ionen sehr lange und konstant bei ein und derselben Temperatur abgibt , und jetzt bestatigt sich auch die Angabe von Thomsonl) , daB Aluminiumphosphat den besten Effekt zeigt. Er gluhte bei den Versuchen mit den Metallen auch immer erst die Drahte lange Zeit, um eine mog- lichst konstante Ionenabgabe zu erhalten. Die Messungen wurden nach der Kondensatormethode ausgefuhrt, und zwar bei positivem und negativem Feld, welches beide Male wieder 514 Gauss betrug. Die angelegte Spannung war die gleiche wie bei den vorigen Messungen, niimlich 60 Volt. Der Null- groBere Anzahl von Ionen abgegeben als von den Metallen, wenn sie die gleiche Zeit gegluht hatten. Daraus geht hervor, daB das Phosphat Ionen sehr lange und konstant bei ein und derselben Temperatur abgibt , und jetzt bestatigt sich auch die Angabe von Thomsonl) , daB Aluminiumphosphat den besten Effekt zeigt. Er gluhte bei den Versuchen mit den Metallen auch immer erst die Drahte lange Zeit, um eine mog- lichst konstante Ionenabgabe zu erhalten. Die Messungen wurden nach der Kondensatormethode ausgefuhrt, und zwar bei positivem und negativem Feld, welches beide Male wieder 514 Gauss betrug. Die angelegte Spannung war die gleiche wie bei den vorigen Messungen, niimlich 60 Volt. Der Null- Die Emissim von Elektronen und posifircn Ionen usiti. 845 punkt lag bei diesen Versuchen bei dem Teilstrich 59,6 der Kreisskala. Fig. 12 gibt die graphische Darstellung der er- haltenen Elektrometerausschliige und Drehungswinkel in Zenti- metern wieder. Aluminiumphosphat. Nach den Beobachtungen liegen die Minima, von denen bei den Versuchen drei gefunden wurden, bei den positiven und negativen magnetischen Feldern an Stellen, die nach- folgenden Drehungswinkeln entsprachen: H = +314 Gauss H = -314 Gauss a) 2,5 cm = 2O 23'24" a) 2.8 crn = 2O40'48" b) 10,8 ,, = lOO20'24" b) 10,6 ,, = 10°9' C) 15,9 ,, = 16' 13' 12" C) 15,l ,, = 14O27'36" Diesen Winkeln entsprechen wieder nach Gleichung (11) fur elm und m / H die Werte: H = +314 Gauss H = - 314 Gauss x) elm = 283; m/H = 34,1 a) elm = 346; mlH = 27,9 b) elm = 4809; mlH = 2,O b) elm = 4646; m/H = 2,O c) elm = 9808; mfH = 1,0 c ) elm = 8797; m/H = 1,0 Die b-Bande liiBt eine Zahl fur elm erkennen, die dem Wasser- stoffmolekul entspricht, und die c-Bande eine solche des Atoms von Wasserstoff. Die a-Bande ergibt einen Wert fur elm, bei dem wieder eine ganze Reihe von Stoffen liegen konnen, und zwar wird das auch der Fall sein, da es sich bei Venuchen mit der Stoppuhrmethode ergab, daB im Ver- hiiltnis zu ,den Wasserstoffbanden die a-Bande sehr stark ausgebildet war. 1 ) A. Wehnelt, Ann. d. Phys. 14. p. 441. 1904. Caloiurnoxyd. %urn SchluB untersuchte ich CaO auf seine Abgabr von positiven Ionen. Auch hier wurde wieder als Trliger ein Platindraht verwendet. Gluhte ich den Draht, so erhielt ich keinen Ausschlag des Elektrometers. Daraus schloB ich, dalj CaO uberhaupt keine positiven Ionen aussendet und be- statigte damit zugleich Versuchr von A. Wehnelt'), d' lt? er 1 ) A. Wehnelt, Ann. d. Phys. 14. p. 441. 1904. W. Huttemum. 846 mit Calciumoxyd im Jahre 1904 anstellte, wobei er zu dem Resultat kam, daB das gluhende Calciumoxyd im Vakuum keine nennenswerte Anzahl von positiven Ionen aussendet. Dieses Resultat erklarte ich dahin, daB Calciumoxyd bei den hier benutzten Temperaturen weder zerstiiubt noch verdampft, sondern nur mechanisch von dem Platindraht abplatzt. Allgemeine Betrechtungen. beiden Resultate, dnp Metallionen und Wasserstoffhen in den Strahlen gefunden wurden, sind im wesentlichen die Hauptergebnisse der Arbeit, zugleich ntit der Tatsache, dap ein Versuchapparat konstruiert wurde, der ES zula/?t, diese beiden Ionenarten auf eine einfache Art festzustellen. Allgemeine Betrechtungen. Nach den Nessungen konnte also eine Entscheidung daruber getroffen werden, was fur Substanzen die positiven Ionen der Anodenstrahlen ausmachen. Bei samtlichen Ver- suchen wurden fur elm Werte gefunden, die sehr gut bekannten Atomen und Molekulen entsprachen. DaB bis jetzt noch nicht eine Entscheidung daruber getroffen wurde, was fur positive Ionen aus dem gluhenden Versuchsdraht herauskommen, liegt wohl zum Teil an der Wahl des Versuchsapparates. Richard- son benutzte z. B. in seiner hier zugrunde liegenden Arbeit einen Apparat, der in seinem Innern statt Drehzylinder zwei gegenuberliegende Platten hatte, die mit Schliteen ~ versehen waren. Der Abstand der Platten betrug bei ihm nur wenige Nillimeter, und die vorzunehmende Verschiebung einer Platte betrug beim Anlegen eines magnetischen Feldes noch nicht 1 mm. Aus diesen sehr kleinen Dimensionen konnten sich also schon Ungenauigkeiten ergeben. Er beobachtete , wie schon mehrfach erwahnt worden ist, don Draht erst, wenn er langere Zeit gegluht hatte, und dann wurden die Wasser- stoffionen und die Ionen von Platin - letztere sind ja schon nach 10 Minuten bei ein und derselben Temperatur nicht mehr vorhanden - nur noch so minimal abgbgeben, daS seine angewandte Elektrometerempfindlichkeit wohl nicht ausreichte, dieselben festzustellen. Nein Versuchsapparat is t auf ganz anderen Dimensionen aufgebaut; z. B. gehort, um bei einem verh8;ltnismaBig schwachen magnetischen Felde Ionen von Wasserstoffatomen festzustellen, eine Drehung des inneren Zylinders von ca. 15 cm auf der Kreisskala dazu. huch ist meine Methode der Drehzylinder aus dem Grunde viel vorteilhafter, weil beim Anlegen eines magnetischen Feldes, wie bewiesen worden war, die Ablenkung der positiven Ionen sich auf einem Kreise vollzieht, wahrend bei R i char ds o n die Bahn eine viel liompliziertere Kurve darst,ellt. Durch I& Emission von Elektronen unrl positiven Ionen UYIO. 8-27 I& Emission von Elektronen unrl positiven Ionen UYIO. 8-2 8-27 meine Drehzylindermethode ist es mir also vor allen Dingen gelungen, den Wasserstoff, der in den Driihten immer vor- handen ist, festzustellen. Bei den Messungen war auch wichtig, daB ich mit meiner Methode positive Metallionen fand, deren Vorhandensein Richardson bei seiner Arbeit entgangen ist. Den entschiedensten Beweis hierfur liefert Wolfram und Tantal, bei welchen mit der etakten Kondensatormethode deutliche Bandcn von Metallionen festgestellt werden konnten. Auch Platin zeigt einen Beweis hierfur, und zwar nach der S toppuhrme t hode. Diese Fes ts tellungen veranlaBten micb dann auch zu der Behauptung, daB in der starken Bande von m/H = 30 bei Aluminiumphosphat positive Ionen von dem Metal1 vorhanden sind. Diesc! 6. Bei den Beobachtungen wurden fiir elm stets Werte gefunden, die diese angegebenen Arten von Ionen darstellen. h c k von Metz~eer d Wittlg in hipzig. (Eingegangen 1 5. Juni 191 7.) Beaultate. 1. Platin, Wolfram, Tantal und Aluminiumphosphat zeigen alle vier Abgabe von positiven Ionen, wahrend Calciumoxyd diesen Effekt nicht hat. 2. Platin, Wolfram und Tantal geben beim Anfang des Gllihzustandes mehr positive Ionen ab als Aluminiumphosphat ; jedoch nimmt dieser Effekt bei den drei Metallen nach einiger Zeit stark ab, wahrend bei dem Phosphat die Ionenabgabe lange und konstant stattfindet. 3. Bei siimtlichen untersuchten Drahten, die Ionen- emission zeigten, wurden positive Ionen sowohl vou Wasser- stoffatomen als auch von Wassentoffmolekulen gefunden. 4. Es wurden bei allen Drghten, die Ionenabgabe zeigten, Metallionen gef unden. 4. Es wurden bei allen Drghten, die Ionenabgabe zeigten, Metallionen gef unden. 5. Bei den Drkhten war eine Bande m/H=SO vorhanden; es liegen hierbei gewisse Vermutungen zugrunde, daB sie aus Sauerstoff und zum Teil aus Kohlenoxyd besteht. 6. Bei den Beobachtungen wurden fiir elm stets Werte gefunden, die diese angegebenen Arten von Ionen darstellen. 6. Bei den Beobachtungen wurden fiir elm stets Werte gefunden, die diese angegebenen Arten von Ionen darstellen. 048 W . Hiittemann. Die Emission von E l e k t r m usw. 7. Weiter wurde gezeigt, daB es bei den Versuchen am vorteilhaftesten ist, gleich die Drahte am Anfang des Gliih- eustandes zu beobachten. 7. Weiter wurde gezeigt, daB es bei den Versuchen am vorteilhaftesten ist, gleich die Drahte am Anfang des Gliih- eustandes zu beobachten. 8. Die Temperatur der Drlihte wurde am besten bei schwacher Rotglut gewiihlt. 8. Die Temperatur der Drlihte wurde am besten bei schwacher Rotglut gewiihlt. Die Arbeit ist auf Anregung und unter Leitung VOD Hrn. Pr.ofessor Dr. A. Wehnelt ausgefiihrt. Es sei mir ge- stattet, fiir die vielfache Unterstiitzung Hm. Professor W ehnel t meinen aufrichtigsten Dank auseusprechen. (Eingegangen 1 5. Juni 191 7.)
https://openalex.org/W3197768567	https://isprs-archives.copernicus.org/articles/XLVI-M-1-2021/147/2021/isprs-archives-XLVI-M-1-2021-147-2021.pdf	English	null	INVESTIGATION OF THE UNDERGROUND BUILDING HERITAGE AND THE MECHANISM OF WATER FLOWING IN <i>QANĀTS</i> IN PALERMO THROUGH INNOVATIVE SURVEYING TECHNIQUES	The international archives of the photogrammetry, remote sensing and spatial information sciences/International archives of the photogrammetry, remote sensing and spatial information sciences	2,021	cc-by	7,303	The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China 1. INTRODUCTION considerable complexity since it represents a unicum in terms of typology and construction. The valorization, protection and preservation policies for the underground building heritage are often difficult to implement due to an inadequate knowledge of hypogeal constructions. The fragmented and incomplete documentation is, often, due to the lack of systematization of multidisciplinary studies carried out in a unique typological context and related to only apparently episodic discoveries; to the difficulties to access to underground architectures and their complexity; to problems related to safety that require careful exploration programs. The indications formulated and codified in the Athens Charter and, more recently, in the Krakow Charter, define the guidelines for the conservation and enhancement of cultural heritage, emphasizing the strategic importance of multidisciplinary scientific approaches to the management of interventions in cultural heritage sites (De Naeyer, 2000). In different urban contexts like in Palermo, indeed, the presence of numerous underground cavities of anthropogenic origin that remain unknown could determine formation of chasms, collapses of streets or buildings above them and consequent disasters and/or inundation phenomena with possible loss of human lives. In particular, the system of groundwater connected conveyance tunnels are of particular interest in Palermo. A detailed survey of the underground systems is extremely important to properly know underground building heritage before eventual disaster can happen. However, the investigation of this underground aqueduct system is, therefore, of The valorization, protection and preservation policies for the underground building heritage are often difficult to implement due to an inadequate knowledge of hypogeal constructions. The fragmented and incomplete documentation is, often, due to the lack of systematization of multidisciplinary studies carried out in a unique typological context and related to only apparently episodic discoveries; to the difficulties to access to underground architectures and their complexity; to problems related to safety that require careful exploration programs. The indications formulated and codified in the Athens Charter and, more recently, in the Krakow Charter, define the guidelines for the conservation and enhancement of cultural heritage, emphasizing the strategic importance of multidisciplinary scientific approaches to the management of interventions in cultural heritage sites (De Naeyer, 2000). A renewed interest emerged from the studies carried out in the last years. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. ABSTRACT: The valorisation, protection and preservation policies for the underground building heritage are often difficult to implement due to an inadequate knowledge of hypogeal constructions. The complex and widespread underground structures of the vast “Cavo” Heritage (“horizontal wells”, “shelf wells” or “well tunnels”), so called qanāts, hidden underground and built over the centuries in Palermo, representing an evocative testimony to the history of water culture in the ancient city. Through the historical and constructive analyses and the implementation and development of measurement and 3D representation and visualization, first actions have been carried out. The paper will present the first results of the restoration project and the path of re-introduction in the fruition network of the qanāt “Gesuitico alto”, developed also in the field of “iHeritage. Mediterranean Platform for UNESCO Cultural Heritage” project, financed by ENI CBC MED Programme 2014-2020. The paper presents an experimentation of a procedural workflow of data acquisition, analysis and subsequent 3D virtual navigation of hypogeal environments. The methodology used is the SLAM with a last generation WMLS. The platform of virtual reality visualization, within UnReal Engine, allows the user to immerse and navigate in the anthropic environment by engaging it with a set of infographics that highlight the virtual visit. INVESTIGATION OF THE UNDERGROUND BUILDING HERITAGE AND THE MECHANISM OF WATER FLOWING IN QANĀTS IN PALERMO THROUGH INNOVATIVE SURVEYING TECHNIQUES R. Corrao 1, F. Di Paola 1, D. Termini 2, C. Vinci 1 1 DARCH, Department of Architecture, 90128 Palermo, Italy - (rossella.corrao, francesco.dipaola, calogero.vinci)@unipa.it 2 DING, Department Engineering, 90128 Palermo, Italy - donatella.termini@unipa.it KEY WORDS: Underground building heritage, qanāt, SLAM, wearable mobile laser system, 3D digitalization, visualization, cultural heritage. Corresponding author 1.1 Case Study: the qanāt “Gesuitico alto” The network of qanāts in Palermo develops in three different levels, both for the underground bottom depth and for the typology and construction techniques. These aspects, probably, depends on the progressive lowering of the water level and thus to the necessity to reach deeper underground depths to intercept the water. When the lowering of the water level was very rapid (e.g. earthquake in 1693), it was preferred to directly dig a new tunnel at a lower level, also with different construction techniques. This paper presents the preliminary results of the restoration project and the path of re-introduction in the fruition network of “Gesuitico alto” qanāt, an intricate underground architecture made of tunnels simply carved into the rock and/or defined with squared ashlars. Figure 1. “Corso Gesuitico Alto”. From the left, morphology of level 1, level 2 and level 3 sections of tunnels. Figure 1. “Corso Gesuitico Alto”. From the left, morphology of level 1, level 2 and level 3 sections of tunnels. Figure 2. “Corso Gesuitico Alto” in the urban context. (Survey: Gruppo Speleo CAI Palermo. Graphic restitution: INS Salvatore Sammataro, INS Silvia Sammataro - 2014). As often happens, in hypogeal buildings, the most superficial level (level 1) of tunnels is the oldest. The unaltered segment of the latter develops to the north for a length of about 155 m, to the south for 77 m, at a depth of about 6 m. The width, the height and the shape of the section of these tunnels are very variable. An extensive part of the qanāts was almost completely filled with material from the excavation of the lower tunnel. The tunnel level 2, at a depth of 8,50 m, has a length of about 110 m; the section is rectangular, it has variable height and its walls are without lining. Tunnel Level 3 is at a depth of 10 m and is 502 m long; the path partially overlaps that of level 1. For this reason, when the distance between the two tunnels became very small, they were joined into a single section with a height varying between 2.50 and 3 m. The tunnel path in levels 1 and 2 is moderately tortuous and deviates in correspondence with more compact rock masses. Level 3 has long straight stretches in which even the most compact rock masses have in many cases been excavated and demolished. 1. INTRODUCTION Today, this system results partially destroyed because of the construction of the new buildings of modern city that often did not properly considering its presence underground; for these 147 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China reasons its surviving traces are very vulnerable and they need to be preserved and valorized. reasons its surviving traces are very vulnerable and they need to be preserved and valorized. Under the umbrella of “iHERITAGE. Mediterranean Platform for UNESCO Cultural Heritage” project, financed by ENI CBC MED Program 2014-2020, a multidisciplinary investigation started with the aim to re-discovery and systematize all existing fragments, visible and “invisible”, epigean and hypogeal, of the historical system of water management of ancient Palermo. With the logistical and scientific support of “CAI Palermo” speleological group, which for years has been collaborating with the Superintendence of cultural and environmental heritage for the exploration of qanāt, the iHeritage research group is working for the development of an innovative tools that will allow the virtual fruition of qanāt also to people with disabilities that usually cannot have access to qanāt. truncated conical brick segments) of different sizes; barrage systems and numerous historical interventions of restoration and consolidation (figs. 1-2). Under the umbrella of “iHERITAGE. Mediterranean Platform for UNESCO Cultural Heritage” project, financed by ENI CBC MED Program 2014-2020, a multidisciplinary investigation started with the aim to re-discovery and systematize all existing fragments, visible and “invisible”, epigean and hypogeal, of the historical system of water management of ancient Palermo. With the logistical and scientific support of “CAI Palermo” speleological group, which for years has been collaborating with the Superintendence of cultural and environmental heritage for the exploration of qanāt, the iHeritage research group is working for the development of an innovative tools that will allow the virtual fruition of qanāt also to people with disabilities that usually cannot have access to qanāt. 2. MATERIAL AND METHODS The survey process cannot disregard the exploration and the non-invasive digital acquisition of underground environments that face the many logistical challenges of capture (presence of humidity, restricted and narrow areas, lack of light, absence of GPS signal, inability to stand or survey, etc.) to detect complex surfaces with high geometric resolution (Zlot et al., 2013; Roncat et al., 2011). Figure 1. “Corso Gesuitico Alto”. From the left, morphology of level 1, level 2 and level 3 sections of tunnels. 1.1 Case Study: the qanāt “Gesuitico alto” 1. INTRODUCTION Through the historical and constructive analyses and the implementation and development of measurement and 3D representation and visualization, first actions have been carried out for revaluating the hypogeal building heritage of Palermo, intended as a resource that can be exploited in a perspective of conservation and sustainable fruition. The complex and widespread underground structures of the vast “Cavo” Heritage (“horizontal wells”, “shelf wells” or “well tunnels”), so called qanāts, hidden underground and built over the centuries, representing an evocative testimony to the history of water culture in the ancient city of Palermo (Todaro, 1988; Vinci, 2018). The system of connected qanāts constituted an underground aqueduct able to transport water from an underground natural aquifer to the surface. It identified an original hydraulic system, very similar to the qanāt of the Middle East culture, allowing over the centuries the water supply to the city. In different urban contexts like in Palermo, indeed, the presence of numerous underground cavities of anthropogenic origin that remain unknown could determine formation of chasms, collapses of streets or buildings above them and consequent disasters and/or inundation phenomena with possible loss of human lives. In particular, the system of groundwater connected conveyance tunnels are of particular interest in Palermo. A detailed survey of the underground systems is extremely important to properly know underground building heritage before eventual disaster can happen. However, the investigation of this underground aqueduct system is, therefore, of Moreover, the network of qanāts represented the invisible part of a more complex combined system including the so called "Scirocco rooms", used in the villas built by the Palermitan aristocracy between XVI and XVIII centuries in the surrounding area of historical city center (the so called “Conca d’Oro”). This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. 1.1 Case Study: the qanāt “Gesuitico alto” This testifies, in addition to the presence of many parts covered with ashlars made of limestone, more advanced excavation and construction techniques. Figure 2. “Corso Gesuitico Alto” in the urban context. (Survey: Gruppo Speleo CAI Palermo. Graphic restitution: INS Salvatore Sammataro, INS Silvia Sammataro - 2014). The study evaluates the potential of the laser system employed, considering several parameters that influence the final quality of the 3D point cloud (identification of critical areas; prediction for loop closure; the speed of movement; the time taken to obtain the 3D point cloud; the density of the point cloud). 3D reality- based surveying instruments and techniques offers new and effective solutions for the 3D modeling of hypogeal environments (Galeazzi et al., 2014). The study evaluates the potential of the laser system employed, considering several parameters that influence the final quality of the 3D point cloud (identification of critical areas; prediction for loop closure; the speed of movement; the time taken to obtain the 3D point cloud; the density of the point cloud). 3D reality- based surveying instruments and techniques offers new and effective solutions for the 3D modeling of hypogeal environments (Galeazzi et al., 2014). In recent years, metric surveying applications with hybrid solutions, such as mobile mapping systems (MMS), have raised great capabilities and possibilities (Nocerino et al., 2017; Vaaja, Virtanen, 2017). In addition to advances in optical sensors, which minimize error propagation, one of the key advances in MMSs is related to spatial reference technology. The lining walls and the covers of the tunnel are made of dry limestone ashlars; this guarantees the stability of the excavation walls in case of not very resistant soils and, at the same time, the drainage of the waters, that occasionally infiltrates from the side walls increasing the flow of the tunnel. The coverage of the channel, in the coated sections, is made as “bocca di forno” type: two ashlars inclined forming a triangular profile. In this way, a pushing structure is created that stabilizes the two side walls of tunnel and it allows to deposit the excavated material on the extrados of the ceiling, avoiding the costly and tiring transport out of the tunnel. In recent years, metric surveying applications with hybrid solutions, such as mobile mapping systems (MMS), have raised great capabilities and possibilities (Nocerino et al., 2017; Vaaja, Virtanen, 2017). 1.1 Case Study: the qanāt “Gesuitico alto” In addition to advances in optical sensors, which minimize error propagation, one of the key advances in MMSs is related to spatial reference technology. In this category of equipment, especially in complex contexts with enclosed and difficult-to-access spaces, which are common morphological features in Cultural Heritage, wearable mobile laser systems (WMLS) present themselves as a potential solution for mapping indoor environments, as a 3D point cloud p “Corso Gesuitico alto” is, furthermore, a rare testimony of the ancient Sicilian water culture; in fact, it preserves many characteristic elements: the pit for the senia at the end of level 2; the serial aeration pits; a repertoire of catusi (pipes with 148 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China walks through the area of interest. This wearable system combines laser scanning technology (2D time-of-flight laser scanner) and an inertial measurement unit (IMU) in a portable equipment that can be managed by an operator while walking around the site. The sensor captures moving point clouds using Simultaneous Localization and Mapping (SLAM) algorithms (Bosse et al., 2012) without the need for global navigation satellite system (GNSS) support (fig. 4). With a 360° vertical field of view and 60-80 m maximum range under indoor conditions (which was reduced to 5-10 m under real-world working circumstances), the operator moved within the hypogeal section capturing about 300.000 points per second. Regarding accuracy, the manufacturer stated it to be 1-3 cm in relative terms and 1-30 cm in absolute positioning for a 10 min scan with a single loop closure. Table 1 shows additional technical specifications. of the environment can be obtained with an accuracy of cm precision. However, this accuracy could be strongly affected by trajectory characteristics, such as the speed of movement or the path followed (Lagüela et al., 2018). In this specific field of investigation, the paper presents an experimentation of a procedural workflow of data acquisition, analysis and subsequent 3D virtual navigation of hypogeal environments in Palermo. In the following paragraphs will be described the workflow tested, and the considerations that determined the parameter settings and survey strategies. Figure 4. Main components of the wearable mobile laser systems (WMLS) used for data acquisition. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. 1.1 Case Study: the qanāt “Gesuitico alto” The phases of the acquisition process, 3D data analysis, management and optimization of the digital model set up will be described to obtain a realistic scenario of the hypogeal architectural system for the creation of a virtual tour in gaming modality (Kalay et al., 2007). Computer Graphics, Web Design and Gaming technologies and with high-performance devices in VR, AR and Mixed Reality allow to generate applications for high-resolution visualization of three-dimensional environments with the ability to navigate, analyse and query virtual models in real time, allowing a navigation experience even for users with physical or perceptual disabilities or special pathologies. Moreover, now more than in the past it is possible to download VR and AR on mobile devices not only for gaming or Cultural Heritage (CH) dissemination, but also for supporting decision-making processes and for planning intervention actions to manage and monitor sites in danger, such as the case study related to Palermo qanāts. Figure 4. Main components of the wearable mobile laser systems (WMLS) used for data acquisition. The paper presents a prototype of the virtual reality visualization platform, implemented within the environment UnReal Engine (by Epic Games), allows the user to immerse and navigate in the anthropic environment by engaging it with a set of infographics that highlight the virtual visit by explaining construction techniques and materials (fig. 3). Figure 3. Methodological phases of the procedural pipeline. Zeb Horizon features Parameters Value Range 100 m Protection class IP54 Processing Post Data logger carrier Backpack or shoulder strap Payload 3,7 Kg Scanner points per second 300,000 No. of sensors 16 Relative accuracy 1-3 cm Raw data file size 100-200MB a minute Colourised point cloud ● Referenced imagery ● Automated scanner start ● Field of view 360° x 270° Operating temperature 0° C to +50° C Table 1. Technical specification of the Geo Simultaneous Localization and Mapping (SLAM) ZEB-HORIZON device. Figure 3. Methodological phases of the procedural pipeline. Table 1. Technical specification of the Geo Simultaneous Localization and Mapping (SLAM) ZEB-HORIZON device. Table 1. Technical specification of the Geo Simultaneous Localization and Mapping (SLAM) ZEB-HORIZON device. 2.1 Equipment: The methodology used is the SLAM (Simultaneous Localization and Mapping) with a last generation wearable mobile laser scanner. The WMLS tested in the case study is the Zeb Horizon, commercialized by GeoSLAM (Di Filippo et al., 2018). The instrument provides a rapid means of capturing 3D point cloud data. The data is captured as the user 2.2 Acquisition and survey design: The analysis of this complex underground building heritage requires the planning of a specific methodology of investigation. The first step required a preliminary check carried out with a series of inspections, aimed at identifying potential critical areas This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. 149 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China scanning process (recommended at a maximum of 30 minutes) (figs. 6-7). In addition to trajectory calculation, prior planning was done to place the mobile device in an environment that had suitable and safe morphological characteristics. during the scanning process (obstructed accesses during the path; flooding; danger of collapse; undercuts; prohibitive conditions in relation to the SLAM instrument range). The geometric-formal complexity of the site (rocky and jagged structure with few identifiable landmarks; scarcity of light and operating space; presence of water at different depths) and the type of articulated and practicable path in a single direction of travel have imposed a strategy that would allow to close the loop and, at the same time, to detect the undercut areas. The tunnel has an almost constant width of about 70 cm, while the average height is more than 2 m, but with stretches high only 1 m and a half, which forces to continue the tour with the bust tilted forward. Figure 6. Survey design with the WMLS. Strategy of "round- trip" trajectory to close the loop and to detect the undercut areas. For a correct processing, it is, in fact, required that the operator starts and ends the survey in the same position to ensure the closure of the loop. Before starting the acquisition phases, simulations were carried out to evaluate some factors that could have affected the quality of the data flow (speed of movements during the path, variable heights and obstacles that would have slowed down the operator's movements) (fig. 5). Figure 5. The geometric-formal complexity of the site (Photographic archives CAI Palermo Speleological Group). Figure 6. Survey design with the WMLS. Strategy of "round- trip" trajectory to close the loop and to detect the undercut areas. Figure 7. Some phases of the survey in “Gesuitico alto” qanāt. Figure 5. The geometric-formal complexity of the site (Photographic archives CAI Palermo Speleological Group). During scanning phase, great care was taken with the forward speed to ensure good coverage and high-resolution data. Actually, it is known, that if the forward motion is too fast, there may not be enough repeated feature scans for the SLAM algorithm to process the raw laser data into a point cloud. In addition, to ensure high data density and not to incur errors while transitioning through survey areas of the qanāt, the scan head was kept stationary for short periods of time to acquire difficult and limited access points. The SLAM algorithm used to process the raw laser scanning data into a 3D point cloud relies on there being elements in the scanned environment that are repeatedly scanned as the operator progresses along the path. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. This contribution has been peer-reviewed. For an element to be significant, the ratio of its size to its range must be about 1:10, e.g. at 5m for an element to be significant it must have a size >0.5m. In the case study under consideration, excessively narrow spaces or smooth walls are classified as "element-poor" environments. Under these circumstances, there may not be sufficient elements in the direction for the SLAM algorithm to determine forward motion. So, to avoid algorithm processing errors, a route was planned that involved repeated scanning of feature-poor elements. Figure 7. Some phases of the survey in “Gesuitico alto” qanāt. In addition, the coexistence of confined spaces and complex larger areas required special attention to instrument management in the field. To avoid the recording of large files and reduce possible error propagation, we tried to calibrate, also, the speed of the movements with the duration of the 150 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China Increasing these parameters solved some processing errors, but at the expense of processing time. 2.2.1 Data Processing and Registration: As mentioned above, the instrument captures raw laser distance and inertial data. The GeoSLAM Draw solution for efficient processing of point clouds to create detailed 2D plans and raster views was used to visualize the first results (at depths of field different from the section plane). This initial graphical setup enabled practical functions for measuring and querying the hypogeal system. This data must be processed using GeoSLAM's SLAM algorithm to convert the raw data into a 3D point cloud. At registration, the data is processed using the processing application GeoSLAM Hub. To understand the performance of the GeoSLAM algorithm, it is appropriate to describe the general characteristics of the algorithm and its operation. 2.2.2 Post-Processing: After processing the dataset in GeoSLAM Hub, the next step of the elaboration process was to export the point cloud data in LAS format to be processed in other point cloud processing software. It is important to specify that the format chosen for the export does not support the calculation of the points normals and, therefore, this operation, that is fundamental for the creation of the final model mesh, was carried out later (fig. 10). The trajectory reproduced by the software describes the position of the sensor during data acquisition. The 3D SLAM algorithm combines 2D laser scanning data with IMU data, returning accurate 3D point clouds. Data processing requires an incremental procedure (segments are recorded one by one), like the well-known iterative nearest point (ICP) algorithm (Di Filippo et al., 2018). The registration phase has imported and processed the captured dataset within the proprietary software. This operation allowed the 3D point cloud to be visualized and post-processed. Given the critical nature of the unique and typologically singular scan area, it was necessary to initiate re-processing options that compensated for drifts and slips caused by inconsistent data due to the presence of scanned areas with few significant features in the 10 m range. Figure 10. Workflow processing and data integration. Some parameters (Local and Global Registration Options) have been set to optimize the final data and compensate for any scanning errors. Two important modified parameters are reported: “Convergence Threshold” and “Voxel Density”. Figure 10. Workflow processing and data integration. The first one grows the maximum number of iterations per processing step and reduces the convergence threshold during the local registration phase. The second one, however, controls the size of the voxels (cubes) into which the data is divided. Increasing the voxel density decreases the voxel size. Increasing the voxel density will allow more detail to be detected at close range and used during SLAM processing (figs. 8-9). The next step after the first data recording was to process the point cloud in the well-known point cloud processing software, CloudCompare (2.12 alpha). The software was mainly used to optimize the cloud, applying the tools (segmentation, distance computation, spatial operators, colours, normal compute, scalar fields) and plugins, managing the classic octree structure of the program. The main objective was to extrapolate vector profiles in cross-sections orthogonal to the trajectory and longitudinal (fig. 11). Figure 8. GeoSLAM Draw interface. Top view in orthogonal projection of the point cloud of the “Gesuitico Alto” qanāt. Figure 11. Scalar Fields gradient output and histogram dialog. Figure 8. GeoSLAM Draw interface. This contribution has been peer-reviewed. archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. CloudeCompare processing Parameters Value Edit Subsample Method space Min space between points 0,04 Tools Clean SOR Filter section 1 section 2 section 3 Average distance 100 nSigma 5 section 4 Average distance 100 section 6 nSigma 10 section 5 Average distance nSigma 100 8 Table 2. Point cloud processing in CloudCompare. Segmentation, applied tools and set parameters. Figure 13. Graphic restitution of the different types of hypogeal sections. 2.2.4 Surface mesh reconstruction: Mesh surface reconstruction from oriented points presents several difficulties that affect the result. The sampling of the points is not uniform. Table 2. Point cloud processing in CloudCompare. Segmentation, applied tools and set parameters. Table 2. Point cloud processing in CloudCompare. Segmentation, applied tools and set parameters. The directions of the normals are not homogeneous due to the imprecision of the sampling. Significant logistical difficulties during survey operations and the presence of undercut and inaccessible areas resulted in some regions of the surface being poor in information. 2.2.3 Plan and Section Restitution: Having still maintained a high density for the reference cloud, longitudinal profiles and a plan profile were extracted. Here again, as with all other procedures, they were performed on a section-by-section basis. Instead to define multiple orthogonal section profiles, it would have been more convenient to define a single path and automatically generate sections orthogonal to the trajectory, travelled by the operator during the scan, at regular intervals (Tools -> Segmentation -> Extract sections). 2.2.3 Plan and Section Restitution: Having still maintained a high density for the reference cloud, longitudinal profiles and a plan profile were extracted. Here again, as with all other procedures, they were performed on a section-by-section basis. The procedural steps of post-processing and tessellation of the polygonal mesh were processed within the Leios software (developed by an Italian company, EGS). Using the cloud management and correction tools, overlapping and redundant vertices were detected and eliminated. Given these unfavourable conditions, different methods with different algorithms (CloudCompare: Poisson Surface Reconstruction and Leios) were employed to reconstruct the surface topology, evaluating the most reliable results in terms of accuracy of the resulting model geometry. To maintain more control, it was preferred to use the "cross section" command which segments the selected clouds into smaller parts separated by an assigned distance of 1 m (in total 103 profiles were extracted). This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. Top view in orthogonal projection of the point cloud of the “Gesuitico Alto” qanāt. Figure 8. GeoSLAM Draw interface. Top view in orthogonal projection of the point cloud of the “Gesuitico Alto” qanāt. Figure 9. GeoSLAM Draw interface. Side section view in orthogonal projection of the point cloud of “Gesuitico Alto” qanāt. Figure 11. Scalar Fields gradient output and histogram dialog. To manage the amount of data more efficiently, it was appropriate to segment the cloud into six sections, determined in relation to the morphological characteristics of the entire path. To filter the data, some specific algorithms were used, following a methodological procedure with values and processing criteria for all the portions identified with the aim of eliminating duplicate points and “outliers” by reducing redundant points, while maintaining the cloud with a high information density (the procedures adopted allowed to reduce the cloud from 123,790,232 points to 72,680,052 points). In specific, two commands were used, respectively, for the above operations: “subsample a point cloud” and the “SOR filter”, in the section tools clean. Figure 9. GeoSLAM Draw interface. Side section view in orthogonal projection of the point cloud of “Gesuitico Alto” qanāt. This contribution has been peer-reviewed. 151 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China Figure 13. Graphic restitution of the different types of hypogeal sections. Table 2 shows the values applied for each of the sections. It should be noted that it was not necessary to use the Noise Filter algorithm, capable of reducing noise, since the raw cloud produced by the SLAM methodology was well structured. CloudeCompare processing Parameters Value Edit Subsample Method space Min space between points 0,04 Tools Clean SOR Filter section 1 section 2 section 3 Average distance 100 nSigma 5 section 4 Average distance 100 section 6 nSigma 10 section 5 Average distance nSigma 100 8 Table 2. Point cloud processing in CloudCompare. Segmentation, applied tools and set parameters. p https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. This contribution has been peer-reviewed. It was not possible to establish this distance with the first procedure mentioned, that gave only the option to determine the number of sections to be made. Our interest was to get more sections at the same time, so we operated through the command “export multiples slices”. Obtained file was finally processed in Rhinoceros to obtain the plan and the cross sections of the of the underground building (figs. 12-13). Considering the surface geometry (curvature, contiguity edges and polygonal mesh density) the spatial structure of the model undergoes corrections and some artifacts due to the scanning process are eliminated. The process eliminates recurring topological errors in the geometry. 2.2.5 The platform of virtual reality visualization: The current procedures of study and preservation of CH have as a scientific reference the acquisition of data and their digitization, aimed at the development of applications for use and knowledge. In this field, the video game industry has contributed in a surprising and attractive way to communicate CH with visual and interactive tools, involving the public. The so-called “serious games” are an effective tool, especially for the new generations, which elaborates, in a single context, the technological and historical-cultural issues of a topic. Therefore, the video game is one of the valuable resources essential to transmit knowledge and culture. The game environment needs continuous contributions of platform implementation and technological and procedural development. Especially in the case study addressed the design of a graphical and interactive navigation interface, allows to overcome physical, logistical and perceptual barriers, making everyone able to enjoy the underground heritage (Di Paola et al., 2019). Figure 12. The plan and the cross sections of the qanāt. Figure 12. The plan and the cross sections of the qanāt. The interface set up, within the environment UnReal Engine (by Epic Games), allows navigation in two interaction modes. 152 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China the urban macro-scale, where the presence of the cavity may cause collapses and/or disruption to overlying structures, and the small scale related to safety within the cavity itself. 3. DISCUSSION Paradoxically, in urban areas, the discovery of many hypogeal buildings is most often due to localized instability or collapses that appear on the street surface, to fortuitous discoveries following excavations and, only more rarely, to targeted research aimed at verifying references found in archival and bibliographic sources. The detailed reconstruction of the qanāts network used for water supply not only has a strong historical interest but it is especially important to reconstruct the water flowing conditions also preventing disaster events. Figure 16. Deterioration due to sidewall collapse and degradation due to root action (Photographic archives CAI Palermo Speleological Group). If the typological-functional features of qanāts and serial pits have already been the subject of numerous studies and publications, the constructive aspects and the evaluation of recurrent degradations and instabilities have not been sufficiently investigated. The typological and functional recognition of a historical hypogeal buildings allows to foresee possible risks during exploration, study and eventual fruition, and allows to identify, interpret or predict some recurrent degradation and instability phenomena and possible risks for primary and secondary safety. The assessment of primary safety for hypogeal structures can be evaluated at two different scales: The procedure will allow to capture the depth relationship between the underground structures and the ground. Orientation is being recorded using topographic and GPS techniques, taking advantage of the entrances of the two entrance shafts. Figure 16. Deterioration due to sidewall collapse and degradation due to root action (Photographic archives CAI Palermo Speleological Group). Figure 15. UnReal Engine interface, gaming navigation mode inside the qanāt. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. The first one allows the geo-referencing of the 3D model with infographics and 2D documentation (images, archive photos, diagrams, and drawings), through reference tags. The user can interact with the implemented environmental context with heterogeneous infographics. The second mode allows the user to immerse himself in a realistic scenario and navigate inside the qanāt in game mode, simulating, for example, the light of the protective hat (figs 14-15). Contrary to underground caves of considerable size, qanāts are generally characterized by a more linear planimetric pattern and smaller cross sections. With regard to instabilities, these can derive either from intrinsic causes (such as congenital weaknesses in the construction phase) or from indirect causes due to environmental conditions changes (such as water level variations, inflowing water dispersed by aqueducts and sewers) or to anthropic alterations. In the latter case the alteration can be either of direct type on the artefact (robbery quarries, interference with new excavations, deep foundation construction), or because of lack of maintenance (silting up of the tunnels), or of indirect type determined by a variation of the channels shape (variation of loads due to new construction, planting of tall trees with invasive root systems) (Cristiano, 2017; Ferriello, 2006). Figure 14. UnReal Engine interface, integrated navigation mode. The geo-referencing of the 3D model with infographics and 2D documentation, through reference tags. The paper contributes to the definition of an innovative methodology, aimed at a multidisciplinary reading for the protection and conservation of this fragile and precious invisible Heritage (fig. 16). By using the collected data the reconstruction of the qanāt “Gesuitico alto” has been operated identifying very narrow cross-sections. It should be noted that the shape of the cross-section could strongly affect the flow velocity and, consequently, the presence of sand/sediments deposited on the bottom. Sediments deposition can determine a progressive reduction of the effective flowing section and the transition to under pressure flow motion. The detailed identification of the longitudinal channel’s bottom slope is also important both for identifying flow velocity and for evaluating the potential interactions with monitoring operations. Figure 14. UnReal Engine interface, integrated navigation mode. The geo-referencing of the 3D model with infographics and 2D documentation, through reference tags. Figure 15. UnReal Engine interface, gaming navigation mode inside the qanāt. The georeferencing phase of the acquired data of the hypogeal system with the surface terrain is in progress. 2/W15, 421–428, https://doi.org/10.5194/isprs-archives-XLII-2- W15-421-2019. 2/W15, 421–428, https://doi.org/10.5194/isprs-archives-XLII-2- W15-421-2019. recovery that takes into account, where feasible, the possible rehabilitation of the original function and leads to an enhancement of this heritage as a resource, even in a perspective of sustainable use. Although less easy to visit, the qanāts, especially the “Gesuitico Alto” one, are another attraction for many users who are simply attracted by the fascinating similarity with Middle Eastern culture, and for scholars from different disciplines who see their interests converge around the theme of the relationship between city and water. With respect to this theme, the valorisation is oriented towards the rediscovery and systematization of all the surviving elements, visible and invisible, of the historical “system” of water management in Palermo, that, often, fed the fishponds of many Normans palatial buildings. In this case, the need for conservation is not determined by the “value” of the single artifact, but by a broader vision of the complex relationship between Norman buildings (that already exist), the city and water resources, also in relation to the temporal and cultural transversality that characterizes it. In the field of iHeritage project the investigation of qanāts and hypogeal building heritage of Norman palaces in Palermo will be further implemented and development of measurement and 3D representation and visualization will be carried out with the aim to spread knowledge of Norman epigeal and hypogeal building heritage and allow to many people as possible to visit them with the support of VR and/or AR tools. Ferriello, G., 2006. L'estrazione delle acque nascoste. Scientific and technical treatise di Karaji. Torino: Kim Williams Books. Galeazzi F, Moyes, H., Aldenderfer, M., 2014. 3D Documentation in Archaeology: Recording Las Cuevas Site, Chiquibul Reserve, Belize. In: Archaeology in the Digital Era Volume II, Amsterdam University Press, Amsterdam, pp. 350- 362. GeoSLAM Technology, ZEB-Horizon Solution Brochure. Available online: https://geoslam.com/wp-content/uploads/2020/07/ZEB- Horizon-product-card.pdf (accessed on 25 June 2021). Kalay, Y.; Kvan, T.; Affleck, J., 2007. New Heritage: New Media and Cultural Heritage, 1st ed.; Routledge: London, UK, ISBN 978-0415773560. Lagüela, S., Dorado, I., Gesto, M., Arias, P., González- Aguilera, D., Lorenzo, H., 2018. Behavior Analysis of Novel Wearable Indoor Mapping System Based on 3D-SLAM. Sensors, 18, 766. https://doi.org/10.3390/s18030766. Lehtola, V.V., Kaartinen, H., Nüchter, A., Kaijaluoto, R., Kukko, A., Litkey, P.; Honkavaara, E., Rosnell, T., Vaaja, M.T., Virtanen, J.-P., 2017. Comparison of the selected state-of- the-art 3d indoor scanning and point cloud generation methods. Remote Sens. 2017, 9, 796. ACKNOWLEDGEMENTS The Authors thank the Microgeo Srl Company (www.microgeo.it) who provide us the ZEBHorizon system, in particolar thank to Francesco Battimelli e Gianfranco Lupo. The Authors thank, also, the Speleo Team CAI Palermo for its precious assistance in the measurement campaign. Nocerino, E., Menna, F., Remondino, F., Toschi, I., Rodríguez- Gonzálvez, P., 2017. Investigation of indoor and outdoor performance of two portable mobile mapping systems. In Videometrics, Range Imaging, and Applications XIV; International Society for Optics and Photonics: Munich, Germany. REFERENCES De Naeyer, A., Arroyo, S.P., and Blanco, J.R., 2000. Krakow Charter 2000: Principles for Conservation and Restoration of Built Heritage. Available online: De Naeyer, A., Arroyo, S.P., and Blanco, J.R., 2000. Krakow Charter 2000: Principles for Conservation and Restoration of Built Heritage. Available online: Roncat A., Dublyansky Y., Spotl, C., Dorninger P., 2011. Full- 3D surveying of caves: A case study of Marchenhohle (Austria). Proc. IAMG2011 Conference, pp. 1393-1403. http://hdl.handle.net/1854/LU-128776 (accessed on 25 June 2021). http://hdl.handle.net/1854/LU-128776 (accessed on 25 June 2021). Todaro, P., 1988. Il sottosuolo di Palermo, Dario Flaccovio Editore, Palermo. Beraldin, J.A., Picard, M., Bandiera, A., Valzano, V., Negro, N., 2011. Best Practices for the 3D Documentation of the Grotta dei Cervi of Porto Badisco, Italy. Proc. SPIE, Vol. 7864, pp. 78640J-78640J-15. Vinci, C., 2018. Il patrimonio "cavo". Architetture ipogee e spazio idraulico nella piana di Palermo. In ReUSO 2018. L'intreccio dei saperi per rispettare il passato, interpretare il presente, salvaguardare il futurro, pp. 997-1004. Roma, Gangemi Editore International. Bosse, M., Zlot, R., Flick, P. Zebedee, 2012. Design of a spring- mounted 3-d range sensor with application to mobile mapping. IEEE Trans. Robot, 28, 1104–1119. Zlot, R., Bosse, M., Greenop, K., Jarzab, Z., Juckes, E., Robert, J., 2013. Efficiently capturing of large, complex Cultural Heritage sites with a handheld 3D mobile laser mapping system. In: Journal of Cultural Heritage, Vol. 15(6), pp. 670-678. Cristiano, M., 2017. La conoscenza del sottosuolo di Napoli per la mitigazione del rischio di crolli e dissesti. In: Proceedings of Colloqui.AT.e. Demolition or reconstruction. Monfalcone: EdicomEdizioni. 4. CONCLUSIONS During the 20th century, the lack of maintenance and the loss of interest in the “invisible” built heritage and the ancient water supply system of Palermo – considered unhygienic – have physically erased, and even from memory, the existence of many hypogeal buildings. Today a new interest seems to emerge from the studies produced in recent years. These studies direct towards a 153 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLVI-M-1-2021 28th CIPA Symposium “Great Learning & Digital Emotion”, 28 August–1 September 2021, Beijing, China This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. APPENDIX Di Filippo, A., Sánchez-Aparicio, L.J., Barba, S., Martín- Jiménez, J.A., Mora, R., González Aguilera, D., 2018. Use of a Wearable Mobile Laser System in Seamless Indoor 3D Mapping of a Complex Historical Site. Remote Sensing, MDPI, 10 (12), https://doi.org/10.3390/rs10121897. Author Contributions: All authors conceived and designed the experimental campaign; all authors wrote the introduction, the discussion and the conclusions; F.D.P. performed and wrote the pre-processing and post-processing steps. Di Paola, F., Inzerillo, L., and Alogna, Y., 2019. A gaming Approach for Cultural Heritage knowledge and dissemination, Int. Arch. Photogramm. Remote Sens. Spatial Inf. Sci., XLII- Funding: This paper was also framed within “iHERITAGE. ITC Mediterranean Platform for UNESCO. Cultural Heritage” project (B_A.2.1_0056), financed by ENI CBC MED Program 2014-2020. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLVI-M-1-2021-147-2021 \| © Author(s) 2021. CC BY 4.0 License. 154
https://openalex.org/W4224261128	https://iris.unige.it/bitstream/11567/1080126/1/smartcities-05-00027-v2.pdf	English	null	Harmonic and Supraharmonic Emissions of Plug-In Electric Vehicle Chargers	Smart cities	2,022	cc-by	16,344	smart cities smart cities Citation: Mariscotti, A. Harmonic and Supraharmonic Emissions of Plug-In Electric Vehicle Chargers. Smart Cities 2022, 5, 496–522. https://doi.org/10.3390/ smartcities5020027 Academic Editors: Emilio José Palacios-García and Antonio Moreno-Munoz Received: 1 February 2022 Accepted: 6 April 2022 Published: 9 April 2022 Citation: Mariscotti, A. Harmonic and Supraharmonic Emissions of Plug-In Electric Vehicle Chargers. Smart Cities 2022, 5, 496–522. https://doi.org/10.3390/ smartcities5020027 Keywords: distortion; electric vehicles; electric vehicle charger; harmonics; low-voltage grid; power quality; supraharmonics Article Andrea Mariscotti Dipartimento di Ingegneria Navale, Elettrica, Elettronica e delle Telecomunicazioni—DITEN, University of Genova, 16145 Genova, Italy; andrea.mariscotti@unige.it Abstract: Electric vehicle (EV) charging represents a relevant electric load with a rapid evolution in terms of number, power rating and distortion, in particular, considering the connection to the low-voltage public grid: available short-circuit power may be limited and particularly susceptible loads may co-exist in the same grid portion. Standards can partially address the problem cover- ing only the harmonic interval, but they necessitate signiﬁcant extension and improvement in the supraharmonic range. In addition, EV chargers have been observed to violate in some scenarios the applicable harmonic limits, so that the mechanisms of emission and distortion should be better understood and evaluated, including phenomena of mutual inﬂuence between EV chargers and with pre-existing grid distortion. Although models can help simulate large-scale scenarios in terms of fundamental frequency phenomena, such as power ﬂow, voltage ﬂuctuation and imbalance, sub- stantial and reliable information can come from experimental results, providing measured harmonic and supraharmonic emissions, accompanied by details on loads mix, grid characteristics and EV charger operating conditions. This work thus deﬁnes the applicable constraints in terms of limits and compatibility levels for public and light industrial low-voltage grids, discusses the available experimental results and datasets, analyzing the typical distortion behavior and providing indication of sources of information for further studies. Citation: Mariscotti, A. Harmonic and Supraharmonic Emissions of Plug-In Electric Vehicle Chargers. Smart Cities 2022, 5, 496–522. https://doi.org/10.3390/ smartcities5020027 Academic Editors: Emilio José Palacios-García and Antonio Moreno-Munoz Received: 1 February 2022 Accepted: 6 April 2022 Published: 9 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1. Introduction g , , g p ) In particular, the following technical aspects have been evaluated in the available literature: g g p ) In particular, the following technical aspects have been evaluated in the available literature • Voltage unbalance, troublesome in the case of numerous, albeit low-power, single- phase charging loads; • Voltage unbalance, troublesome in the case of numerous, albeit low-power, single- phase charging loads; p g g • Voltage ﬂuctuation, in particularm caused by fast charging and uncoordinated charg- ing sessions [5,6]; • Veriﬁcation of suitability of LV distribution elements, such as MV/LV cabin transform- ers and feeders; • Dynamic peak shaving and exploitation of renewables and smart storage [7], including integration with other electriﬁed transportation systems [8,9]; g p y • Balancing power demand by economic incentives and regulations to push the peaks of exploitation to more attractive times of the day. Many of the works addressing even the most technical and low-level interfaces and phenomena are based on simulated data, for an obvious reason of availability and readi- ness [10–13]. It is recognized that simulated charging patterns and proﬁles on the one hand may be realistic, representative and quite close to the measured data available, and on the other hand, may allow the application of induced artiﬁcial variability, probabilistic behavior and statistical dispersion, which would not otherwise be possible with purely experimental data. The accuracy and reliability of simulated data and synthetic time series are discussed among others in [14,15], focusing on EV load, charge proﬁle and locations, without exploring the details of the EV electric behavior. This was carried out in [16], integrating EV and LV distribution grid models from a probabilistic perspective. g g g p p p The impact on the electric distribution network, however, should be also evaluated and discussed in Power Quality (PQ) terms, as EV charging represents a signiﬁcant source of distortion. Especially for integration with renewable source and storage and for decoupling from the public AC grid, DC grids are a signiﬁcant viable solution [17–19], that deserves attention in terms of enhancing PQ and efﬁciency; this is a solution currently under investigation for ultra-fast charging. 1. Introduction It is established that the number of electric vehicles (EVs) for private and public mobility is increasing, not only in absolute terms, but also compared to the total ﬂeet of circulating vehicles [1,2]. This has been fostered also by economic incentives, a slight reduction of prices and the incoming publicized ban for vehicles running on conventional fossil fuels: some cities announced circulation prohibition for such vehicles around 2030, as well as entire countries announcing a stop of sales, following the “green package” that the European Union disclosed on 14 July 2021 [3]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. y EV advantages are well recognized mainly as [4]: EV advantages are well recognized mainly as [4]: • Ecologically friendly with negligible direct polluting emissions; • Indirect emissions are also lower, if more efﬁcient generation at the source and the possibility of exploiting renewables are considered; • Higher energy efﬁciency and better performance than conventional fossil-fuel vehicles; • Reduced noise, especially at low speed amid cities. Copyright: © 2022 by the author. 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/). On the other hand, disadvantages reside in a still uncertain duration of batteries and the way they can be recycled or disposed of, the convenience and duration of a recharge operation compared to conventional refuel at the gas station, and the overall impact on the existing electric network, in addition to the necessary improvements in terms of planning, infrastructure and operation. On this latter aspect, there have been signiﬁcant efforts for https://www.mdpi.com/journal/smartcities Smart Cities 2022, 5, 496–522. https://doi.org/10.3390/smartcities5020027 Smart Cities 2022, 5 497 simulating and evaluating the power demand, its dynamic behavior and the adequacy of the electric infrastructure (in particular the Low-Voltage, LV, distribution network and the Medium-Voltage, MV, feeding points). simulating and evaluating the power demand, its dynamic behavior and the adequacy of the electric infrastructure (in particular the Low-Voltage, LV, distribution network and the Medium-Voltage, MV, feeding points). 2. EV Charging Modes and Architectures From a general high-level standpoint, we may distinguish, ﬁrst of all, between Con- ductive Charging (CC) and Wireless Charging (WC), or more generally, Wireless Power Transfer (WPT) to include feeding of vehicle during run, besides charging its on-board battery. In terms of performance, both approaches require a large amount of deployed power, or that charging times be compatible with typical acceptable waiting times at the gas station for the former and acceptable vehicle speed when running over the WPT coil system for the latter. Charging at home during, e.g., the whole night, of course, does not need such power level, also to necessarily accommodate for the lower available power at the feeding point (higher short-circuit impedance of residential LV distribution). The CC high-level advantages and disadvantages are well summarized in Table 1 of [13]: • Different charging power levels may be negotiated during one charge session thanks to the communication channel between the intelligent socket and the on-board logic; it is observed that, for example, fast charging may be allowed only when grid conditions are suitable, such as when there is no risk of abnormal voltage ﬂuctuations and grid instability; • Communication supports also the V2G (vehicle-to-grid) mode [24], particularly useful when the EV is available connected for long times (such as a work day or an entire night at ofﬁce and home, respectively); the V2G operation, however, reduces expected battery life; • Higher efﬁciency compared to, e.g., wireless power transfer at the cost of a direct injection of distortion into the grid. Focusing on CC, a distinction may be drawn between off-board and on-board charging conﬁgurations. The latter is the most relevant, with on-board chargers differing from vehicle to vehicle and connecting directly to the LV distribution grid. The off-board charger has instead an optimized installation for the dedicated feeder it is connected to, which is then more tolerant to distortion and conducted disturbance thanks to the much lower short-circuit impedance. p In agreement with IEC 61851-1 [25], charging modes range from 3.7 kW to 43 kW, with an additional 200 kW ultra-fast charge mode (mode 4) [17,26]. The ﬁrst three modes are compatible with AC feeding points: single phase of 16 A and 32 A (corresponding to mode 1 and 2, with 3.7 and 7.4 kW, respectively) and three-phase socket of 32 A and 63 A (corresponding to mode 2 and 3, with 22 and 43 kW, respectively). 1. Introduction However, considering an ubiquitous, direct and presently ready implementation of EV charging, the connection to the public AC grid is the most relevant interface, for which some points should be taken in due consideration: • On-board chargers may differ signiﬁcantly among EV models, especially considering the span of fabrication date and of power levels, from small vehicles to sport cars and to public transportation means; • Distortion patterns may change during the charging process as a function of the state of charge, of the absorbed current intensity and the charging mode; • For the widespread use of power converters and smart conversion methods to enhance ﬂexibility and efﬁciency, distortion patterns are not only various, but extend signiﬁ- cantly in the supraharmonic range (namely, 9–150 kHz), where switching components and their harmonics are easily visible; y • Superposition of low- and high-frequency distortion and secondary emissions (distor- tion caused by a converter subject to network voltage distortion, such as caused by other converters) are more important as the mix of connected EV chargers becomes more complex. All such points can be effectively addressed with extensive and comprehensive mea- surements, carried out with adequate hardware and methods for both harmonic and supraharmonic frequency intervals and phenomena. There is, in particular nowadays, a common agreement on the relevance of supraharmonics as a distortion phenomenon with signiﬁcant penetration and non-negligible intensity, relevant for ageing of network components, disturbance to connected loads and network stability [18,20–23]. Smart Cities 2022, 5 498 This work thus evaluates PQ issues and the impact of the charging process, discussing various ﬁndings both for harmonic and supraharmonic phenomena, including identifying available repositories of experimental data. p p The paper continues with Section 2, where charging architectures are brieﬂy reviewed, together with a description of typical distortion phenomena. Section 3 provides an overview of available experimental results and a quantitative assessment of the relevance of distortion caused by EV charging; it includes elements such as superposition and aggregation, preex- isting network distortion, comparison of harmonic and supraharmonic limits, discussing the formulation of suitable emission limits for the latter. Section 4 reports selected works providing extensive sets of data, which are compared and cross-checked for consistency and relevance. 2. EV Charging Modes and Architectures EV charging possibilities are made more complex by a wide range of connectors [4], integrating AC pins for the ﬁrst three modes, DC pins in some models for additional mode 4 ultra-fast charge and a set of communication pins. The design of the on-board charger conversion system aims ﬁrst of all at energy efﬁciency and reduced space, weight and number of components, besides a suitable elec- tric interface to the AC grid with unity power factor (pursuing minimization of reactive power at fundamental and reduction of low-order harmonics, providing a fairly sinu- soidal current waveform). Bidirectional power ﬂow is an additional feature to support e.g., V2G functionality. Smart Cities 2022, 5 499 The front-end converter for connection to the AC grid is a rectiﬁer (AC–DC converter) that in general is based on a traditional diode bridge or on an active bidirectional rectiﬁer in half or totally controlled arrangement, featuring MOS or IGBT devices (the latter shown in Figure 1a). The reasons for the latter solution are: lower voltage drop across active devices than using diodes and better control of input power factor and waveshape (power factor correction), largely reducing reactive power and low-order harmonics compared to a diode rectiﬁer. Although in general passive and active power factor correction (PFC) exist, all modern power converters use active PFC, generically indicated as PFC in the following: • Single-phase diode rectiﬁers are followed by a full-rated boost converter (DC–DC step-up converter); • The solution is not directly suitable for three-phase bridges (so for higher power levels), although three interleaved PFC boost converters may be used; • The active rectiﬁer solution can control voltage and current vectors over a wide range of conditions and is equally applicable to single- and three-phase connections; however, being usually decoupled with series inductors to the grid, necessitates a compensating inner current loop to improve speed of response [27]; • An attractive solution that allows downrating the PFC circuit is the use of a shunt connected active power ﬁlter, which is sized for approximately 40% of the rated power of the diode rectiﬁer [28]. More elaborated architectures, especially ones achieving higher efﬁciency and better sharing of higher voltage among devices, can be based with neutral-point clamped (NPC) converters [29,30], exempliﬁed in Figure 1b,c. (c) (b) (c) Figure 1. Typical AC–DC converter schemes (three-phase versions shown): (a) two-level active rectiﬁer, (b) ﬂoating capacitor converter, (c) neutral point clamped converter. 2. EV Charging Modes and Architectures The impact of PFC on the AC grid materializes as typical pulse width modulation bands for active rectiﬁer implementations, or oscillating pulses, named zero-crossing oscillations in [36]: such pulses have frequency occupation in approximately the 2–10 kHz range [37,38] and can cause excessive stress and interference in equipment and components, with consequential malfunction and damage [36]. The increase of the switching frequency values of modern converters and the extensive use of the PFC have increased emissions in the supraharmonic range causing higher levels of distortion and network pollution. This has been favored by the absence of limits of emissions as it will be discussed in Section 3. Reduction of such supraharmonic emissions is surely possible by the effect of differential ﬁlters of the LCL type (preferable to the CLC ones so as to prevent network instability and to keep network impedance at higher values, hindering then supraharmonic components propagation). The adoption of such ﬁlters is, however, avoided when not compulsory, as they add weight and increase losses, in particular, due to the two series inductors. p DC bus regulation downstream is implemented by means of a range of DC–DC converter types: • Buck, buck–boost, Cuk, Sepic for non-isolated architectures; • Single or dual active bridge (SAB and DAB), or even dual half bridge (DHB), providing galvanic isolation. Galvanic isolation at basic insulation level is mandatory as per ISO 6469-3 [39] to ensure electrical safety, possibly extended to a double or reinforced insulation, as one of the measures covering both basic and fault protection. Whatever the DC–DC converter downstream, its input current ripple will be reﬂected upstream on the AC network, mixing its switching components to those of the input rectiﬁer. The typical shape of the series inductor scheme is a triangular waveform, whose Fourier spectrum decreases rapidly; if designed for 10% ripple at the switching frequency, the ﬁrst harmonic of order 3 is about 1% and negligible. In case of resonant operation, there will be an almost sinusoidal current that in some operating intervals (e.g., high power) may be distorted by even components [40]. 2. EV Charging Modes and Architectures (b) (a) (b) (a) (c) Figure 1. Typical AC–DC converter schemes (three-phase versions shown): (a) two-level active rectiﬁer, (b) ﬂoating capacitor converter, (c) neutral point clamped converter. The basic distortion of three-level inverters using various types of optimized PWM schemes for harmonic distortion reduction, besides efﬁciency improvement, is in the order of 15–25% for the line voltage, and about 1/3 to 1/5 for line current distortion, considering normal values of resistive-inductive feeding impedance [31–33]. For high power levels, interleaving of elementary converters brings along harmonic reduction thanks to the reduced ripple and increased equivalent switching frequency. Alternative advanced solutions to harmonic distortion reduction require increasing the number of levels, implementing an asymmetrical structure, where dc voltages are not equally shared among modules, as well as the switching frequency [34]. The proposed modulation in fact provides high-frequency switching only for few cells with lower dc voltage providing a kind of ﬁne regulation of the voltage shape, while reducing overall Smart Cities 2022, 5 500 switching losses. The attained THD is less than 25% of a standard NPC converter and about 60% to 90% of the THD of a symmetric voltage converter. The studied frequency range extends to the 150th harmonic, which for the 50 Hz mains corresponds to 7.5 kHz, making it clear that harmonically related emissions extend nowadays well above the conventional 2 kHz boundary [35]. y The basic PWM scheme has been subjected to several optimizations for reduction of harmonic distortion, such as: • Zero dead-time PWM that improves the THD by about a factor of two [32] without exposing the converter to the shoot-through risk, since the implemented switching tt d t ll t ti t ti l t it h f th l • Zero dead-time PWM that improves the THD by about a factor of two [32] without exposing the converter to the shoot-through risk, since the implemented switching pattern does not allow to activate consecutively two switches of the same leg; p y g • Random PWM, which avoids the peaks of emission at the switching frequency har- monics, but increases the remaining spectrum signiﬁcantly, de facto providing only a partial solution. With the extensive use of PFC, low-frequency harmonic distortion problems have been largely mitigated, shifting distortion to higher frequency, as a byproduct of waveform manipulation to achieve PFC and mixing with the distortion of the DC–DC converter downstream for DC bus regulation. 2. EV Charging Modes and Architectures In general, current distortion is limited to a few % and only the fundamental at the resonant frequency can be retained; the portion reaching the AC network upstream is quite limited by the local DC link capacitor, whose action is facilitated by the extremely large resonant frequency, an order of magnitude higher than the switching frequency of non-resonant DC–DC converters. The DAB converter is made by operating in resonant conditions to achieve high efﬁciency and reduced size, although it is difﬁcult to maintain a zero voltage or current switching condition for a wide range of load levels. Resonant behavior is achieved by adding a capacitor in series with each of the two windings of the intermediate transformer, Smart Cities 2022, 5 501 possibly including a separate series inductor to better control the resonance frequency and to bring it to lower values. g Often, a designer’s attention is focused on high loading levels to achieve fast charging rates, with EV connection lasting for a short time. If, conversely, home and ofﬁce charging is considered, then the connection may last much longer and reduced loading levels may be used. The optimal design of an EV on-board charger must reconcile then these two opposite scenarios, especially in terms of efﬁciency and distortion. Battery cell equalization is another operation that contributes to high-frequency distor- tion and emissions: various arrangements may be implemented, employing buck–boost or switched-capacitor elementary converters, exchanging energy between cells from higher- voltage ones to lower-voltage ones [41]. Equalization may be started at any time by the battery management system (BMS), asynchronously and not related to any particular state of charge. It will then last for time intervals that may range from some to several tens of minutes depending on the level of unbalance and the adopted stop criteria. Although cell equalization does not have a remarkable inﬂuence on harmonic distortion, it does in terms of conducted emissions at higher frequency. Based on personal experience, it causes a signiﬁcant variability of conducted emissions in the tens of kHz to a few MHz, impacting on the repeatability of otherwise identical test sequences. 3. Distortion Limits and Assessment This section discusses the limits applicable to EV chargers for the harmonic and supra- harmonic frequency intervals, also providing information on the so-called compatibility levels, so the expected maximum disturbance levels to expect in a given environment (in the present case, a type of distribution grid, such as residential, light industrial, etc.) It is observed that the correct process proceeds from the deﬁnition of tolerable levels of disturbance (in terms of voltage) and then, based on three factors, limits for individual loads are stipulated (in terms of current). The three factors are: • Propagation or attenuation of emissions from the respective connection points along the network; • Characteristics of phase angle distribution and degree of superposition and compensation; • Network impedance transforming current emissions of loads into network voltage distortion. It is immediately clear that all three elements above have widely different behavior for harmonics and supraharmonics, and for the latter, in particular, over a considerably wide frequency interval. Attenuation depends signiﬁcantly on frequency; phase distribution of emissions depends on the power conversion process, the degree of synchronization to the network fundamental, etc.; impedance curves are then shaped by resonances and anti-resonances and have a general increase of amplitude and ﬂattening of peaks with increasing frequency. 3.1.1. Harmonic Frequency Interval 3.1.1. Harmonic Frequency Interval 3.1.1. Harmonic Frequency Interval For public LV distribution networks, compatibility levels are indicated in the IEC 61000-2-2 [42] which summarizes the tolerable voltage distortion for the harmonic interval up to the 40th order, as shown in Figure 2. (a) (a) (b) Figure 2. Harmonic distortion compatibility levels for public LV distribution grid (Table 1 of IEC 61000-2-2), with a prescribed voltage THD level of 11%: (a) numeric values, (b) graphical form. (b) Figure 2. Harmonic distortion compatibility levels for public LV distribution grid (Table 1 of IEC 61000-2-2), with a prescribed voltage THD level of 11%: (a) numeric values, (b) graphical form. Figure 2. Harmonic distortion compatibility levels for public LV distribution grid (Table 1 of IEC 61000-2-2), with a prescribed voltage THD level of 11%: (a) numeric values, (b) graphical form. As the scenarios of EV charging extend to both residential and ofﬁce environments, possibly including light industrial environment (in case charging is carried out at small factories and workshops), the IEC 61000-2-4 standard [43] should be considered as well. The IEC 61000-2-4 deﬁnes three classes of environments: • Class 1: protected supplies with compatibility levels lower than public networks, representing, e.g., laboratories with measurement and scientiﬁc equipment; • Class 2: light industrial environment that may be without (class 2a, ofﬁce) or with (class 2b, ofﬁce + light industry) industrial power electronic equipment; in case of class 2b, there is no separation by a supply transformer between the ofﬁce-like and the light industrial parts of the network; g p • Class 3: heavy industrial environment with power converters, welding machines, motors starting and stopping frequently. Our discussion focuses mainly on the class 2 environment, being sensible that, in case of EV charging, this is connected before the local protected supply, thus, to a public (IEC 61000-2-2) or light industrial (IEC 61000-2-4, class 2) network. The compatibility levels for voltage distortion are shown in Figure 3. Our discussion focuses mainly on the class 2 environment, being sensible that, in case of EV charging, this is connected before the local protected supply, thus, to a public (IEC 61000-2-2) or light industrial (IEC 61000-2-4, class 2) network. The compatibility levels for voltage distortion are shown in Figure 3. 3.1. Harmonics and Supraharmonics Limits Distorting loads are in general subject to regulatory limits for what regards ﬁrst of all harmonics, and then overall harmonic distortion and emissions at higher frequency. There is a dichotomy for which compatibility levels for the electric network the load is connected to are speciﬁed as tolerable voltage distortion and then apportioned to each load in terms of current distortion limits. This process assumes certain characteristics of the network and a certain mix of loads: the latter is bound to change with the increase of distorting loads that are diffusely connected to LV distribution nowadays, as in the present case of EVs. The various compatibility levels and limits of emission are reviewed in the following, distinguishing the more consolidated harmonic interval (up to the 40th harmonic order, namely, 2 kHz or 2.4 kHz for 50 Hz and 60 Hz mains fundamental, respectively), and the supraharmonic interval above it. Smart Cities 2022, 5 502 3.1.1. Harmonic Frequency Interval The class 2a values, as expected, correspond to those indicated in the previous Figure 2 regarding the IEC 61000-2-2, but not completely: low-order even harmonics are twice as large for the former. 503 Smart Cities 2022, 5 (a) (b) Figure 3. Harmonic distortion compatibility levels for light/heavy industrial networks (Table 2 of IEC 61000-2-4), with a prescribed voltage THD level of 11%: (a) numeric values, (b) graphical form. (a) (a) (b) Figure 3. Harmonic distortion compatibility levels for light/heavy industrial networks (Table 2 o IEC 61000-2-4), with a prescribed voltage THD level of 11%: (a) numeric values, (b) graphical form (a) (b) Figure 3. Harmonic distortion compatibility levels for light/heavy industrial networks (Table 2 of IEC 61000-2-4), with a prescribed voltage THD level of 11%: (a) numeric values, (b) graphical form. Figure 3. Harmonic distortion compatibility levels for light/heavy industrial networks (Table 2 of IEC 61000-2-4), with a prescribed voltage THD level of 11%: (a) numeric values, (b) graphical form. For LV low-power equipment limits are established in the IEC 61000-3-2 standard [44], whose scope encompasses all electrical equipment (both single-phase and three-phase) absorbing no more than 16 A (charging mode 1) and connected to the LV public network. For larger current levels, the reference standard is the IEC 61000-3-12 [44] for electrical equipment absorbing more than 16 A and up to 75 A, thus encompassing the other two charging modes 2 and 3. g g The applicability of IEC 61000-3-2 for chargers with Mode 1, i.e., with absorbed current up to 16 A, is not so clear and straightforward, as the standard was not originally conceived for such equipment and thus all reported examples and considerations are applicable to other types of equipment, such as portable electric tools, lighting devices, electric appliances, and so on. However, the product standard IEC 61851-21-1 [45] makes explicit reference to both IEC 61000-3-2 and to IEC 61000-3-12 with a short-circuit ratio of 33. In addition, authors in [21] conﬁrm the compulsory applicability of IEC 61000-3-2 (of course in its CENELEC equivalent version) before marketing of chargers within the European Union. In case of charging through a private electric network that is in turn connected to the public network at the Medium- or High-Voltage level, the standard to take into considera- tion is the IEC 61000-3-6. 3.1.1. Harmonic Frequency Interval This latter case is representative of a charging facility, such as a modern gas station, and allows an overall compensation of the aggregated distortion of the connected loads downstream the connection to the public grid. It will not be further considered, because it is a special case covered by harmonic ﬂow studies and speciﬁc control measures, depending on the available short-circuit ratio Rsc at the feeding point. The focus of this work, instead, is on the impact on the public network at the LV level, as for residential and ofﬁce charging of EVs. Smart Cities 2022, 5 504 Regarding harmonic distortion, the IEC 61000-3-12 [46] indicates a total current har- monic distortion (THC) and a weighted version, that is deﬁned starting from order 14, and it is thus called partial weighted harmonic current (PWHC): THC = v u u t 40 ∑ h=2 I2 h PWHC = v u u t 40 ∑ h=14 hI2 h (1) (1) The limits of current distortion for the direct connection to the public network are reported in Table 1 for the IEC 61000-3-2 [44] (class A, applicable to 16 A chargers) and in Figures 4 and 5 for the IEC 61000-3-12 [46]. Table 1. Harmonic distortion limits as per Table 1 of IEC 61000-3-2 standard. Table 1. Harmonic distortion limits as per Table 1 of IEC 61000-3-2 standard. Harmonic Order h Limit (A) 2 1.08 3 2.30 4 0.43 5 1.14 6 0.30 7 0.77 9 0.40 11 0.33 13 0.21 8 ≤h ≤40 0.23 8 h 15 ≤h ≤39 0.15 15 h (a) (b) Figure 4. Harmonic distortion limits for unbalanced loads as a function of short-circuit ratio Rsc (Table 2 of IEC 61000-3-12): (a) numeric values, (b) graphical form. Harmonic Order h Limit (A) 2 1.08 3 2.30 4 0.43 5 1.14 6 0.30 7 0.77 9 0.40 11 0.33 13 0.21 8 ≤h ≤40 0.23 8 h 15 ≤h ≤39 0.15 15 h (a) (a) (b) Figure 4. Harmonic distortion limits for unbalanced loads as a function of short-circuit ratio Rsc (Table 2 of IEC 61000-3-12): (a) numeric values, (b) graphical form. (a) (b) (b) Figure 4. Harmonic distortion limits for unbalanced loads as a function of short-circuit ratio Rsc (Table 2 of IEC 61000-3-12): (a) numeric values, (b) graphical form. Smart Cities 2022, 5 505 (a) (b) Figure 5. 3.1.1. Harmonic Frequency Interval Harmonic distortion limits for balanced three-phase loads as a function of short-circuit ratio Rsc (Table 3 of IEC 61000-3-12): (a) numeric values, (b) graphical form. (a) (b) Figure 5. Harmonic distortion limits for balanced three-phase loads as a function of short-circuit ratio Rsc (Table 3 of IEC 61000-3-12): (a) numeric values, (b) graphical form. (a) (b) Figure 5. Harmonic distortion limits for balanced three-phase loads as a function of short-circuit r (a) (b) ( ) Figure 5. Harmonic distortion limits for balanced three-phase loads as a function of short-circuit ratio Rsc (Table 3 of IEC 61000-3-12): (a) numeric values, (b) graphical form. A speciﬁc table of the IEC 61000-3-12 standard (Table 5) speciﬁes a separate limit of 1% for even harmonics above order 12, instead of including them all comprehensively in the Total Harmonic Distortion (THD) for current (THC). This particular condition applies to equipment with either particularly low 5th and 7th harmonics (less than 3%), or with a 5th harmonic phase evenly distributed over the whole 0–360◦or consistently located in a favorable 150–210◦sector. Observing the stipulated compatibility levels and current limits for the harmonic inter- val, it is possible to reason on how the three factors at the beginning of this Section 3 have been taken into account. This is a useful exercise carried out in Section 3.2 covering supra- harmonics, for which limits of emissions are not available, but only compatibility levels. 3.1.2. Supraharmonic Frequency Interval The 2–150 kHz interval has been recently extensively considered for the distortion and commutation byproducts of a wide range of devices and equipment, such as LED lamps, ﬂuorescent lamps (CFLs) and Switched Mode Power Supplies, that are quite widespread in residential, ofﬁce and light-industrial environments. The relevance of emission components in this frequency range is built around the following points: • Large penetration in supply networks, undergoing marginal attenuation similar to high-order harmonics; • Potential disturbance to metering and control equipment, as well as to power line carrier (PLC) devices; • Possible excitation of network resonances, in particular considering the small LV grids of limited extension. The IEC 61000-2-2 [42] addresses supraharmonic distortion with compatibility levels added in the new 2017 version, ranging between 2 kHz and 150 kHz, speciﬁcally for Smart Cities 2022, 5 506 nonintentional emissions (as mains signaling is considered separately). Limit values and graphical representation are shown in Figure 6. (a) (a) (b) Figure 6. Supraharmonic distortion compatibility levels for public LV distribution grid (Table 2 of IEC 61000-2-2): (a) numeric values, (b) graphical form. (a) ( ) (b) Figure 6 Supraharmonic distortion compatibility levels for public LV distribution grid (Table 2 (b) Figure 6. Supraharmonic distortion compatibility levels for public LV distribution grid (Table 2 of IEC 61000-2-2): (a) numeric values, (b) graphical form. Similarly, the IEC 61000-2-4 deﬁnes compatibility levels for equipment connected to LV distribution, distinguishing between unbalanced loads (that cover single-phase EV chargers rated in the range of 16 to 32 A) and balanced three-phase loads. They are reported in Figure 7, distinguishing between Industrial, Residential and Special environments, the most suitable one being the second for EV charging at home or ofﬁce. Such levels are deﬁned using a 200 Hz bandwidth invoking the IEC 61000-4-7 [47] grouping for frequencies between 2 and 9 kHz, and in line with measurements for non- narrowband phenomena, regulated by CISPR 16-1-1 [48].The standard, however, does not go into detail regarding the deﬁnition of such bandwidth, i.e., as resolution bandwidth of a frequency domain scan measurement or frequency resolution of a Fourier-transformed time-domain signal [49,50]. 3.1.2. Supraharmonic Frequency Interval Limits are again applied above 150 kHz for what is referred to as radio-frequency conducted emissions, what is worth recalling are unsym- metrical voltages, measured with respect to ground, and not differential-mode line-to-line voltages as for harmonics and supraharmonics The IEC 61000-2-4 indicates that according • The “same bandwidth interval of 200 Hz” could exclude the combination of different sources of emission, but in a real scenario the number of different EV models is not so large (9 BEVs were considered in [51] and a few had similar emissions). (a) (a) (b) Figure 7. Supraharmonic distortion limits for LV distribution grids (distinguishing Protected, class 1 Residential/Ofﬁce, class 2, and Industrial, class 3, environments, as in Tables 4 and 5 of IEC 61000-2-4) (a) (b) Figure 7. Supraharmonic distortion limits for LV distribution grids (distinguishing Protected, class 1, Residential/Ofﬁce, class 2, and Industrial, class 3, environments, as in Tables 4 and 5 of IEC 61000-2-4): (a) numeric values, (b) graphical form. Unfortunately, there are no supraharmonic limits applicable to LV equipment, and in particular, EV charging converters. Limits are again applied above 150 kHz for what is referred to as radio-frequency conducted emissions, what is worth recalling are unsym- metrical voltages, measured with respect to ground, and not differential-mode line-to-line voltages, as for harmonics and supraharmonics. The IEC 61000-2-4 indicates that, according to CISPR 16-2-1 [52], suitable emission limits are 6 dB lower than those for differential-mode emissions. Regarding RF emissions, in fact, the IEC 61851-21-1 refers to the residential/light- industry limits of the IEC 61000-6-3. As anticipated, supraharmonics have only recently received the attention of the scien- tiﬁc community and standardization is still lagging, having established some compatibility levels in the IEC 61000-2-4 [43], including indications for compatibility with PLC technology in the EN 50065-1 [53]. 3.1.2. Supraharmonic Frequency Interval An important point regards the implicit assumptions for the deﬁnition of such levels as stated by the IEC 61000-2-2: at a given location, the disturbance level in a same bandwidth interval of 200 Hz is assumed not to result from more than two pieces of equipment generating nonintentional emissions close to the emission limit at the same time. Now there are two terms that deserve some further attention: • The concept of location should be better deﬁned, as for LV distribution, it may mean the same part of the LV grid, or the entire LV grid that is fed from the same MV/LV transformer, representing a decoupling point; in both cases, it is easy to see that several EVs can be plugged at the same time (during the day at the ofﬁce, during the night at home) in a small portion of the LV network; • The concept of location should be better deﬁned, as for LV distribution, it may mean the same part of the LV grid, or the entire LV grid that is fed from the same MV/LV transformer, representing a decoupling point; in both cases, it is easy to see that several EVs can be plugged at the same time (during the day at the ofﬁce, during the night at home) in a small portion of the LV network; Smart Cities 2022, 5 507 • The “same bandwidth interval of 200 Hz” could exclude the combination of different sources of emission, but in a real scenario the number of different EV models is not so large (9 BEVs were considered in [51] and a few had similar emissions). (a) (b) Figure 7. Supraharmonic distortion limits for LV distribution grids (distinguishing Protected, class 1, Residential/Ofﬁce, class 2, and Industrial, class 3, environments, as in Tables 4 and 5 of IEC 61000-2-4): (a) numeric values, (b) graphical form. Unfortunately, there are no supraharmonic limits applicable to LV equipment, and in particular, EV charging converters. 3.2. Extrapolation of Supraharmonic Limits In order to attempt an estimation of suitable current emission limits in the suprahar- monic range, a comparison between harmonic and supraharmonic compatibility levels may be carried out by quantifying a proportion factor, to transfer then to the emission limits, starting from the existing harmonic current limits. A point to consider is the identiﬁcation Smart Cities 2022, 5 508 of bandwidth values for homogeneous comparison of compatibility levels: together with the said BWsupr = 200 Hz bandwidth, a harmonic bandwidth BWharm = 5 Hz may be identiﬁed, considering the 200 ms time widow used by the IEC 61000-4-7. The amplitude conversion factor ks−h between the two types of evaluations (or “measurements”) is then ks−h = s BWsupr BWharm = 6.32 (2) (2) It is thus possible to verify that the ratio of compatibility levels is close to this ks−h value: referring to Figure 2, the average amplitude values are 0.458 V or 0.384 V, taking the last ten and ﬁve harmonics, respectively; supraharmonic levels in Figure 6 account for 3.22 V. Multiplication of harmonic levels by ks−h gives values of 2.89 V and 2.43 V that are be- low the 3.22 V value by 10 % and 24.6 %, respectively. This could be justiﬁed only assuming a higher immunity to disturbance in the lower supraharmonic range, an assumption that is not well grounded; in fact, there is a tiny difference of less than one octave between the two regimes, positioned at 1.5–2.0 kHz and 2–3 kHz for harmonics and supraharmonics, respec- tively. The conclusion is that tolerable supraharmonic levels seem slightly overestimated by 1 to 3 dB approximately. By extension, harmonic current limits suitable for public LV network can be projected onto the supraharmonic interval, covering scenarios of ofﬁce and light industrial networks of class 2a, and possibly covering at some extent class 2b. Harmonic compatibility levels (see Figure 6) expressed in voltage units can be compared to current emission limits (see Table 1), to derive, in order, an estimate of the “impedance effect” and margins for multiple sources assumed by the standardization committees. 3.2. Extrapolation of Supraharmonic Limits It is also worth noting that the estimated network impedance ˆZ′ is in the order of 3 to 5 Ωat 2 kHz, in line with the reference impedance values of IEC 61000-4-7 [47] and compatible with a 5 ΩLISN value [54], applicable yet to unsymmetrical impedance starting at 9 kHz. The simple linear extrapolation with frequency of the reactive part of the reference impedance of the IEC 61000-3-11 [55] yields an excess of impedance (the purple curve in Figure 8) not in agreement with the general observed behavior. In fact, in addition, a conﬁrmation comes from [56], where impedance values similar to the above appear, although resonance phenomena are also pointed out, which may increase locally impedance values by a factor of 2–3, depending on losses. It is remarkable that the number of sources of distortion connected to the same small portion of the grid (e.g., one home, ofﬁce or small group of them) has recently increased well above the assumed “10” or thereabout, and that EVs will add on top a signiﬁcant contribution. Figure 8. Harmonic impedance for selected groups: odd harmonics multiple of 3 (green dots), even harmonics (blue dots) and odd characteristic harmonics (red dots); recalculated network impedance ˆZ′ for even ( ˆZ′e, light blue) and odd characteristic ( ˆZ′oc, light brown) harmonics (triangles are for the n = 10 case, squares for n = 15). The violet curve is the linear extrapolation with harmonic order of the reference impedance of the IEC 61000-3-11. Figure 8. Harmonic impedance for selected groups: odd harmonics multiple of 3 (green dots), even harmonics (blue dots) and odd characteristic harmonics (red dots); recalculated network impedance ˆZ′ for even ( ˆZ′e, light blue) and odd characteristic ( ˆZ′oc, light brown) harmonics (triangles are for the n = 10 case, squares for n = 15). The violet curve is the linear extrapolation with harmonic order of the reference impedance of the IEC 61000-3-11. When aggregating supraharmonic emissions, a random phase distribution may be certainly assumed, given the mechanisms of generation, not synchronized to the mains. It is also sensible to presume that the number of sources responsible for a speciﬁc emission component is smaller than for the harmonic interval, where emission components are rigidly located at harmonic frequencies, synchronized to the mains fundamental. 3.2. Extrapolation of Supraharmonic Limits The simple ratio below includes effects of network impedance and multiple sources and is thus indicated as ˆZ′: ˆZ′ = CL EL (3) ˆZ′ = CL EL (3) where CL and EL denote the compatibility level and the emission limit for LV public networks ˆ p y p Such ˆZ′ values are shown in Figure 8 for the harmonic interval where both CL and EL are available. Odd and even harmonics have been separated, and among odd ones, those named “characteristic” are distinguished from the noncharacteristic ones. Even and odd characteristic harmonics are then selected to assign them a speciﬁc behavior for what regards aggregation: for a given number of alike sources n, it is assumed that partial cancellation may occur for the even ones, which in principle have a random phase distribution (not being closely related to the power conversion process mechanisms), whereas characteristic ones are assumed to sum in phase (that is correct for a wide range of loads, e.g., featuring front-end rectiﬁers, as demonstrated later in Section 4.2). The consequence is that the total current value at each harmonic frequency increases with √n and n for the two categories of harmonics, respectively. Even and odd char- acteristics harmonics were selected for this quite different behavior with respect to ag- gregation: such simplifying assumption has some arbitrariness, but allows to keep two well-separated behaviors as reference and is justiﬁed for the objective of extrapolating to the supraharmonic range. ˆZ′ oc = ˆZoc n ˆZ′ e = ˆZe √n (4) (4) The recalculated impedance considering a number n of aggregated harmonic sources is also shown in Figure 8, where the n = 10 value (corresponding to the triangle symbol) was selected to have the two recalculated impedance curves for even ( ˆZe) and odd characteristic ( ˆZoc) harmonics almost overlap. A second larger n = 15 value is shown for comparison (square symbols). Approximate overlap is a condition supported by the assumption that the real network impedance is continuous and cannot take too many different values for adjacent frequency bins. It turns out to be reasonable then that a total of about n = 10 Smart Cities 2022, 5 509 sources including some margin was assumed when stipulating limits. If the assumption of in-phase sum does not fully hold for characteristic harmonics, then a slightly larger number n could be applied. 3.2. Extrapolation of Supraharmonic Limits As for network impedance, values typical of the harmonic interval cannot increase steadily with frequency and may undergo signiﬁcant variations as a consequence of network resonances and anti-resonance. Referring again to [56], impedance values keep approximately at the 2 kHz value or increase slightly under speciﬁc conﬁgurations of network parameters in the ﬁrst decade or so. Stiegler et al. [57] show two main points: the IEC 61000-4-7 impedance is the envelope of the largest measured impedance values in four different countries (Austria, Switzerland, Czech Republic and Germany) and reaches values even larger than those provided by the LISN impedance curve [54]; the 95th percentile proﬁle instead has a much more reasonable behavior, whilst still providing a robust estimate. Such a 95th curve has Smart Cities 2022, 5 510 the following values: 3 Ωat 2 kHz, 6 Ωat 20 kHz, 12 Ωat 50 kHz, 18 Ωat 100 kHz and 18 Ω at 150 kHz, for what is shown in [57]. Compatibility values in Figure 6 can thus be translated into current limits for the supraharmonic range, based on the measured LV public network impedance values pro- vided in [57] and assuming n = 10 randomly distributed sources with emissions occurring in the same measuring RBW interval. The result is shown in Figure 9. Figure 9. Extrapolated supraharmonic current limits based on CLs, estimated network impedance ˆZsh and number n = 10 of concomitant sources with randomly distributed phase: overall limit with n = 1 (brown squares), individual limit with n = 10 (blue triangles). Figure 9. Extrapolated supraharmonic current limits based on CLs, estimated network impedance ˆZsh and number n = 10 of concomitant sources with randomly distributed phase: overall limit with n = 1 (brown squares), individual limit with n = 10 (blue triangles). 3.3. Distortion Measurement and Assessment Standards discussed so far to limit and establish a reference for harmonic and supra- harmonic distortion rarely specify processing methods and settings by which spectral components should be derived and their amplitude estimated. Another aspect that de- serves attention regarding the accuracy of the assessment of emissions is the method with which time-domain signals are processed and evaluated, not only in terms of the time interval to report averaged values (200 ms, 3 s and 10 min with reference to IEC 61000- 4-7 [47]), but also regarding spectral leakage control (in particular with the adoption of speciﬁc smoothing windows [58]) and the inﬂuence on spectral amplitude estimate [49]. 3.3.1. Time Window, Frequency Resolution and Grouping Data compression, when going beyond simple lossless algorithms, unavoidably requires a choice for results representation, namely what should be included and what left out. Examples of condensed indexes are average values and maxima, percentiles, etc., over given time intervals. At a ﬁner scale, considering frequency spectrum values obtained from time samples with a Short-Time Fourier Transform (STFT) approach, a trade-off is necessary between a small and descriptive frequency resolution on the one hand, and an as coherent as possible behavior of spectrum portions along the time axis on the other, achieved by grouping of adjacent components resulting in a coarser frequency representation. Grouping is indicated in two widely used standards for harmonic and supraharmonic measurements, the IEC 61000-4-7 [47] and IEC 61000-4-30 [61], respectively. For the harmonic interval up to 2 kHz grouping solves the problem of interharmonics, caused, e.g., by variable speed drives emissions, occurring in between harmonics of the fundamental. Processing by Discrete Fourier Transform would require a long time window, longer than the fundamental cycle, as the greatest common divisor of all component frequencies might be very small. The measurement process is thus simpliﬁed by adopting a standard frequency resolution of 5 Hz (namely, a time window of 200 ms) and grouping adjacent spectrum components as harmonic and interharmonic groups and sub-groups, with overall amplitude determined by rms summation (square root of the sum of squares) of components falling inside each grouping interval. Grouping brings along a better stability of the amplitude estimation, more robust to residual spectral leakage. p p g For the wider supraharmonic interval, grouping is applied collecting spectrum compo- nents falling in a wider bandwidth of 2 kHz, sweeping between 2 and 150 kHz. (As a note, formal difﬁculties are encountered with the 2–9 kHz sub-interval that is covered by the method of IEC 61000-4-7, Annex B, that differs from that of the IEC 61000-4-30, which indi- cates in its Annex C.2 that incorporation of this sub-interval is under consideration.) The use of a B = 2 kHz bandwidth is justiﬁed by the wider frequency band, keeping roughly the same proportionality of 5 Hz compared to the harmonic interval, and a sufﬁciently ﬁne frequency resolution at the same time. 3.3.1. Time Window, Frequency Resolution and Grouping Especially for static converters of signiﬁcant power rating, which use a relatively large switching frequency and techniques to improve efﬁciency above other exigencies (such as minimization of conducted emissions), the characteristics of differential- and common- mode emissions may be such to require wise selection of settings to improve amplitude estimate uncertainty and reproducibility. The AC input quantities waveshape is in fact characterized by the superposition of power factor control stage byproducts and ringing phenomena occurring at each switching, possibly adopting zero-voltage or zero-current resonant techniques, so in that case, leaking also the main resonant component. All such components have different dynamics and time durations, so as to require different pro- cessing parameters selection [49]. Longer time intervals T with ﬁner frequency resolutions from a Fourier-transform perspective (δ f = 1/T) provide reduced amplitude estimates of faster transient components, which have a shorter duration and become “diluted” over the time interval T [37,49]. Two problems are common to all types of measurements and are related to data collection and representation. They become evident by looking at what is shown in [59,60]. • First, whatever the uncertainty of the measuring equipment and probes and having even minimized background noise sources, a signiﬁcant deal of variability of emissions • First, whatever the uncertainty of the measuring equipment and probes and having even minimized background noise sources, a signiﬁcant deal of variability of emissions Smart Cities 2022, 5 511 is expected both with respect to time and while ideally sweeping the frequency axis, passing from one spectral component to the adjacent one. is expected both with respect to time and while ideally sweeping the frequency axis, passing from one spectral component to the adjacent one. p g p p j • Second, the amount of data, if stored unaltered as time-domain samples, is huge since the required sampling rate is in the order of 500 kHz or larger for the supraharmonic range. Data compression, when going beyond simple lossless algorithms, unavoidably requires a choice for results representation, namely what should be included and what left out. Examples of condensed indexes are average values and maxima, percentiles, etc., over given time intervals. • Second, the amount of data, if stored unaltered as time-domain samples, is huge since the required sampling rate is in the order of 500 kHz or larger for the supraharmonic range. 3.3.1. Time Window, Frequency Resolution and Grouping Authors in [59] are cautious on the interpretation of the potential interference effect of grouped components over, in that case, a 4 kHz bandwidth, stating that the impact will not be that of a single component of same intensity; on this, it is possible to object that interference is to be weighted with the concept of victim bandwidth and also that spectrum components quite close to each other may give rise to modulation and beating effects that one single component is not able to. This is conﬁrmed by [22], who report unwanted tripping of residual current devices due to disturbance occurring when many EVs of the same type (but not switching exactly at the same frequency) were plugged in. In view of evaluating supraharmonic emissions to compare with limits and to limit disturbance to connected devices, B should be interpreted as the bandwidth of the victim equipment, as all spectral components falling within the same bandwidth B will contribute to the same disturbance term. For a uniform approach, however, the bandwidth B should not be changed depending on victim devices, but uniﬁed to a standard value (as the proposed 2 kHz), from which disturbance occurring with larger bandwidth may be simply estimated by, e.g., rms summation. Authors in [59] are cautious on the interpretation of the potential interference effect of grouped components over, in that case, a 4 kHz bandwidth, stating that the impact will not be that of a single component of same intensity; on this, it is possible to object that interference is to be weighted with the concept of victim bandwidth and also that spectrum components quite close to each other may give rise to modulation and beating effects that one single component is not able to. This is conﬁrmed by [22], who report unwanted tripping of residual current devices due to disturbance occurring when many EVs of the same type (but not switching exactly at the same frequency) were plugged in. 3.3.1. Time Window, Frequency Resolution and Grouping The inﬂuence of the selected bandwidth on the estimate of spectral components is exempliﬁed in [35,64]: • Tested bandwidths were 5 Hz, 200 Hz and 2 kHz, in line with the discussed standards, representing well a narrow, medium and large bandwidth for the supraharmonic frequency range; q y g • Effects of bandwidth on the amplitude estimate of spectral components can be divided, as known, between narrowband and broadband components: the inﬂuence is larger for broadband components, where the increase of amplitude follows ideally the square root of the ratio of the bandwidth, namely, 16 dB passing from 5 Hz to 200 Hz, and 10 dB from 200 Hz to 2 kHz. Even apparently narrowband components subjected to some frequency instability or modulation undergo variability of amplitude estimate, as it was shown in Figure 10 of [64]. In that ﬁgure, curves at 5 Hz and 200 Hz have a difference of about 7 dB, reduced to about 1 dB passing from 200 Hz to 2 kHz: this conﬁrms the inadequacy of too-narrow bandwidth values (namely 5 Hz or so), when dealing with real emissions, which seem stably evaluated by bandwidth values of hundreds of Hz or few kHz; • When facing transient components for which a larger bandwidth allows tracking a fast-changing spectrum, the attention is drawn in [35] on the worsening of the amplitude estimate accuracy, due to the increased superposed background noise, and widening of spectral lines (visible in the same Figure 10 of [64]). For a matter of compactness of reported results, in general, supraharmonic emissions are represented by a total supraharmonic distortion factor (TSHC), which for the type of measurement is referred to as current. This factor is the translation to the supraharmonic range of the total harmonic distortion for current already shown in (1) and in Section 3.1.1. TSHC = v u u t SH ∑ h=1 I2 h (5) (5) The summation takes supraharmonic terms Ih evaluated over bandwidth B, which, if set to 200 Hz, leads to SH = 740. The summation starts at 2 kHz, taken conventionally as h = 1. 3.3.1. Time Window, Frequency Resolution and Grouping Such bandwidth B was deemed appropriate when writing down the IEC 61000-4- 30, but its value is in reality somewhat arbitrary and subject to different exigencies and constraints for its ﬁne tuning: • As most supraharmonic emission sources are switching power converters with emissions mostly of the pulse-width-modulated (PWM) type, attention must be paid to the width of each PWM carrier (or switching frequency multiple k fs) and the side bands, spaced by even multiples of the modulation frequency m fm. Each switching+modulation PWM group of components is characterized by some amount of power that spectral grouping should try to capture and preserve in the resulting representation [62]. From this, the exigency to set B to a value bracketing most of such side-band components belonging to the same PWM group. However, such justification should be accompanied by the determination of the switching carriers and their harmonics, so as to align the grouping bandwidth B [62], which complicates spectrum evaluation; In a scenario of several EV chargers of the same type, whose emissions overlap with plausible shifts of the switching frequency due to normal tolerance of components and settings and related drift over time, a sufﬁcient bandwidth value should be set to correctly compose alike spectral components and observe the expected compensation, if phase displacement occurs. This is demonstrated in [63], where a 1 kHz bandwidth puts together slightly different peaks around the nominal 10 kHz switching frequency, resulting in mutual compensation, although beating phenomena also take place. Smart Cities 2022, 5 512 In view of evaluating supraharmonic emissions to compare with limits and to limit disturbance to connected devices, B should be interpreted as the bandwidth of the victim equipment, as all spectral components falling within the same bandwidth B will contribute to the same disturbance term. For a uniform approach, however, the bandwidth B should not be changed depending on victim devices, but uniﬁed to a standard value (as the proposed 2 kHz), from which disturbance occurring with larger bandwidth may be simply estimated by, e.g., rms summation. 3.3.1. Time Window, Frequency Resolution and Grouping Other proposed indexes are simple variations of the simple formulation above, where the underlying starting point is the extension of the summation to the entire suprahar- monic interval, adding then weighting proportional to frequency (as losses and aging of dielectrics are approximately proportional to frequency) or establishing the ratio to a reference frequency (the so-called K-factor). Smart Cities 2022, 5 513 3.3.2. Supply Voltage, Grid Distortion and Superposition of Emissions When considering an EV charger (as any other distorting load subject to assessment), a signiﬁcant difference of results may be expected for measurements carried out at laboratory and in a real scenario (namely in a real grid, with other connected loads, under variable operating conditions). p g Inﬂuence of the supply voltage level on emissions was preliminarily studied for CFLs [65] and switched-mode power supplies [66], and represents an overlooked fac- tor, affecting variability of emissions for a wide range of distorting loads, including power converters. p Results in [67] demonstrate that the supply voltage level plays a signiﬁcant role: • Two EVs (EV1 and EV2) show a completely different behavior of their Irms, with very constant values in one case and a change of about ±1.25 A around a mean value of about 14 A for an excursion of the supply voltage of 230 V ±10%; • Individual harmonics feature a wide range of values for the same supply voltage ex- cursion: EV1 has the ﬁfth spreading +200% and −60% around the reference amplitude of 11 A, whereas the most variable harmonic for EV2 is the ninth varying by +110% and −10%; in both cases, the increase of harmonic amplitude is observed at the largest power supply levels (241 V and 253 V); p pp y • Phase angle variations are also dramatic with both harmonics considered above spanning 120◦. • Phase angle variations are also dramatic with both harmonics considered above spanning 120◦. It is clear that any deal of compensation and partial cancellation occurring at some harmonic is compromised by any, even small, change of the supply voltage level, as the same harmonics of different EVs may vary quite differently. It is interesting to remark that such supply voltage changes may originate in other ports of the network, such as upstream, but may also be the consequence of local load change, e.g., when new EVs connect for charging, or, the other way round, when they suddenly disconnect. 3.3.1. Time Window, Frequency Resolution and Grouping As for low-frequency distortion (harmonics), the grid impedance plays a major role also for supraharmonics. In particular, EV charger supraharmonic emissions will ﬂow easily in the ﬁlters of other connected loads (such as PWM output ﬁlters of inverters, EMI ﬁlters, etc.), setting up a low-impedance path between loads connected to the same LV grid up to a considerable distance (hundreds of meters), that is a large portion of the typical extension of LV feeders stemming from the secondary of a MV/LV transformer. g y As the EV charger is connected to a real grid with preexisting distortion, three factors must be duly considered: • Superposition of spectral components from different sources occurring at the same frequency or, better, with a difference less than the resolution bandwidth. Examples are not only multiple EV chargers, but also for coexistence with other power converters, such as photovoltaic inverters and various types of power drives. Superposition, depending on the speciﬁc phase relationship, may have the positive effect of the reduction of the overall distortion components. This in general is more likely to occur for harmonics, whose frequency is ﬁxed by the grid fundamental, rather than supraharmonics; quite interestingly in [63] the intensity of the 10 kHz switching component at the feeding point reduces signiﬁcantly with the number of vehicles n, more than the expected √n assuming random phase and identical frequency. The amount of harmonic cancellation may be expressed simply by the coefﬁcient ηcanc, calculated for a variable number of sources N. Combined emissions of EVs and household loads were evaluated in [68], showing a non-obvious relationship between low-order harmonics and the amount of connected EVs (see Section 4); • Superposition of spectral components from different sources occurring at the same frequency or, better, with a difference less than the resolution bandwidth. Examples are not only multiple EV chargers, but also for coexistence with other power converters, such as photovoltaic inverters and various types of power drives. Superposition, depending on the speciﬁc phase relationship, may have the positive effect of the reduction of the overall distortion components. This in general is more likely to occur for harmonics, whose frequency is ﬁxed by the grid fundamental, rather than supraharmonics; quite interestingly in [63] the intensity of the 10 kHz switching component at the feeding point reduces signiﬁcantly with the number of vehicles n, more than the expected √n assuming random phase and identical frequency. 3.3.1. Time Window, Frequency Resolution and Grouping The amount of harmonic cancellation may be expressed simply by the coefﬁcient ηcanc, calculated for a variable number of sources N. Combined emissions of EVs and household loads were evaluated in [68], showing a non-obvious relationship between low-order harmonics and the amount of connected EVs (see Section 4); ηcanc = 1 − Ih,tot ∑N n=1 \|Ih(n)\| (6) (6) • The inﬂuence of low-frequency harmonic distortion on higher-frequency emissions, such as in the supraharmonic range. Such inﬂuence was observed for various types Smart Cities 2022, 5 514 of converters, where, in general, supraharmonic emissions depend on both the AC supply voltage level and low-order distortion (affecting the local wave shape and thus high-frequency emissions localized along the fundamental waveform). This is discussed further in Section 4; of converters, where, in general, supraharmonic emissions depend on both the AC supply voltage level and low-order distortion (affecting the local wave shape and thus high-frequency emissions localized along the fundamental waveform). This is discussed further in Section 4; The so-called secondary emissions, where the charger is passively subject to grid distortion components, letting ﬂow a corresponding current at the same frequency of the said components. This is particularly evident when small distortion voltages can cause a signiﬁcant current intensity to ﬂow through the low EMI (electromagnetic in- terference) ﬁlter impedance at such frequencies; in such cases, minimizing the amount of capacitance at the grid side is a necessity, not only used to prevent zero-sequence current leakage. Figure 2 in [64] demonstrates that three supraharmonics spectra taken at two ﬁeld locations and one laboratory are quite different with broadband and narrowband components occurring at 35 and 100 kHz for the two ﬁeld measurements, respectively. The so-called secondary emissions, where the charger is passively subject to grid distortion components, letting ﬂow a corresponding current at the same frequency of the said components. This is particularly evident when small distortion voltages can cause a signiﬁcant current intensity to ﬂow through the low EMI (electromagnetic in- terference) ﬁlter impedance at such frequencies; in such cases, minimizing the amount of capacitance at the grid side is a necessity, not only used to prevent zero-sequence current leakage. Figure 2 in [64] demonstrates that three supraharmonics spectra taken at two ﬁeld locations and one laboratory are quite different with broadband and narrowband components occurring at 35 and 100 kHz for the two ﬁeld measurements, respectively. 4. Experimental Data This section reports experimental evidence of distortion characteristics of EV charging operation, in some cases referring to the same publications and results that were used for clariﬁcation and exempliﬁcation in Section 3. 3.3.1. Time Window, Frequency Resolution and Grouping The effect of the grid voltage waveshape (in turn affected by low-order harmonics) was explored in [64], where a photovoltaic inverter, supplied by a ﬂat or pointed top sinusoidal voltage, has harmonics larger by an order of magnitude compared with when it is fed by a sinusoidal voltage. 4.1. Arrangement and Results of the Systematic Review An extensive search was carried out using the major journals databases (Elsevier, IEEE, MDPI) and the Google search engine (including thus journals and conferences not sponsored by the major publishers, and ResearchGate), using keywords such as “electric vehicle”, “charge”, “distortion”, “harmonic”, “supraharmonic”, “measurement”, in AND combination of at least 3 terms. In journal databases, such terms were searched almost always within “abstract”, then “full text”, excluding things such as the name of the confer- ence (if using “metadata”), but avoiding limiting the search to the title only. References in found articles were then added, until relevant hits happened to be already found. As observed in [36] (almost at the same time of this work), long-term datasets providing EV charging measurements extended to the harmonic and supraharmonic range are not yet available. The search proceeded with a ﬁrst set of hits based on the keywords above (set1), that was then cleaned up for all the works evidently just including the sought keywords, but not providing any relevant information (set2). The works belonging to this second set were then inspected more closely, verifying if they provide results and their quality, and in particular the use of experimental data (contrasted to models and simulation data). For Google search results, the large amount of hits that makes set1 was examined by removing duplicates, focusing on hits with a linked ﬁle, and limiting the operation to the ﬁrst 50 pages. The number of search hits are reported in Table 2, distinguishing two search schemes, using either “supraharmonic” or “harmonic” in conjunction with “electric vehicle” and “measurement”, applied to the entire text of the sources. The objective is to show what is the progress in the research regarding supraharmonics, compared to the more classical topic of harmonics, albeit applied to the emerging sector of electric vehicles. We may observe that Elsevier and MDPI have fewer hits, but an intense publishing activity of pertinent works: they consistently have an almost triple percentage of supraharmonics-related publications, compared to IEEE, that reﬂects the general trend represented by Google, of about 0.5–0.6%. Smart Cities 2022, 5 515 Table 2. Results of the hits of bibliographic search (set1): columns “Supraharmonic” and “Harmonic” refer to the use of the two respective keywords. 4.1. Arrangement and Results of the Systematic Review Publisher/Search Engine “Supraharmonic” “Harmonic” Ratio Elsevier 24 1511 1.6% IEEE 78 12,910 0.6% MDPI 15 1072 1.4% Google 5440 1,050,000 0.5% The analysis of the search results is completed by showing some characteristics of the selected references that make set3: type of source (conference, journal, technical report, website), origin (Elsevier, IEEE, MDPI, others) and the year of publication. A graphical representation appears in Figure 10. (a) (b) (c) Figure 10. Overview of sources of experimental harmonic and supraharmonic data for electric vehicles: (a) publication type versus grouped years, (b) publisher versus grouped years, (c) topic (harmonics, supraharmonics, counting when both) versus grouped years. (a) (b) (b) (a) (c) (a) (b) (c) Figure 10. Overview of sources of experimental harmonic and supraharmonic data for electric vehicles: (a) publication type versus grouped years, (b) publisher versus grouped years, (c) topic (harmonics, supraharmonics, counting when both) versus grouped years. The number of works providing reusable experimental data has evidently increased, by almost 50% the average of the works published between 2013 and 2018, and testifying the increased interest for this topic. Supraharmonics are gaining attention, doubling the number of works in the last 3-year period, with the same amount now as for harmonics. Looking at the types of publications, the shift to journal publications is remarkable, from the prevailing conferences of the two 3-year periods between 2013 and 2018. In this, MDPI represents a signiﬁcant pole of attraction in the most recent period (including two months of 2022), sharing equally with IEEE and the rest of the reachable sources. It is remarked that many works still speak of loading and not of distortion, propose models, possibly suitable for statistical analysis, but not measurements. Their quantity is more than ﬁve times greater, as indicated by the size of set2 (in the order of 150) compared to that of set3 (27), as derivable from Figure 10. g Selected works providing experimental data and technical judgment have been in- cluded in the references, used in the previous Section 3 to discuss methods of processing and assessment; those with substantial sets of data are analyzed in more detail in the rest of this section. 4.2. Discussion of Publications and Experimental Data A signiﬁcant set of data regarding harmonics and supraharmonics for 9 EVs in use in The Netherlands at the end of 2018 is reported by [51]. The results show that four out of nine vehicles fail to comply with the IEC 61000-3-2 limits for harmonics ranging from the 13th to the 37th: the degree of noncompliance amounts to 20% to 60% of the limit. Three EVs capable of charging above 16 A were tested against IEC 61000-3-12 limits and found instead compliant. This situation highlights the problem of pollution speciﬁc to LV grids for residential and ofﬁce loads, but at the same time, casts a doubt on the appropriateness of IEC 61000-3-2 limits, formulated more than 15 years ago and unchanged in the course Smart Cities 2022, 5 516 of three revisions. The reason is that the more and more frequent and widespread use of distorting loads in home and ofﬁce scenarios would necessitate a new evaluation of limits. of three revisions. The reason is that the more and more frequent and widespread use of distorting loads in home and ofﬁce scenarios would necessitate a new evaluation of limits. g Supraharmonic emissions for the 9 EVs are quite diversiﬁed in terms of frequency (switching fundamental ranging from 10 to 50 kHz) and amplitude (from 27 to 1080 mA). Emissions have not, however, been statistically characterized, nor correlated to the state of charge or charging current. It is worth underlining that signiﬁcant emissions in the audible frequency range have caused several cases of acoustic noise emitted by the EV and charger, but also by other loads in the same grid portion (such as induction cooking plates). Another relevant set of data is provided in [69], where ﬁve chargers (ranging from a small single-phase electric bike charger to a three-phase 11 kW car charger) were subjected to measurement. The work not only reports results on the harmonic behavior of each, but goes into detail on the waveshape and power factor, distortion power factor and displacement factor, showing that some deal of compensation between distorting loads is possible, although at large EV penetration compensation is compromised and distortion is relevant. Comparison of measurements carried out at off-peak (4:30 am) and on-peak (6:00 pm) hours conﬁrms larger current distortion, but a lower resulting voltage distortion, for the former. 4.2. Discussion of Publications and Experimental Data The reason is the lack of compensation with other distorting loads (leading to the larger current distortion), but at the same time, the grid is almost unloaded and the resulting voltage drops are lesser. g g p A similar opinion is expressed in [68], where a mix of EVs and household loads is con- sidered, showing some compensation of the 5th harmonic and an overall lower distortion for a speciﬁc amount of EVs, that, however, have a prevailing distortion contribution when their number is increased. A signiﬁcant compensation with external load (heat pump) is reported in [70], where the constant increase of the 7th harmonic intensity is effectively reduced by the switching on of a heat pump; when the second heat pump is switched on overcompensation occurs and the contribution of the latter prevails; the observed EVs had a negligible contribution of 5th harmonic, so that the switching on of the ﬁrst heat pump does not produce any beneﬁcial effect. Iqbal et al. [71] report a low variability of EV harmonic phase displacement for the 5th and 7th harmonics, spreading at most 45◦for the vast majority of the examined EVs (80% of a set of 11 EVs), preventing thus a substantial compensation between different EVs. In addition, it is observed that when comparing the harmonic phase angles of [70,71], they could be in agreement by applying a sign rever- sal, which raises a concern that many studies do not indicate clearly the polarity of the measured voltage and current quantities (e.g., voltage sign reversal as in German notation, preference for incoming or outgoing current). In [68], results regarding supraharmonic emissions are also provided, showing switch- ing frequencies of 27, 37 and 100 kHz with quite a wide range of amplitude, from 335 mA for the ﬁrst one, and only 10 mA for the last, and highest, switching frequency. One impor- tant point is also that tests conducted at a laboratory and on-site do not fully match, with one EV in particular undergoing a 4.3% shift of the main switching component and a 31% change of amplitude. 4.2. Discussion of Publications and Experimental Data Such values of supraharmonic emissions are conﬁrmed in [36], where the results of three parking lots are reported with low mean values (proﬁles of about 80 to 50 dBµA for 2 to 150 kHz) and a maximum proﬁle (less than 5% probability) 20 dB above, with sporadic peaks for a few switching components above 0.1 Arms. Similarly, the spectra up to 50 kHz reported in [21] show a couple of vehicles with clear emissions at 10 kHz and 27 kHz with peaks in amplitude of about 0.1 and 1 A rms, but also two proﬁles of emissions that do not have clear peaks of the fundamental switching frequency, thus cleverly achieving a reduction of supraharmonic emissions, limited to 10 mA rms. Regarding the positioning of emissions over the 2–150 kHz interval, the most common behavior corresponds to narrowband emissions between about 10 kHz (a common emission frequency among EVs) and 50 kHz [51]. A particular case is mentioned in [59] with broadband emissions between 2 and 6 kHz, that are probably of the same kind as those observed in [37] and result from the main pulse of the PFC stage. This impulsive nature is Smart Cities 2022, 5 517 conﬁrmed by [36] providing high-pass ﬁltered time-domain traces, rather than a frequency- domain analysis; in particular, its Figure 9c provides a waveform quite close to those analyzed in [37]. conﬁrmed by [36] providing high-pass ﬁltered time-domain traces, rather than a frequency- domain analysis; in particular, its Figure 9c provides a waveform quite close to those analyzed in [37]. y It should also be noted that the particular waveshape of the grid voltage has inﬂuence on the harmonic emissions of the connected load; in particular, a ﬂattened sinusoid (typical of residential LV grids with third harmonic pollution) may cause positive or negative changes of amplitude of some low-order harmonics by a signiﬁcant amount (between 30% and 300%), also depending on the type of EV charger [68]. In addition, the extensively mentioned unbalance at the fundamental caused by single- phase chargers is transferred also to harmonics and supraharmonics, as shown by [36], where differences of up to a factor of 2 between average values of supraharmonic total distortion can be observed. 4.2. Discussion of Publications and Experimental Data This is conﬁrmed by [59], where the supraharmonic current proﬁle (grouped on a bandwidth of 800 Hz or 4 kHz, the latter for the mentioned 2–6 kHz broadband emission) has almost a 2:1 difference between phase A and C. As anticipated, “unexpected” components, not visible when an EV charger is indi- vidually tested, e.g., in laboratory conditions, may arise due to two mechanisms that are discussed in [22]: intermodulation and beating. They both occur between two or more EV chargers: • Beating takes place when harmonic or supraharmonic emissions exist with a small frequency difference and they mix within the grid to which the EV chargers are connected. The small frequency difference may be justiﬁed by normal tolerance, drift and ageing of two otherwise identical chargers. The result is a low-frequency pulsation after superposition of individual emission signals as difference of the two frequency values, thus, mathematically identical to a second-order intermodulation discussed below. The example provided in [22] indicates a pulsation of 10% amplitude, resulting from two “identical” EVs with a frequency difference of 2.4 Hz out of the two respective 10 kHz switching frequencies; p g q • Intermodulation distortion instead occurs even with spectral components of emission that are located farther apart and is in general caused by nonlinear elements, such as the power converters themselves. The example in [22] clariﬁes the extent to which such a phenomenon can occur: taking two components at f1 = 56.2 kHz and f2 = 60.5 kHz of a measured spectrum, in the spectrum itself a second-order intermodulation term (f2 −f1 = 4.3 kHz) and two third-order intermodulation terms (2f2 −f1 = 64.8 kHz and 2f1 −f2 = 51.9 kHz) are clearly visible. 5. Conclusions The articulated problem of harmonic and supraharmonic emissions of plug-in EV chargers was analyzed, focusing ﬁrst on the typical power converter architecture imple- menting EV chargers and on the normative requirements and reference levels. The typical mechanisms of emission, the spectral behavior and the superposition of emissions from different chargers and with existing LV grid distortion were then discussed, with extensive reference to existing experimental data, that unfortunately are limited to fewer than a dozen works in the last ten years or so. There are some signiﬁcant elements to consider for standardization purposes (e.g., to identify suitable limits for a new category of highly dynamic loads), for the design of lines feeding the charging points, and when preparing a measurement campaign and evaluating then the measurement results: • Harmonic distortion is still quite variable between EV models, above IEC limits in some conditions, although PFC should be extensively applied; • Superposition of harmonics can occur with a wide range of distorting loads, such as home and ofﬁce appliances and equipment, characterized by a signiﬁcant penetration into the AC grid; Smart Cities 2022, 5 518 • Some major low-order harmonics may be quite dependent on the waveshape of the grid voltage, in particular for cases of ﬂat-top voltage affected thus by third harmonic pollution; this exempliﬁes the necessity of testing reﬂecting the real conditions of use; • The supraharmonic interval contains emissions from the PFC stage and power con- verters downstream inside the EV, in the form of a main switching component and its harmonics; broadband emissions may occur in the lowest frequency interval, below, e.g., 10 kHz, excited by the PFC impulses; g y p • A signiﬁcant mutual inﬂuence between different chargers connected to the same grid was observed, pointing out the inﬂuence of ﬁeld conditions contrasted with laboratory qualiﬁcation results of single standalone EV; such inﬂuence materializes in a signiﬁcant deal of beating and intermodulation phenomena; • At a different level, mutual inﬂuence was also reported, consisting of the reciprocal interference between EVs passing through different charging conditions and switching on and off the charging process abruptly, causing changes of grid response and transient disturbance; interference resulted in EV charger disconnection, repeated charging attempts and the necessity of manual intervention. 5. Conclusions Lessons learned would suggest, among others, three lines of intervention for the im- provement of the assessment of the impact of EV charging on LV grids and connected loads: • Modeling and accounting for superposition of components and resulting intermodulation; • Consistent test of emissions to reﬂect real conditions of use for grid impedance (in- cluding the effect of other loads) and pre-existing distortion; g p g • Immunity test of EVs to supraharmonics, zero-crossing oscillatory signals and tran- sient events caused by EV interactions and concomitant charging. A thorough understanding of the response of the grid in its entirety to an increasing number of connected EVs should go beyond the estimate of the resulting electrical load and impact in terms of voltage drop and voltage imbalance. This is particularly important for the reported episodes distortion and interference in the most recent papers and in view of the green transition to an exclusively electric vehicle production in the next ten years or so. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Conﬂicts of Interest: The author declares no conﬂict of interest. References 1. Energy Technology Policy (ETP) Division. Nordic EV Outlook 2018, Directorate of Sustainability, Technology and Outlooks (STO) of the International Energy Agency (IEA); Technical Report; International Energy Agency (IEA): Paris, France, 2018. gy g y p gy g y ( ) 2. Energy Technology Policy (ETP) Division. Global EV Outlook 2019, Directorate of Sustainability, Technology and Outlooks (STO) of the International Energy Agency (IEA); Technical Report; International Energy Agency (IEA): Paris, France, 2019. 2. Energy Technology Policy (ETP) Division. Global EV Outlook 2019, Directorate of Sustainability, Technology and Outlooks (STO) of the International Energy Agency (IEA); Technical Report; International Energy Agency (IEA): Paris, France, 2019. 3. European Union. European Green Deal: Commission Proposes Transformation of EU Economy and Society to Meet Climate Ambi- tions. 2021. Available online: https://ec.europa.eu/commission/presscorner/detail/en/IP_21_3541 (accessed on 28 January 2022). 3. European Union. European Green Deal: Commission Proposes Transformation of EU Economy and Society to Meet Climate Ambi- tions. 2021. Available online: https://ec.europa.eu/commission/presscorner/detail/en/IP_21_3541 (accessed on 28 January 2022). 4. Sanguesa, J.A.; Torres-Sanz, V.; Garrido, P.; Martinez, F.J.; Marquez-Barja, J.M. A Review on Electric Vehicles: Technologies and Challenges. Smart Cities 2021, 4, 372–404. [CrossRef] p p p ( J y ) 4. Sanguesa, J.A.; Torres-Sanz, V.; Garrido, P.; Martinez, F.J.; Marquez-Barja, J.M. A Review on Electric Vehicles: Technologies and Challenges. Smart Cities 2021, 4, 372–404. [CrossRef] g 5. Dharmakeerthi, C.; Mithulananthan, N.; Saha, T. Impact of electric vehicle fast charging on power syste Electr. Power Energy Syst. 2014, 57, 241–249. [CrossRef] 6. Auer, M.; Kaffe, E.; La Fauci, R. Impact of fast charging and home charging infrastructure for electric vehicles on the power quality of the distribution grid. In Proceedings of the 25th International Conference on Electricity Distribution, Madrid, Spain, 3–6 June 2019. gy 8. Fernández-Rodriguez, A.; Fernández-Cardador, A.; Cucala, A.; Falvo, M. Energy Efﬁciency and Integration of Urban Electrical Transport Systems: EVs and Metro-Trains of Two Real European Lines. Energies 2019, 12, 366. [CrossRef] Smart Cities 2022, 5 519 9. Smith, K.; Hunter, L.; Galloway, S.; Booth, C.; Kerr, C.; Kellett, M. Integrated Charging of EVs Using Existing LVDC Light Rail Infrastructure: A Case Study. In Proceedings of the 2019 IEEE Third International Conference on DC Microgrids (ICDCM), Matsue, Japan, 20–23 May 2019. , J p , y 10. Akhavan-Hejazi, H.; Mohsenian-Rad, H.; Nejat, A. Developing a Test Data Set for Electric Vehicle Applications in Smart Grid Research. References In Proceedings of the 2014 IEEE 80th Vehicular Technology Conference (VTC2014-Fall), Vancouver, QC, Canada, 14–17 September 2014. p 11. Gaete-Morales, C.; Kramer, H.; Schill, W.P.; Zerrahn, A. An open tool for creating battery-electric vehicle time series from empirical data, emobpy. Sci. Data 2021, 8, 1–18. [CrossRef] [PubMed] 12. Haben, S.; Arora, S.; Giasemidis, G.; Voss, M.; Greetham, D.V. Review of low voltage load forecasting: Methods, applications, and recommendations. Applied Energy 2021, 304, 117798. [CrossRef] 13. Arif, S.M.; Lie, T.T.; Seet, B.C.; Ayyadi, S.; Jensen, K. Review of Electric Vehicle Technologies, Charging Optimization Techniques. Electronics 2021, 10, 1910. [CrossRef] p q 14. Mies, J.; Helmus, J.; van den Hoed, R. Estimating the Charging Proﬁle of Individual Charge Sessions of Electric Vehicles in The Netherlands. World Electr. Veh. J. 2018, 9, 17. [CrossRef] 15. Amara-Ouali, Y.; Goude, Y.; Massart, P.; Poggi, J.M.; Yan, H. A Review of Electric Vehicle Load Open Data and Models. Energies 2021, 14, 2233. [CrossRef] 16. Gruosso, G. Analysis of impact of electrical vehicle charging on low voltage power grid. In Proceedings of the 2016 International Conference on Electrical Systems for Aircraft, Railway, Ship Propulsion and Road Vehicles & International Transportation Electriﬁcation Conference (ESARS-ITEC), Toulouse, France, 2–4 November 2016 17. Abraham, D.S.; Verma, R.; Kanagaraj, L.; Raman, S.R.G.T.; Rajamanickam, N.; Chokkalingam, B.; Sekar, K.M.; Mihet-Popa, L. Electric Vehicles Charging Stations’ Architectures, Criteria, Power Converters, and Control Strategies in Microgrids. Electronics 2021, 10, 1895. [CrossRef] [ ] 18. Mariscotti, A. Power Quality Phenomena, Standards, and Proposed Metrics for DC Grids. Energies 2021, 14, 6453. [CrossRef] 18. Mariscotti, A. Power Quality Phenomena, Standards, and Proposed Metrics for DC Grids. Energies 2021, 14, 6453. [CrossRef] 19. Raﬁ, M.A.H.; Bauman, J. A Comprehensive Review of DC Fast-Charging Stations with Energy Storage: Architectures, Power Converters, and Analysis. IEEE Trans. Transp. Electrif. 2021, 7, 345–368. [CrossRef] y p g 19. Raﬁ, M.A.H.; Bauman, J. A Comprehensive Review of DC Fast-Charging Stations with Energy Storage: Architectures, Power Converters, and Analysis. IEEE Trans. Transp. Electrif. 2021, 7, 345–368. [CrossRef] y p f 20. Rönnberg, S.K.; Castro, A.G.; Bollen, M.H.J.; Moreno-Munoz, A.; Romero-Cadaval, E. Supraharmonics from power electronics converters. In Proceedings of the 9th International Conference on Compatibility and Power Electronics (CPE), Costa da Caparica, Portugal, 24–26 June 2015; pp. 539–544. g pp 21. Meyer, J.; Mueller, S.; Ungethuem, S.; Xiao, X.; Collin, A.; Djokic, S. Harmonic and supraharmonic emission of on-board electric vehicle chargers. References In Proceedings of the IEEE PES Transmission & Distribution Conference and Exposition-Latin America (PES T&D-LA), Morelia, Mexico, 21–24 September 2016. ´ p 22. Slangen, T.; van Wijk, T.; ´Cuk, V.; Cobben, S. The Propagation and Interaction of Supraharmonics from Electric Vehicle Chargers in a Low-Voltage Grid. Energies 2020, 13, 3865. [CrossRef] 23. Blum, J. Evaluating Supraharmonics Up to 150 kHz in Electric Vehicles at the University of Applied Sciences 2021. Available online: https://powerquality.blog/2021/11/05/evaluating-supraharmonics-up-to-150 at the university of applied sciences bingen (accessed on 28 January 2022) 23. Blum, J. Evaluating Supraharmonics Up to 150 kHz in Electric Vehicles at the University of Applied Sciences Bingen; Technical Report; 2021. Available online: https://powerquality.blog/2021/11/05/evaluating-supraharmonics-up-to-150-khz-in-electric-vehicles- at-the-university-of-applied-sciences-bingen (accessed on 28 January 2022). 24. Lee, B.K.; Kim, J.P.; Kim, S.G.; Lee, J.Y. An Isolated/Bidirectional PWM Resonant Converter for V2G(H) EV On-Board Charger. IEEE Trans. Veh. Technol. 2017, 66, 7741–7750. [CrossRef] c Vehicle Conductive Charging System—Part 1: General Requirements. IEC: Geneva, Switzerland, 2017. 25. IEC 61851-1; Electric Vehicle Conductive Charging System—Part 1: General Requirements. IEC: G 26. Triviño, A.; González-González, J.M.; Aguado, J.A. Wireless Power Transfer Technologies Applied to Electric Vehicles: A Review. Energies 2021, 14, 1547. [CrossRef] g 27. Zhang, Z.; Xu, H.; Shi, L.; Li, D.; Han, Y. A unit power factor DC fast charger for electric vehicle charging station. In Proceedings of the 7th International Power Electronics and Motion Control Conference, Harbin, China, 2–5 June 2012. 28. Kuperman, A.; Levy, U.; Goren, J.; Zafranski, A.; Savernin, A. Modeling and Control of the PFC Stage for a 50 kW EV Fast Battery Charger. Proc. World Congr. Eng. 2011, 2, 5–9. 29. Ramakrishnan, H.; Rangaraju, J. Power Topology Considerations for Electric Vehicle Charging Stations; Technical Report; Texas Instruments: Dallas, TX, USA, 2020. 30. Piasecki, S.; Zaleski, J.; Jasinski, M.; Bachman, S.; Turzy´nski, M. Analysis of AC/DC/DC Converter Modules for Direct Current Fast-Charging Applications. Energies 2021, 14, 6369. [CrossRef] 31. Das, S.; Narayanan, G. Analytical Closed-Form Expressions for Harmonic Distortion Correspond Sequences for Neutral-Point-Clamped Inverters. IEEE Trans. Ind. Electron. 2014, 61, 4485–4497. [CrossR , G. Analytical Closed-Form Expressions for Harmonic Distortion Corresponding to Novel Switching ral-Point-Clamped Inverters. IEEE Trans. Ind. Electron. 2014, 61, 4485–4497. [CrossRef] 32. Kan, Y.; Hyun, S.W.; Hong, S.J.; Won, C.Y. Zero dead-time PWM implementation method for reducing total harmonic distortion in 3-level NPC inverter. In Proceedings of the 2015 18th International Conference on Electrical Machines and Systems (ICEMS), Pattaya City, Thailand, 25–28 October 2015. References y y 33. Feng, Z.; Zhang, X.; Wang, J.; Yu, S. A High-Efﬁciency Three-Level ANPC Inverter Based on Hybrid SiC 2020, 13, 1159. [CrossRef] Wang, J.; Yu, S. A High-Efﬁciency Three-Level ANPC Inverter Based on Hybrid SiC and Si Devices. Energies sRef] 34. Zambra, D.A.B.; Rech, C.; Pinheiro, J.R. Comparison of Neutral-Point-Clamped, Symmetrical, and Hybrid Asymmetrical Multilevel Inverters. IEEE Trans. Ind. Electron. 2010, 57, 2297–2306. [CrossRef] Smart Cities 2022, 5 520 35. Mariscotti, A. Experimental characterisation of active and non-active harmonic power ﬂow of AC rolling stock and interaction with the supply network. Electr. Syst. Transp. 2021, 11, 109–120. [CrossRef] pp y y p 36. Streubel, T.; Kattmann, C.; Eisenmann, A.; Rudion, K. Characterization of Supraharmonic Emission from Three Different Electric Vehicle Charging Infrastructures in Time and Frequency Domain. Energies 2022, 15, 394. [CrossRef] g g q y 37. Sandrolini, L.; Mariscotti, A. Signal Transformations for Analysis of Supraharmonic EMI Caused by Switched-Mode Power Supplies. Electronics 2020, 9, 2088. [CrossRef] 38. Sandrolini, L.; Mariscotti, A. Waveform and Spectral Characteristics of Supraharmonic Unsymmetrical Conducted EMI of Switched-Mode Power Supplies. Electronics 2022, 11, 591. [CrossRef] pp 39. ISO 6469-3; Electrically Propelled Road Vehicles—Safety Speciﬁcations. Part 3: Electrical Safety. ISO: G 39. ISO 6469-3; Electrically Propelled Road Vehicles—Safety Speciﬁcations. Part 3: Electrical Safety. ISO: Geneva, Switzerland, 2017. 40. Liu, C. Analysis, Design and Control of DC-DC Resonant Converter for On-Board Bidirectional Battery Charger in Electric V hi l Ph D Th i U i it f Sh fﬁld Sh fﬁld UK 2017 A il bl li htt // th hit k/17528/1/ 39. ISO 6469-3; Electrically Propelled Road Vehicles—Safety Speciﬁcations. Part 3: Electrical Safety. ISO: Geneva, Switzerland, 2017. 40. Liu, C. Analysis, Design and Control of DC-DC Resonant Converter for On-Board Bidirectional Battery Charger in Electric Vehicles. Ph.D. Thesis, University of Shefﬁeld, Shefﬁeld, UK, 2017. Available online: https://etheses.whiterose.ac.uk/17528/1/ Ch h iLi Th i Fi l 2017 05 30 df ( d 28 J 2022) 40. Liu, C. Analysis, Design and Control of DC-DC Resonant Converter for On-Board Bidirectional Battery Charger in Electric Vehicles. Ph.D. Thesis, University of Shefﬁeld, Shefﬁeld, UK, 2017. Available online: https://etheses.whiterose.ac.uk/17528/1/ ChaohuiLiu_Thesis_Final_2017-05-30.pdf (accessed on 28 January 2022). p ( J y ) 41. Liu, K.; Yang, Z.; Tang, X.; Cao, W. Automotive Battery Equalizers Based on Joint Switched-Capacitor and Buck-Boost Converters. IEEE Trans. Veh. Technol. 2020, 69, 12716–12724. [CrossRef] 42. References IEC 61000-2-2; Electromagnetic Compatibility (EMC)—Part 2-2: Environmen—Compatibility Levels for Low-Frequency Con- ducted Disturbances and Signalling in Public Low-Voltage Power Supply Systems. IEC: Geneva, Switzerland, 2019. 43. IEC 61000-2-4; Electromagnetic Compatibility (EMC)—Part 2-4: Environment—Compatibility Levels In Industrial Plants for Low-Frequency Conducted Disturbances. IEC: Geneva, Switzerland, 2020. q y 44. IEC 61000-3-2; Electromagnetic Compatibility (EMC)—Part 3-2: Limits – Limits for Harmonic Current Emissions (Equipment Input Current ≤16 A Per Phase). IEC: Geneva, Switzerland, 2019. p 45. IEC 61851-21-1; Electric Vehicle Conductive Charging System—Part 21-1: Electric Vehicle on-Board Ch for Conductive Connection to an AC/DC Supply. IEC: Geneva, Switzerland, 2017. pp y 46. IEC 61000-3-12; Electromagnetic Compatibility (EMC)—Part 3-12: Limits–Limits for Harmonic Currents Producedby Equipment Connected to Public Low-Voltage Systems with Input Current >16 A and ≤75 A per Phase. IEC: Geneva, Switzerland, 2011. g y p p 47. IEC 61000-4-7; Electromagnetic Compatibility (EMC)—Part 4-7: Testing and Measurement Techniques—General Guide on Harmonics and Interharmonics Measurements and Instrumentation, for Power Supply Systems and Equipment Connected Thereto. IEC: Geneva, Switzerland, 2002. 48. CISPR 16-1-1; Speciﬁcation for Radio Disturbance and Immunity Measuring Apparatus and Methods—Part 1-1: Radio Distur- bance and Immunity Measuring Apparatus—Measuring Apparatus. IEC: Geneva, Switzerland, 2019. y g pp g pp 49. Sandrolini, L.; Mariscotti, A. Impact of short-time Fourier transform parameters on the accuracy of EMI spectra estimates in the 2–150 kHz supraharmonic interval. Electr. Power Syst. Res. 2021, 195, 107130. [CrossRef] 50. Klatt, M.; Meyer, J.; Schegner, P. Comparison of measurement methods for the frequency range of 2 kHz to 150 kHz. In Proceedings of the International Conference on Harmonics and Quality of Power, Bucharest, Romania, 25–28 May 2014; pp. 818–822. 51. Slangen, T.M.H.; van Wijk, T.; Cuk, V.; Cobben, J.F.G. The Harmonic and Supraharmonic Emission of Battery Electric Vehicles in The Netherlands. In Proceedings of the 2020 International Conference on Smart Energy Systems and Technologies (SEST), Istanbul, Turkey, 7–9 September 2020. y p 52. CISPR 16-2-1; Speciﬁcation for Radio Disturbance and Immunity Measuring Apparatus and Methods—Part 2-1: Methods of Measurement of Disturbances and Immunity—Conducted Disturbance Measurements. IEC: Geneva, Switzerland, 2014. EN 50065-1; Signalling on Low-Voltage Electrical Installations in the Frequency Range 3 kHz to 148,5 Requirements Frequency Bands and Electromagnetic Disturbances CENELEC: Brussels Belgium 2012 0065-1; Signalling on Low-Voltage Electrical Installations in the Frequency Range 3 kHz to 148,5 kHz P 53. References EN 50065-1; Signalling on Low-Voltage Electrical Installations in the Frequency Range 3 kHz to 148,5 kHz Part 1: General Requirements, Frequency Bands and Electromagnetic Disturbances. CENELEC: Brussels, Belgium, 2012. 53. EN 50065 1; Signalling on Low Voltage Electrical Installations in the Frequency Range 3 kHz to 148,5 kHz Part 1: General Requirements, Frequency Bands and Electromagnetic Disturbances. CENELEC: Brussels, Belgium, 2012. Requirements, Frequency Bands and Electromagnetic Disturbances. CENELEC: Brussels, Belgium, 2012. 54. CISPR 16-1-2; Speciﬁcation for Radio Disturbance and Immunity Measuring Apparatus and Methods—Part 1-2: Radio Dis- turbance and Immunity Measuring Apparatus—Coupling Devices for Conducted Disturbance Measurements. IEC: Geneva, Switzerland, 2014. 55. IEC 61000-3-11; Electromagnetic Compatibility (EMC)—Part 3-11: Part 3-11: Limits—Limitation of Voltage Changes, Voltage Fluctuationsand Flicker in Public Low-Voltage Supply Systems—Equipment Withrated Current ≤75 A and Subject to Conditional Connection. IEC: Geneva, Switzerland, 2019. 56. Busatto, T.; Larsson, A.; Ronnberg, S.K.; Bollen, M.H.J. Including Uncertainties From Customer Connections in Calculating Low-Voltage Harmonic Impedance. IEEE Trans. Power Deliv. 2019, 34, 606–615. [CrossRef] l h k l h d l l k f k d h f 57. Stiegler, R.; Meyer, J.; Schori, S.; Höckel, M.; Scheida, K.; Drápela, J.; Hanžlík, T. Survey of network impedance in the frequency range 2–9 kHz in public low voltage networks in AT/CH/CZ/GE. In Proceedings of the 25th International Conference and Exhibition on Electricity Distribution (CIRED), Madrid, Spain, 3–6 June 2019. y p 58. Harris, F.J. On the use of windows for harmonic analysis with the discrete Fourier transform. Proc. IEEE 1978, 66, 51–83. [CrossRef] d d b l f h ( h ) 59. Darmawardana, D.; David, J.; Perera, S.; Robinson, D.; Meyer, J.; Jayatunga, U. Analysis of High Frequency (Supraharmonics) Emissions Caused by Electric Vehicle Charging. In Proceedings of the 2020 19th International Conference on Harmonics and Quality of Power (ICHQP), Dubai, United Arab Emirates, 6–7 July 2020. y y 60. Streubel, T.; Kattmann, C.; Eisenmann, A.; Rudion, K. Detection and Monitoring of Supraharmonic Anomalies of an Electric Vehicle Charging Station. In Proceedings of the 2019 IEEE Milan PowerTech, Milan, Italy, 27 June 2017. Smart Cities 2022, 5 521 61. IEC 61000-4-30. Electromagnetic Compatibility (EMC)—Part 4-30: Testing and Measurement Techniques—Power Quality Measurement Methods. IEC: Geneva, Switzerland, 2015. 62. Wang, Y.; Xu, Y.; Tao, S.; Siddique, A.; Dong, X. A Flexible Supraharmonic Group Method Based on Switching Frequency Identiﬁcation. IEEE Access 2020, 8, 39491–39501. [CrossRef] 63. Möller, F.; Müller, S.; Meyer, J. 71. Iqbal, M.N.; Kütt, L.; Daniel, K.; Asad, B.; Ghahfarokhi, P.S. Estimation of Harmonic Emission of Electric Vehicles and Their Impact on Low Voltage Residential Network. Sustainability 2021, 13, 8551. [CrossRef] 70. Malano, A.A.; Muller, S.; Meyer, J.; Bachmann, S. Harmonic interaction of electric vehicle chargers in a central charging infrastructure. In Proceedings of the 2016 17th International Conference on Harmonics and Quality of Power (ICHQP), Belo Horizonte, Brazil, 16–19 October 2016. References Impact of Electric Vehicles on Power Quality in Central Charging Infrastructures. In Proceedings of the 1st E-Mobility Power System Integration Symposium, Berlin, Germany, 23 October 2017 y y g y p y 64. Meyer, J.; Bollen, M.; Amaris, H.; Blanco, A.M.; Gil De Castro, A.; Desmet, J.; Klatt, M.; Kocewiak, L.; Ronnberg, S.; Yang, K. Future work on harmonics—Some expert opinions Part II —Supraharmonics, standards and measurements. In Proceedings of y y g y p y 64. Meyer, J.; Bollen, M.; Amaris, H.; Blanco, A.M.; Gil De Castro, A.; Desmet, J.; Klatt, M.; Kocewiak, L.; Ronnberg, S.; Yang, K. Future work on harmonics—Some expert opinions Part II —Supraharmonics, standards and measurements. In Proceedings of International Conference on Harmonics and Quality of Power, ICHQP, Bucharest, Romania, 25–28 May 2014; pp. 909–913. 65. Gil-De-Castro, A.; Medina-Gracia, R.; Ronnberg, S.; Blanco, A.; Meyer, J. Differences in the performance between CFL and LED lamps under different voltage distortions. In Proceedings of the 18th International Conference on Harmonics and Quality of Power (ICHQP), Ljubljana, Slovenia, 13–16 May 2018. ( ) j j y 66. Mariscotti, A.; Sandrolini, L.; Pasini, G. Variability caused by Setup and Operating Conditions for Conducted EMI of Switched Mode Power Supplies over the 2–1000 kHz Interval. IEEE Trans. Instrum. Meas. 2022. [CrossRef] 67. Kutt, L.; Saarijarvi, E.; Lehtonen, M.; Molder, H.; Niitsoo, J. Electric vehicle charger load current harmonics variations due to supply voltage level differences—Case examples. In Proceedings of the 2014 International Symposium on Power Electronics, Electrical Drives, Automation and Motion, Ischia, Italy, 18–20 June 2014. y 68. Möller, F.; Müller, S.; Wald, C.; Isensee, S. Impact of electric vehicle charging on unbalance and harmonic distortion—Field study in an urban residential area. In Proceedings of the 23rd International Conference on Electricity Distribution, Lyon, Franc, 15–18 June 2015. 69. Collin, A.J.; Djokic, S.Z.; Thomas, H.F.; Meyer, J. Modelling of electric vehicle chargers for power system of the 11th International Conference on Electrical Power Quality and Utilisation, Lisbon, Portugal, 17– 70. Malano, A.A.; Muller, S.; Meyer, J.; Bachmann, S. Harmonic interaction of electric vehicle chargers in a central charging infrastructure. In Proceedings of the 2016 17th International Conference on Harmonics and Quality of Power (ICHQP), Belo Horizonte, Brazil, 16–19 October 2016. 71. Iqbal, M.N.; Kütt, L.; Daniel, K.; Asad, B.; Ghahfarokhi, P.S. Estimation of Harmonic Emission of Electric Vehicles and Their Impact on Low Voltage Residential Network. Sustainability 2021, 13, 8551. [CrossRef]
W1984518880.txt	https://journals.iucr.org/e/issues/2008/10/00/ng2495/ng2495.pdf	en		<i>catena</i>-Poly[[(dimethyl sulfoxide-κ<i>O</i>)zinc(II)]-μ-(<i>E</i>)-2-[(2-oxido-1-naphthyl)methyleneamino]propanoato-κ<sup>4</sup><i>O</i><sup>2</sup>,<i>N</i>,<i>O</i><sup>1</sup>:<i>O</i><sup>1′</sup>]	Acta crystallographica. Section E	2,008	cc-by	2,360	metal-organic compounds Acta Crystallographica Section E Experimental Structure Reports Online Crystal data ISSN 1600-5368 catena-Poly[[(dimethyl sulfoxide-jO)zinc(II)]-l-(E)-2-[(2-oxido-1-naphthyl)methyleneamino]propanoato0 j4O2,N,O1:O1 ] Jun You, Bo Liu,* Yan-Qiong Chen, Cui Xiao and Duo-Qi Zhai School of Chemistry and Environment Engineering, Harbin University of Science and Technology, Harbin 150040, People’s Republic of China Correspondence e-mail: liubo200400@vip.sina.com Received 22 September 2008; accepted 23 September 2008 Key indicators: single-crystal X-ray study; T = 291 K; mean (C–C) = 0.003 Å; R factor = 0.024; wR factor = 0.056; data-to-parameter ratio = 17.7. [Zn(C14H11NO3)(C2H6OS)] Mr = 384.74 Monoclinic, P21 a = 9.676 (4) Å b = 7.651 (4) Å c = 11.715 (5) Å = 106.256 (15) V = 832.6 (7) Å3 Z=2 Mo K radiation = 1.62 mm1 T = 291 (2) K 0.31 0.28 0.24 mm Data collection Rigaku R-AXIS RAPID diffractometer Absorption correction: multi-scan (ABSCOR; Higashi, 1995) Tmin = 0.633, Tmax = 0.697 8191 measured reflections 3729 independent reflections 3533 reflections with I > 2(I) Rint = 0.020 Refinement R[F 2 > 2(F 2)] = 0.024 wR(F 2) = 0.056 S = 1.06 3729 reflections 211 parameters 1 restraint H-atom parameters constrained max = 0.25 e Å3 min = 0.19 e Å3 Absolute structure: Flack (1983), 1675 Friedel pairs Flack parameter: 0.006 (8) Table 1 Selected geometric parameters (Å, ). In the title coordination polymer, [Zn(C14H11NO3)(C2H6OS)]n, each ZnII ion is five-coordinated in a slightly distorted trigonal–bipyramidal coordination environment, formed by three O atoms from two 2-[(2-oxido-1-naphthyl)methyleneamino]propanoate ligands, one O atom from a dimethyl sulfoxide molecule and the N atom from the aminopropanoate ligand. The propanoate ligands bridge ZnII ions, forming a zigzag chain parallel to [010]. Related literature For the synthesis of (E)-2-[(2-hydroxynaphthalen-1-yl)methyleneamino]propanoic acid, see: Audriceth et al. (1954). N1—Zn1 O1—Zn1 O2—Zn1 O3ii—Zn1—O1 O3ii—Zn1—N1 O1—Zn1—N1 O3ii—Zn1—O4 O1—Zn1—O4 2.0119 (18) 2.0040 (15) 2.1891 (16) 98.54 (7) 138.57 (7) 88.32 (7) 101.12 (7) 96.09 (7) O3—Zn1i O4—Zn1 N1—Zn1—O4 O3ii—Zn1—O2 O1—Zn1—O2 N1—Zn1—O2 O4—Zn1—O2 1.9560 (15) 2.0520 (17) 118.82 93.89 166.10 78.22 87.52 (7) (7) (6) (6) (7) Symmetry codes: (i) x þ 1; y þ 12; z þ 1; (ii) x þ 1; y 12; z þ 1. Data collection: RAPID-AUTO (Rigaku, 1998); cell refinement: RAPID-AUTO; data reduction: CrystalStructure (Rigaku/MSC, 2002); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97. This work was supported by the National Natural Science Foundation of China (No. 20272011). Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: NG2495). References Audriceth, L. F., Scott, E. S. & Kipper, P. S. (1954). J. Org. Chem. 19, 733–741. Flack, H. D. (1983). Acta Cryst. A39, 876–881. Higashi, T. (1995). ABSCOR. Rigaku Corporation, Tokyo, Japan. Rigaku (1998). RAPID-AUTO. Rigaku Corporation, Tokyo, Japan. Rigaku/MSC (2002). CrystalStructure. Rigaku/MSC Inc., The Woodlands, Texas, USA. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. m1338 You et al. doi:10.1107/S1600536808030614 Acta Cryst. (2008). E64, m1338 supporting information supporting information Acta Cryst. (2008). E64, m1338 [doi:10.1107/S1600536808030614] catena-Poly[[(dimethyl sulfoxide-κO)zinc(II)]-µ-(E)-2-[(2-oxido-1-naphthyl)methyleneamino]propanoato-κ4O2,N,O1:O1′] Jun You, Bo Liu, Yan-Qiong Chen, Cui Xiao and Duo-Qi Zhai S1. Comment The continuous interest in designing and making novel Schiff base ligand and transition-metal complexes have persisted because of their impressive catalytic property. In this paper, we report the new title compound, (I), synthesized by the reaction of (E)-2-((2-hydroxynaphthalen-1-yl)methyleneamino)propanoic acid ligands and Zn(OAc)2 in an aqueous solution. As shown in Fig. 1, ZnII ion is five-coordinate in a slightly distorted trigonal-bipyramidal coordination environment, formed by four O atoms and one N atom. Each quadridentate Schiff base ligand bridge two different CuII ions, resulting in a one-dimensional polymeric structure chain (Fig. 2). S2. Experimental (E)-2-((2-Hydroxynaphthalen-1-yl)methyleneamino)propanoic acid was prepared of L-alanine acid and 2-hydroxy-1naphthaldehyde in aqueous solution (Audriceth et al., 1954). (E)-2-((2-Hydroxynaphthalen-1-yl)methyleneamino)propanoic acid (0.243 g, 1 mmol) and Zn(OAc)2 (0.190 g, 1 mmol) dissolved in hot aqueous solution (20 ml) then refluxed for 1 huor. After cooling to room temperature the solution was filtered, the residue was recrystaled in DMSO and methanol (10/1, V/V) solution, several days latter, a suitable for X-ray diffraction yellow crystal was obtained. S3. Refinement H atoms bound to C atoms were placed in calculated positions and treated as riding on their parent atoms, with C—H = 0.93 Å (Caromatic); C—H = 0.96 Å (methyl); C—H = 0.98 ° A (methine), and with Uiso(H) = 1.2Ueq(C). Acta Cryst. (2008). E64, m1338 sup-1 supporting information Figure 1 The molecular structure of (I), showing displacement ellipsoids at the 30% probability level for non-H atoms. [Symmetry code: (I) -x + 1, 1/2 + y, -z + 1]. Acta Cryst. (2008). E64, m1338 sup-2 supporting information Figure 2 A partial packing view, showing the one-dimensional chain structure. H atoms have been omitted for clarity. catena-Poly[[(dimethyl sulfoxide-κO)zinc(II)]- µ-(E)-2-[(2-oxido-1-naphthyl)methyleneamino]propanoatoκ4O2,N,O1:O1′] Crystal data [Zn(C14H11NO3)(C2H6OS)] Mr = 384.74 Monoclinic, P21 Hall symbol: P 2yb a = 9.676 (4) Å b = 7.651 (4) Å c = 11.715 (5) Å β = 106.256 (15)° Acta Cryst. (2008). E64, m1338 V = 832.6 (7) Å3 Z=2 F(000) = 396 Dx = 1.535 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 7870 reflections θ = 3.2–27.5° µ = 1.62 mm−1 sup-3 supporting information T = 291 K Block, yellow 0.31 × 0.28 × 0.24 mm Data collection Rigaku R-AXIS RAPID diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω scans Absorption correction: multi-scan (ABSCOR; Higashi, 1995) Tmin = 0.633, Tmax = 0.697 8191 measured reflections 3729 independent reflections 3533 reflections with I > 2σ(I) Rint = 0.020 θmax = 27.5°, θmin = 3.2° h = −11→12 k = −9→9 l = −15→15 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.024 wR(F2) = 0.056 S = 1.06 3729 reflections 211 parameters 1 restraint Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0259P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.25 e Å−3 Δρmin = −0.19 e Å−3 Absolute structure: Flack (1983), 1675 Friedel pairs Absolute structure parameter: 0.006 (8) Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) C1 C2 C3 C4 H4 C5 H5 C6 H6 C7 H7 C8 x y z Uiso/Ueq 0.7743 (2) 0.66925 (19) 0.6855 (2) 0.5858 (3) 0.5020 0.6085 (3) 0.5400 0.7336 (3) 0.7504 0.8305 (3) 0.9131 0.8094 (2) −0.1178 (3) −0.0224 (3) 0.0055 (4) 0.0965 (3) 0.1392 0.1243 (4) 0.1837 0.0636 (3) 0.0859 −0.0276 (4) −0.0694 −0.0613 (3) 0.23967 (18) 0.15494 (15) 0.03564 (15) −0.0557 (2) −0.0416 −0.1655 (2) −0.2243 −0.1883 (2) −0.2613 −0.10433 (19) −0.1209 0.00897 (19) 0.0288 (4) 0.0272 (4) 0.0304 (4) 0.0429 (6) 0.051 0.0477 (6) 0.057* 0.0483 (6) 0.058* 0.0445 (6) 0.053* 0.0340 (5) Acta Cryst. (2008). E64, m1338 sup-4 supporting information C9 H9 C10 H10 C11 H11 C12 H12 C13 H13A H13B H13C C14 C15 H15A H15B H15C C16 H16A H16B H16C N1 O1 O2 O3 O4 S1 Zn1 0.9098 (2) 0.9890 0.8947 (2) 0.9628 0.5465 (2) 0.4778 0.3897 (2) 0.3676 0.2635 (2) 0.2422 0.1808 0.2879 0.4141 (2) 0.7228 (3) 0.6852 0.7508 0.6501 0.9720 (3) 1.0605 0.9930 0.9161 0.52124 (17) 0.77231 (16) 0.51906 (16) 0.32074 (15) 0.82733 (16) 0.87374 (6) 0.66361 (2) −0.1605 (4) −0.2075 −0.1897 (3) −0.2568 0.0524 (3) 0.1043 0.1430 (3) 0.2387 0.0163 (5) −0.0219 0.0738 −0.0829 0.2180 (3) 0.4372 (3) 0.3618 0.5467 0.4571 0.2924 (4) 0.2349 0.3998 0.2180 0.0554 (2) −0.1464 (2) 0.1680 (2) 0.3268 (2) 0.1620 (2) 0.33792 (8) −0.00892 (3) 0.0950 (2) 0.0756 0.2055 (2) 0.2595 0.18248 (18) 0.1198 0.29272 (17) 0.2349 0.2663 (3) 0.1851 0.2788 0.3182 0.41799 (18) 0.3802 (3) 0.3132 0.3534 0.4202 0.3760 (3) 0.4161 0.3418 0.3143 0.28544 (14) 0.34856 (12) 0.49919 (13) 0.42825 (13) 0.51567 (15) 0.47907 (5) 0.440772 (16) 0.0418 (5) 0.050* 0.0382 (5) 0.046* 0.0292 (4) 0.035* 0.0307 (4) 0.037* 0.0612 (9) 0.092* 0.092* 0.092* 0.0274 (4) 0.0555 (7) 0.083* 0.083* 0.083* 0.0566 (7) 0.085* 0.085* 0.085* 0.0268 (3) 0.0355 (3) 0.0372 (4) 0.0354 (3) 0.0422 (4) 0.03915 (13) 0.02749 (7) Atomic displacement parameters (Å2) C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16 U11 U22 U33 U12 U13 U23 0.0313 (10) 0.0308 (8) 0.0376 (9) 0.0557 (15) 0.0727 (17) 0.0787 (18) 0.0566 (13) 0.0387 (11) 0.0357 (11) 0.0345 (11) 0.0311 (10) 0.0280 (10) 0.0308 (11) 0.0279 (10) 0.0493 (14) 0.0464 (15) 0.0256 (11) 0.0236 (10) 0.0261 (11) 0.0403 (14) 0.0373 (14) 0.0388 (13) 0.0462 (16) 0.0339 (13) 0.0521 (15) 0.0424 (14) 0.0269 (10) 0.0330 (12) 0.073 (2) 0.0267 (10) 0.0333 (14) 0.0558 (19) 0.0325 (9) 0.0288 (8) 0.0303 (8) 0.0372 (11) 0.0331 (11) 0.0374 (11) 0.0411 (10) 0.0330 (10) 0.0441 (12) 0.0392 (11) 0.0291 (9) 0.0319 (10) 0.0840 (18) 0.0333 (10) 0.088 (2) 0.0764 (19) −0.0010 (8) 0.0005 (10) −0.0042 (11) 0.0075 (12) −0.0006 (13) −0.0155 (13) −0.0114 (14) −0.0073 (9) 0.0053 (11) 0.0104 (10) 0.0025 (8) 0.0043 (9) −0.0102 (14) −0.0044 (8) 0.0012 (11) −0.0036 (13) 0.0141 (9) 0.0113 (7) 0.0141 (8) 0.0204 (12) 0.0146 (12) 0.0323 (14) 0.0308 (11) 0.0162 (9) 0.0221 (11) 0.0130 (10) 0.0076 (9) 0.0096 (9) 0.0225 (12) 0.0181 (9) 0.0255 (15) 0.0319 (15) −0.0017 (8) −0.0007 (10) −0.0031 (10) 0.0064 (10) 0.0055 (10) −0.0064 (10) −0.0094 (12) −0.0070 (8) −0.0068 (11) −0.0003 (10) 0.0001 (7) −0.0071 (9) −0.0487 (19) −0.0043 (8) 0.0115 (12) −0.0019 (15) Acta Cryst. (2008). E64, m1338 sup-5 supporting information N1 O1 O2 O3 O4 S1 Zn1 0.0266 (8) 0.0414 (8) 0.0369 (8) 0.0358 (8) 0.0371 (8) 0.0388 (3) 0.02884 (11) 0.0274 (9) 0.0361 (9) 0.0455 (10) 0.0372 (9) 0.0357 (9) 0.0311 (3) 0.02883 (11) 0.0284 (7) 0.0325 (7) 0.0309 (7) 0.0365 (7) 0.0520 (9) 0.0503 (3) 0.02707 (10) 0.0022 (6) 0.0111 (7) 0.0056 (7) 0.0056 (7) −0.0084 (7) −0.0096 (2) 0.00032 (12) 0.0106 (7) 0.0161 (7) 0.0121 (7) 0.0156 (7) 0.0094 (7) 0.0169 (3) 0.01157 (8) −0.0035 (6) 0.0074 (6) −0.0044 (7) −0.0104 (7) 0.0051 (8) −0.0059 (2) 0.00374 (11) Geometric parameters (Å, º) C1—O1 C1—C2 C1—C10 C2—C11 C2—C3 C3—C4 C3—C8 C4—C5 C4—H4 C5—C6 C5—H5 C6—C7 C6—H6 C7—C8 C7—H7 C8—C9 C9—C10 C9—H9 C10—H10 C11—N1 C11—H11 C12—N1 C12—C13 1.300 (2) 1.410 (3) 1.443 (3) 1.434 (3) 1.465 (2) 1.408 (3) 1.416 (3) 1.381 (3) 0.9300 1.391 (4) 0.9300 1.348 (4) 0.9300 1.422 (3) 0.9300 1.409 (3) 1.361 (3) 0.9300 0.9300 1.296 (2) 0.9300 1.462 (2) 1.521 (3) C12—C14 C12—H12 C13—H13A C13—H13B C13—H13C C14—O2 C14—O3 C15—S1 C15—H15A C15—H15B C15—H15C C16—S1 C16—H16A C16—H16B C16—H16C N1—Zn1 O1—Zn1 O2—Zn1 O3—Zn1i O4—S1 O4—Zn1 Zn1—O3ii 1.531 (3) 0.9800 0.9600 0.9600 0.9600 1.242 (3) 1.259 (3) 1.762 (3) 0.9600 0.9600 0.9600 1.768 (3) 0.9600 0.9600 0.9600 2.0119 (18) 2.0040 (15) 2.1891 (16) 1.9560 (15) 1.5184 (18) 2.0520 (17) 1.9560 (15) O1—C1—C2 O1—C1—C10 C2—C1—C10 C1—C2—C11 C1—C2—C3 C11—C2—C3 C4—C3—C8 C4—C3—C2 C8—C3—C2 C5—C4—C3 C5—C4—H4 C3—C4—H4 C4—C5—C6 C4—C5—H5 124.91 (17) 116.32 (19) 118.77 (17) 121.86 (16) 119.89 (17) 118.24 (18) 116.96 (17) 124.27 (18) 118.77 (19) 122.0 (2) 119.0 119.0 120.1 (2) 120.0 C12—C13—H13B H13A—C13—H13B C12—C13—H13C H13A—C13—H13C H13B—C13—H13C O2—C14—O3 O2—C14—C12 O3—C14—C12 S1—C15—H15A S1—C15—H15B H15A—C15—H15B S1—C15—H15C H15A—C15—H15C H15B—C15—H15C 109.5 109.5 109.5 109.5 109.5 125.85 (18) 119.49 (17) 114.66 (19) 109.5 109.5 109.5 109.5 109.5 109.5 Acta Cryst. (2008). E64, m1338 sup-6 supporting information C6—C5—H5 C7—C6—C5 C7—C6—H6 C5—C6—H6 C6—C7—C8 C6—C7—H7 C8—C7—H7 C9—C8—C3 C9—C8—C7 C3—C8—C7 C10—C9—C8 C10—C9—H9 C8—C9—H9 C9—C10—C1 C9—C10—H10 C1—C10—H10 N1—C11—C2 N1—C11—H11 C2—C11—H11 N1—C12—C13 N1—C12—C14 C13—C12—C14 N1—C12—H12 C13—C12—H12 C14—C12—H12 C12—C13—H13A 120.0 119.9 (2) 120.1 120.1 121.5 (2) 119.2 119.2 119.56 (17) 120.97 (19) 119.5 (2) 122.14 (19) 118.9 118.9 120.8 (2) 119.6 119.6 126.87 (19) 116.6 116.6 111.01 (19) 108.94 (16) 109.48 (17) 109.1 109.1 109.1 109.5 S1—C16—H16A S1—C16—H16B H16A—C16—H16B S1—C16—H16C H16A—C16—H16C H16B—C16—H16C C11—N1—C12 C11—N1—Zn1 C12—N1—Zn1 C1—O1—Zn1 C14—O2—Zn1 C14—O3—Zn1i S1—O4—Zn1 O4—S1—C15 O4—S1—C16 C15—S1—C16 O3ii—Zn1—O1 O3ii—Zn1—N1 O1—Zn1—N1 O3ii—Zn1—O4 O1—Zn1—O4 N1—Zn1—O4 O3ii—Zn1—O2 O1—Zn1—O2 N1—Zn1—O2 O4—Zn1—O2 109.5 109.5 109.5 109.5 109.5 109.5 117.15 (17) 125.29 (13) 116.50 (12) 126.51 (14) 114.09 (12) 126.85 (14) 134.27 (11) 108.15 (11) 106.05 (12) 98.14 (15) 98.54 (7) 138.57 (7) 88.32 (7) 101.12 (7) 96.09 (7) 118.82 (7) 93.89 (7) 166.10 (6) 78.22 (6) 87.52 (7) O1—C1—C2—C11 C10—C1—C2—C11 O1—C1—C2—C3 C10—C1—C2—C3 C1—C2—C3—C4 C11—C2—C3—C4 C1—C2—C3—C8 C11—C2—C3—C8 C8—C3—C4—C5 C2—C3—C4—C5 C3—C4—C5—C6 C4—C5—C6—C7 C5—C6—C7—C8 C4—C3—C8—C9 C2—C3—C8—C9 C4—C3—C8—C7 C2—C3—C8—C7 C6—C7—C8—C9 C6—C7—C8—C3 C3—C8—C9—C10 C7—C8—C9—C10 −0.5 (4) 179.8 (2) 178.2 (2) −1.4 (3) 179.0 (2) −2.2 (4) −1.5 (3) 177.3 (2) −1.7 (4) 177.8 (3) −0.8 (4) 2.2 (4) −1.1 (4) −177.1 (2) 3.4 (3) 2.8 (3) −176.7 (2) 178.4 (2) −1.4 (4) −2.4 (4) 177.7 (2) C13—C12—N1—C11 C14—C12—N1—C11 C13—C12—N1—Zn1 C14—C12—N1—Zn1 C2—C1—O1—Zn1 C10—C1—O1—Zn1 O3—C14—O2—Zn1 C12—C14—O2—Zn1 O2—C14—O3—Zn1i C12—C14—O3—Zn1i Zn1—O4—S1—C15 Zn1—O4—S1—C16 C1—O1—Zn1—O3ii C1—O1—Zn1—N1 C1—O1—Zn1—O4 C1—O1—Zn1—O2 C11—N1—Zn1—O3ii C12—N1—Zn1—O3ii C11—N1—Zn1—O1 C12—N1—Zn1—O1 C11—N1—Zn1—O4 89.0 (2) −150.37 (18) −102.1 (2) 18.5 (2) −20.7 (3) 158.98 (16) −175.97 (16) 4.9 (2) 16.7 (3) −164.18 (13) 23.79 (18) −80.64 (17) 165.79 (17) 26.85 (17) −91.96 (18) 12.5 (4) −122.75 (17) 69.40 (17) −21.72 (17) 170.43 (14) 74.31 (18) Acta Cryst. (2008). E64, m1338 sup-7 supporting information C8—C9—C10—C1 O1—C1—C10—C9 C2—C1—C10—C9 C1—C2—C11—N1 C3—C2—C11—N1 N1—C12—C14—O2 C13—C12—C14—O2 N1—C12—C14—O3 C13—C12—C14—O3 C2—C11—N1—C12 C2—C11—N1—Zn1 −0.6 (4) −177.2 (2) 2.5 (3) 5.6 (4) −173.2 (2) −15.1 (3) 106.5 (2) 165.76 (17) −72.7 (3) 178.6 (2) 10.9 (3) C12—N1—Zn1—O4 C11—N1—Zn1—O2 C12—N1—Zn1—O2 S1—O4—Zn1—O3ii S1—O4—Zn1—O1 S1—O4—Zn1—N1 S1—O4—Zn1—O2 C14—O2—Zn1—O3ii C14—O2—Zn1—O1 C14—O2—Zn1—N1 C14—O2—Zn1—O4 −93.54 (15) 154.80 (18) −13.05 (14) −170.20 (14) 89.82 (14) −1.61 (17) −76.72 (14) −134.50 (15) 19.0 (4) 4.39 (14) 124.51 (15) Symmetry codes: (i) −x+1, y+1/2, −z+1; (ii) −x+1, y−1/2, −z+1. Acta Cryst. (2008). E64, m1338 sup-8
https://openalex.org/W1842388920	https://liberquarterly.eu/article/download/10317/10783	English	null	Microfilm and digitization as choices in preservation	LIBER quarterly	2,003	cc-by	3,540	Microfilm and Digitization as Choices in Preservation by YOLA DE LUSENET The first conference the European Commission on Preservation and Access (ECPA) organized, ‘Choosing to preserve’, took place in Leipzig in 1996. For the keynote lecture we had invited a scholar, Professor Bernhard Fabian. This was a very deliberate choice, as the ECPA subscribed to the view that academic researchers, as users of the resources kept in libraries and archives, have to be involved in discussions about their preservation. The conference had attracted quite a crowd, around 150 people who were of course all there for the opening lecture. We invited Professor Fabian because he was a very good speaker, who could really present a convincing case to an audience, which he did also on that occasion. He devoted a large part of his presentation of 45 minutes to the horrors of microfilm, showing us pictures of illegible film, with bits missing, that was impossible to use. He explained how distressing it was for scholars to be forced to use surrogates that don’t do justice to the originals that they need to study. Recently we at the ECPA heard from a preservation manager in a large library in Europe, that those in charge in his institution were on the point of dismantling the microfilming department and planning to use only digitisation from now on. This somewhat drastic decision was taken because microfilming was considered to be outdated and no longer necessary. In between these two incidents lie seven years in which the scene has radically changed. At the time of the Leipzig conference, we basically had the choice between keeping originals and making microfilm, but now digitisation has entered the stage as the tall, dark and handsome stranger in a play. At the moment the question is whether this attractive hero will indeed carry off the innocent girl and, fickle and restless as he is, abandon her after having squandered her fortune. Or will he prove to be a reliable guy after all and lead her into a solid marriage - and to a happy end of the story? In the more mundane reality of preservation, the choice seems to be between sticking with microfilm as a trusted and proven technology or moving to new technology that seems to offer so many opportunities -and great risks. LIBER QUARTERLY, ISSN 1435-5205 © LIBER 2003. All rights reserved K.G. Saur, Munich, Printed in Germany LIBER QUARTERLY, ISSN 1435-5205 © LIBER 2003. All rights reserved K.G. Saur, Munich, Printed in Germany Microfilm and Digitization as Choices in Preservation Graham Jefcoate has described the new complexity in which the approach of ‘digitising for access, microfilming for preservation’, has started to break down. The problem with the new situation is that as digitisation is essentially not preservation-driven, preservation of originals threatens to become a subsidiary concern. What is digitised will probably be preserved, but what needs to be preserved is selected for digitisation only if there are strong arguments from a perspective of use. We can safely assume that only a fraction of what is available in 112 LIBER QUARTERLY 13: 112-118 YOLA DE LUSENET libraries and archives will be digitised in the foreseeable future. Low-use, specialist materials will not be among the first candidates. And if they happen to be at risk, other measures will still have to be taken for their survival libraries and archives will be digitised in the foreseeable future. Low-use, specialist materials will not be among the first candidates. And if they happen to be at risk, other measures will still have to be taken for their survival Basically no one likes using microfilm. Over the past weeks I discussed the theme of this conference with various people, and every time I heard how they hate microfilm. And, as a librarian once pointed out to me, to make matters worse, users of microfilm are often treated as outcasts and relegated to a bare, unpleasant corner of the library where they cannot disturb the other readers in their struggle with noisy machines. Obviously, most users nowadays would probably prefer or even expect a digital copy. That users have high expectations cannot just be brushed aside. For if users are demanding, they can never be wrong in asking. Advantages for use are a strong argument for favouring digitisation over microfilming. However, what is worrying in the discussion about the advantages of digital images is that use is so often understood as a quantitative concept only. But if we define use in numbers, they may obscure the complexity that lies behind the straightforward lists of figures that can be plotted in a rising graph. Digitisation is a great favourite with politicians and decision makers because by opening up collections more people will be able to use them. So far so good. But there is use and use. Among the visitors of a site there are bound to be many casual users, window-shoppers. Microfilm and Digitization as Choices in Preservation Microfilm and Digitization as Choices in Preservation other satisfactory surrogates are available to users. The distinction is important, because the aim of a digitisation project should determine requirements for quality and maintenance, and requirements for digital preservation masters that can act as substitutes may be different from requirements for user copies. The requirements for digital preservation masters may not be met if the real motivation for digitisation is facilitating use, and it may in fact be an uneconomical decision to create digital preservation masters, to be maintained over time, for originals that are not at risk or that have been microfilmed already. In trying to kill two birds with one stone, one may easily miss one’s target altogether. There are cases where the primary or only concern is to keep the information content of materials, without expectations of extensive use in the near future. This applies not only to the specialist materials mentioned above, but also to unique items, archival materials, or other materials an institution is legally bound to keep for the future, which may even include materials to which access is restricted. When the primary goal is preservation of information for materials that are not frequently used, the advantages of microfilming, which have always been staunchly defended by the preservation community, are still very real. Although I agree wholeheartedly with Graham Jefcoate that because of the widespread introduction of digital born information, solutions to manage digital materials over time must and will be found, they still lack the essential stability that microfilm has. It does make a difference when things are tangible, can be stored safely and left for years or centuries and won’t rot or fade or get corrupted. Microfilm doesn’t require complex equipment to read and it doesn’t become inaccessible of its own accord if it is stored properly. These are undeniable advantages over digital material, which is a nightmare for preservation –not because it cannot be kept accessible, but because keeping it accessible requires a continuing and intensive process of refreshing and converting, with a risk of something going wrong every step of the way. The carriers on which it is stored degrade rapidly, and the machines and the programmes to access the information become outdated within years. Microfilm and Digitization as Choices in Preservation What does it mean if 10,000 people visit a site? How do we interpret user statistics to assess the impact of digitisation? If all we can measure with certainty is frequency of use, there is a risk that increased use becomes the measure of success, irrespective of what this use consists in and who the users are. This favours selection of things that are of some interest to a great many people, over things that are of extreme importance to only a few - scholars, for instance, who are dependent on these materials for their work. A sophisticated concept of use and a thorough understanding of target groups is needed to overcome the pressure of numbers and the risk of promoting trivial use at the expense of serious use. What also confuses the discussion is that digitisation is often presented as a preservation measure because as a surrogating strategy it relieves stress on originals. This is of course true, but in many cases materials are selected for digitisation that are not directly at risk, or that have been microfilmed previously so that preservation is not an issue. The decision to offer a digital surrogate here is in fact not primarily motivated by preservation concerns, but by the desire to channel use. Arguments to digitise are for instance: providing access to treasures from a collection; distant access; a high frequency of use; the possibility to search, annotate or copy materials for research and educational purposes, or to contextualize materials and integrate them into a larger, distributed context. All of these are valid arguments, but none are preservation arguments in themselves. I would regard digitisation of valuable manuscripts as a preservation measure only if these materials are deteriorating because of frequent handling and no 113 Microfilm and Digitization as Choices in Preservation There is no process of slow fading or getting frayed at the edges, no, one day you find it is simply all gone, or the information may still be there, locked in its code, and you can’t get at it – like a hungry castaway on a desert island with a tin of beans but no tin-opener. There is another strong argument in favour of microfilming from a preservation perspective that is not heard much in library circles: authenticity. A microfilm image as a direct image of an original doesn’t allow the information to be changed or otherwise tampered with as is possible with digital materials. Especially in the archival world the discussion on digital preservation centers around the issue of continued access to authentic digital records, because it is felt to be so problematic to keep the information as it was once created. 114 YOLA DE LUSENET YOLA DE LUSENET In short, although digital masters in practice will be created and will be preserved, from a preservation point of view digitising is still a high-risk approach. After all, you solve one preservation problem, saving the information from a deteriorating original, by replacing it with another, more complex one. In short, although digital masters in practice will be created and will be preserved, from a preservation point of view digitising is still a high-risk approach. After all, you solve one preservation problem, saving the information from a deteriorating original, by replacing it with another, more complex one. The research in the so-called hybrid approach was based on the assumption that the strengths of both technologies could be combined. The study at Cornell (Kenney, 1997) evaluated the use of high-resolution bitonal imaging to produce computer output microfilm (COM) that meets preservation standards; the complementary study at Yale (Conway, 1996) studied the production of digital images from microfilm. Both projects relied on high-quality microfilm as the preservation master and produced high-resolution digital images (600 dpi, bitonal). The conclusion of these studies was that in terms of quality and costs it is feasible to produce preservation microfilm from digital images as well as the other way around. The 1999 report by Chapman, Conway and Kenney presents a decision tree for determining whether to scan first or film first. Earlier this year RLG published guidelines on how microfilming can be optimised with a view to subsequent digitisation (Dale, 2003). Microfilm and Digitization as Choices in Preservation I will leave the technical issues these reports discuss for other speakers who will have more to say about technology and quality control. However feasible and safe in theory, the obvious problem with the hybrid approach is that it adds to the cost, not only because one has to do two things instead of one, but also because one has to deal with extra maintenance costs. This issue is not dealt with in the reports, as they focus on production, quality, workflow, and metadata. At the moment there is not enough practical experience in long-term maintenance of digital materials to be sure about anything and certainly not about cost. By way of illustration, I would like to refer to a discussion of relative costs of storage from an article by Steve Chapman. He compares the cost of storing the same amount of information in analog format in the central Harvard Depository and in digital format in the OCLC Digital Archive. I cannot do justice to the discussion in the scope of this paper, and I would highly recommend everyone to read the complete article in order to come to a proper understanding of what the figures in this article reflect exactly. Chapman has made every effort to come to a well-defined comparison, so I think it is justified to mention some of his findings here, but to understand exactly what is covered by these costs and how they have been calculated, the full article should be studied. Chapman presents figures for the relative costs of storing 729,000 pages of text, or 2,202 volumes, in book format, as microfilm - in the vault or in a standard environment - in the Harvard Depository, and as 1-bit page images and ASCII in the OCLC Digital Archive. Harvard charges per square foot, at a standard rate and at a higher rate if the material is kept in the film vault. OCLC charges per gigabyte, at three rates, depending on the total 115 Microfilm and Digitization as Choices in Preservation amount of data deposited per account: the more is deposited by the account holder, the lower the rate per gigabyte. amount of data deposited per account: the more is deposited by the account holder, the lower the rate per gigabyte. Storage in ASCII is the cheapest option, because ASCII is an extremely compact format. Microfilm and Digitization as Choices in Preservation It is, however, a format that would only be used if the appearance of the text, the page layout, was no issue and if there were no illustrations in a text. Storage on microfilm comes second (with the costs doubling when the films are kept in the vault). Third comes storage in book format, then storage as 1-bit page images. Even at the lowest rate (for an account of over 1,000 Gb) storage of digital images is 1,5 times as expensive as storing books, and 2,5 times as storing microfilm in the vault. It is assumed that lossless compression was used for the digital files; uncompressed, the files would become 22 times larger and storing them would become 54 times as expensive as storing the same number of pages on microfilm in the vault. Chapman also compares storage costs for print and various digital formats. As costs are directly related to the number of gigabytes required for storage, file size is the decisive factor, and file size follows from the quality chosen for reformatting: bitonal, or 1-bit, gray or 8-bit, and colour or 24-bit. The effect on costs of storing masters at higher quality is overwhelmingly clear from his calculations: storing the same pages in 8-bit images instead of 1-bits is about 42 as expensive, as 24-bit images storage costs are about 125 times higher compared to 8-bits. It should be borne in mind that in many cases the costs of storing film or digital files, and in the hybrid approach of both, will be additional to the costs of storing print material, as many institutions keep the originals after filming or scanning. And if, in order to limit risks, several copies of the film or the digital files are kept, this adds to the costs as well. Storage is only one factor, and there are others to consider as well, but this comparison of storage costs alone makes clear that producing both film and digital, and maintaining both microfilm and digital preservation masters (which would presumably be high- quality files) increases costs very considerably. This almost inevitably leads to the conclusion that the hybrid approach is a viable option only for the materials that are so central and important that money is no issue. Microfilm and Digitization as Choices in Preservation However, research into the hybrid approach has been valuable for demonstrating how film and digital can be combined, if not in a simultaneous approach than perhaps in a phased approach. If preservation is the primary concern, one can choose to film so that the information is kept. The films, or some of the films, can always be digitised later should there be a need for including them in a digital collection. Or one may digitise from the film on demand, if a user at some point in time wants a digital copy. Conversely, if materials are digitised first, film can still be produced at a later stage, 116 YOLA DE LUSENET YOLA DE LUSENET should the need arise, for instance because it is not considered cost-effective to keep digital images which by then may only be consulted infrequently. should the need arise, for instance because it is not considered cost-effective to keep digital images which by then may only be consulted infrequently. If the originals are stable enough to be kept for the long term, preservation is no issue at all, and quality can be defined on requirements for use. Lower resolutions or compressed files may be adequate and reduce costs. It is often said that requirements will go up in the future, with high-speed networks and new types of monitors, but perhaps it is cheaper to rescan the originals later than invest in uncertain use in the future now. After all, information may be superseded or use may shift. What this comes down to is that the choice of technology and quality requirements differ with the goals one tries to achieve. Preservation of information should be distinguished from preservation of the use of that information, and the technologies can be seen as complementary. User requirements and preservation requirements do not always have to be met with the same technology. And an established technology like microfilm still has a role in a policy for collection management that balances different requirements instead of going for a ‘one size fits all’ solution. When I read Nicholson Baker’s Double Fold (Baker, 2001) I was struck by the parallel between the enthusiasm around the large-scale introduction of microfilm several decades ago and the present spread of digitisation. Microfilm and Digitization as Choices in Preservation With such high hopes placed in a new technology, don’t we run a risk of ignoring what is good about the things that have served us faithfully for years? I like to believe that the old is not simply superseded by the new but that the function of established technology shifts with the arrival of newcomers. Yes, I know, we have lost Morse code and the trusted telegram and punch cards and wax cylinders. And yes, perhaps those in the business of preservation tend to be natural conservatives wishing to keep things as they are – and rightly so! For it does pay to hold on to things that work. Those who bet all their money on the Internet bubble now sit at home and are no doubt happy that there are still books to read! REFERENCES 1. Baker, N. Double Fold: Libraries and the Assault on Paper. Random House, 2001. ISBN: 0375504443. 1. Baker, N. Double Fold: Libraries and the Assault on Paper. Random House, 2001. ISBN: 0375504443. 2. Chapman, S., P. Conway & AR. Kenney. Digital Imaging and Preservation Microfilm: The Future of a Hybrid Approach for the Preservation of Brittle Books. Washington, DC: Council on Library and Information Resources, 1999. http://www.clir.org/pubs/archives/hybridintro.html 2. Chapman, S., P. Conway & AR. Kenney. Digital Imaging and Preservation Microfilm: The Future of a Hybrid Approach for the Preservation of Brittle Books. Washington, DC: Council on Library and Information Resources, 1999. http://www.clir.org/pubs/archives/hybridintro.html 3. Chapman, S.: “Counting the Costs of Digital Preservation: Is Repository Storage Affordable?”. Journal of Digital Information, May 2003. http://jodi.ecs.soton.ac.uk 4. Conway, P.: Conversion of Microfilm to Digital Imagery: A Demonstration Project. Performance Report on the Production Conversion Phase of Project Open Book, Yale University Library, August 1996. See also, Conway, P.: “Yale University Library's Project Open Book: Preliminary Research Findings,’ D-Lib Magazine, February 1996. http://www.dlib.org/dlib/february96/yale/02conway.html 5. Dale, Robin L. RLG Guidelines for Microfilming to Support Digitization. RLG, 2003. http://www.rlg.org/preserv/microsuppl.pdf 5. Dale, Robin L. RLG Guidelines for Microfilming to 2003. http://www.rlg.org/preserv/microsuppl.pdf p g g p pp p 6. Kenney, A.R.: “The Cornell Digital to Microfilm Conversion Project: Final Report 6. Kenney, A.R.: “The Cornell Digital t 6. Kenney, A.R.: “The Cornell Digital to Microfilm Conversion Project: Final Report 7. to NEH”. RLG DigiNews 1(1997)2. htt // l / /di i /di i 2 ht l# 7. to NEH”. RLG DigiNews 1(1997)2. http://www.rlg.org/preserv/diginews/diginews2.html#com 7. to NEH”. RLG DigiNews 1(1997)2. http://www.rlg.org/preserv/diginews/diginews2.html#com Acknowledgements I would like to thank Steve Chapman for allowing me to use his article and providing me with many helpful comments in preparing this paper. I would also like to thank Dennis Schouten, Henriëtte Reerink and Robert Gillesse for sharing their views on some of the issues I discussed. 117 Microfilm and Digitization as Choices in Preservation WEB SITES REFERRED TO IN THE TEXT The European Commission on Preservation and Access (ECPA). http://www.knaw.nl/ecpa/ 118
https://openalex.org/W2071095734	https://www.scielo.br/j/sa/a/FPZDpvGpm6S55SMgQXHdR6k/?lang=pt&format=pdf	Portuguese	null	Determinação de metodologia para oisolamento de protoplastos de tangerina Cleópatra (Citrus reshni Hort.)	Scientia Agrícola	1,995	cc-by	3,066	DETERMINAÇÃO DE METODOLOGIA PARA O ISOLAMENTO DE PROTOPLASTOS DE TANGERINA CLEÓPATRA (Citrus reshni Hort.) R.P. de OLIVEIRA; B.M.J. MENDES; A. TULMANN NETO Centro de Energia Nuclear na Agricultura/USP - C.P. 96- CEP: 13400-900- Piracicaba, SP. ntro de Energia Nuclear na Agricultura/USP - C.P. 96- CEP: 13400-900- Piracicaba, SP. RESUMO: A hibridação somática via fusão de protoplastos vem sendo utilizada no melhoramento de porta-enxertos de citros em diversos países. Nos Estados Unidos, vários estudos demonstram a eficiência de procedimentos no isolamento e cultivo de protoplastos dessa frutífera. O presente trabalho foi realizado com o objetivo de avaliar o efeito do meio de cultivo de calos embriogênicos do porta-enxerto tangerina Cleópatra (Citrus reshni Hort.) sobre o isolamento de protoplastos, bem como sugerir alterações de procedimento. Os resultados mostram a possibilidade do isolamento de 1.4 x 106 a 4.7 x 106 protoplastos por grama de calos da espécie estudada. Verificou-se que, o subcultivo dos calos de tangerina Cleópatra em meio de cultura, sem reguladores de crescimento suplementado com apenas 4% de sacarose e o emprego de um pré-tratamento enzimático com macerozyme a 1% por l hora, sob condições de escuro a 120 rpm, proporcionou maior eficiência de isolamento de protoplastos (4.7 x 106 protoplastos/g de calo). Descritores: tangerina Cleópatra, protoplastos, isolamento Descritores: tangerina Cleópatra, protoplastos, isolamento METHODOLOGY CHOICE FOR PROTOPLAST ISOLATION IN CLEOPATRA MANDARIN (Citrus reshni Hort.) ABSTRACT: Somatic hybridization has been used for citrus rootstock breeding in many countries. In USA, many reports had proved the efficiency of procedures for the isolation and culture of citrus protoplasts. This research was conducted to evaluate the efficiency of procedures of protoplast isolation using embryogenic callus of the the possibility of a protoplast yield of 1.4 to 4.7 x 106 pps/g.f.w. for Cleópatra tangerine rootstock callus. Protoplast yield can be raised to 4.7 x 106 pps/g.f.w. if the embryogenic callus are grown in a medium supplemented only with 4% sucrose and pre-treated with 1% w/v macerozyme for 1 hour, at 120 rpm, in dark, is applied before protoplast isolation. ey Words: Cleópatra mandarin, protoplast, isolation MATERIAIS E MÉTODOS Os experimentos foram realiza- dos na Seção de Radiogenética do Centro de Energia Nuclear na Agricultura (CENA/USP), em Piracicaba-SP, utilizando a espécie tangerina Cleópatra (Citrus reshni Hort.). Efeito do meio de cultivo dos calos na eficiência de isolamento: Os calos embriogênicos de tangerina Cleópatra foram subcultivados por no mínimo 3 cultivos de 3 semanas, nos seguintes meios de cultura: MT (MURASHIGE & TUCKER, 1969) suplementado com 5% de sacarose e 500 mg/1 de extrato de malte (MT), MT suplementado com 5% de sacarose, 500 mg/1 de extrato de malte e 10 mg/1 de benzilaminopurina (MT+BAP) e MT suplementado com 4% de sacarose (MT-S). Em seguida, os protoplastos, isolados a partir de calos subcultivados no meio de cultura MT-S e submetidos a um pré-tratamento enzimático com macerozyme, foram purificados conforme recomendam GROSSER & GMITTER Jr. (1990). A viabilidade desses protoplastos foi estudada através do emprego do corante azul de metileno na concentração final de 0.01% (HOOLEY & McCARTHY, 1980). Foram analisadas a viabilidade de 200 protoplastos após cada isolamento, sendo feitas contagens do numero de protoplastos viáveis e mortos em microscópio invertido. A solução enzimática empregada foi composta por 1.0% de cellulase "Onozuka" RS (Yakult Honsha Co. Ltd., Tokyo, Japan); 1.0% de macerozyme R-10 (Yakult Honsha Co. Ltd., Tokyo, Japan); 0.2% de pectolyase Y-23 (Seishin Pharmaceutical Co. Ltd., Tokyo, Japan); 0.7 M manitol; 12.0 mM de cloreto de cálcio; 1.4 mM de NaH2PO4; e 6.0 mM de MES. O pH dessa solução foi ajustado para 5.6 e a filtração foi realizada em membrana de 0.2 mm (GROSSER & CHANDLER, 1987). INTRODUÇÃO Os resultados foram comparados com um tratamento controle, onde se utilizou o método de digestão da parede celular descrito GROSSER & GMITTER Jr. (1990). As avaliações dos resultados foram realizadas da mesma forma que no experimento anterior, sendo utilizadas 2 repetições por tratamento. Desta forma, o presente traba- lho teve por objetivo avaliar o efeito do meio de cultivo de calos embriogênicos do porta-enxerto tangerina Cleópatra (Citrus reshni Hort.) sobre o isolamento de protoplastos ao utilizar-se o protocolo descrito por GROSSER & GMITTER Jr. (1990), bem como sugerir alterações em seu conteúdo visando aumentar sua eficiência. INTRODUÇÃO et al., 1985 e GROSSER & GMITTER Jr., 1990). Esses híbridos somáticos apresentam a vantagem de serem alotetraplóides, mantendo os genes parentais por não apresentarem segregação meiótica. Assim, as características favoráveis e desfavoráveis controladas por genes dominantes ou codominantes presentes em um dos pais podem se expressar, ao contrário dos genes deletérios recessivos, freqüentemente presentes nas variedades cítricas, que não se expressam (GROSSER & GMITTER Jr., 1990). O Brasil é o maior produtor e exportador de suco cítrico concentrado (FAO, 1992). A diversificação do material genético dos pomares com variedades de enxertos e porta- enxerto tolerantes a pragas, doenças e estresses de natureza abiótica consiste na medida mais racional para sustentação dessa posição no mercado internacional. Nos últimos anos, a hibridação somática via fusão de protoplastos passou a ser empregada no melhoramento de variedades de citros em diversos países, tendo sido obtidos inúmeros híbridos somáticos que, atualmente, estão sendo avaliados a nível de campo (OHGAWARA A hibridação somática de citros vem sendo utilizada como rotina em laboratórios dos Estados Unidos, Israel e Japão, porém trabalhos relacionados ao isolamento, purificação e cultivo de protoplastos são escassos no Brasil. Efeito do pré-tratamento dos calos com macerozyme: Utilizaram-se calos de tangerina Cleópatra com 3 subcultivos de 3 semanas em meio de cultura MT-S. Os tratamentos consistiram em pré-tratamento dos calos com solução enzimática contendo macerozyme R-10 a 1%, por l e 2 horas (BUTTON & BOTHA, 1975), em condições de escuro a 25 ± 2°C, em agitador orbital a 120 rpm. Nessa solução enzimática foram utilizados os estabilizadores osmóticos e de membrana empregados na solução enzimática descrita por GROSSER & CHANDLER (1987). Os resultados foram comparados com um tratamento controle, onde se utilizou o método de digestão da parede celular descrito GROSSER & GMITTER Jr. (1990). As avaliações dos resultados foram realizadas da mesma forma que no experimento anterior, sendo utilizadas 2 repetições por tratamento. Efeito do pré-tratamento dos calos com macerozyme: Utilizaram-se calos de tangerina Cleópatra com 3 subcultivos de 3 semanas em meio de cultura MT-S. Os tratamentos consistiram em pré-tratamento dos calos com solução enzimática contendo macerozyme R-10 a 1%, por l e 2 horas (BUTTON & BOTHA, 1975), em condições de escuro a 25 ± 2°C, em agitador orbital a 120 rpm. Nessa solução enzimática foram utilizados os estabilizadores osmóticos e de membrana empregados na solução enzimática descrita por GROSSER & CHANDLER (1987). RESULTADOS E DISCUSSÃO A análise dos trabalhos de isolamento de protoplastos de citros existentes na literatura permite classificar as soluções enzimáticas em dois grupos com relação ao tempo de digestão enzimática: soluções fortes, em que o isolamento de protoplastos a partir de calo ocorre em 4 horas (GOLDMAN, 1988) e soluções fracas, em que o isolamento ocorre em aproximadamente 16 horas (VARDI et al., 1982; SPIEGEL-ROY & VARDI, 1984). A análise dos resultados do experimento realizado permite incluir, didaticamente, a solução enzimática de GROSSER & CHANDLER (1987) no grupo das soluções fortes. notou-se, para todos os meios de cultivo estudados, que o maior número de protoplastos intactos isolados ocorreu após 4 horas de tratamento enzimático dos calos subcultivados (Figura 1). A análise dos trabalhos de isolamento de protoplastos de citros existentes na literatura permite classificar as soluções enzimáticas em dois grupos com relação ao tempo de digestão enzimática: soluções fortes, em que o isolamento de protoplastos a partir de calo ocorre em 4 horas (GOLDMAN, 1988) e soluções fracas, em que o isolamento ocorre em aproximadamente 16 horas (VARDI et al., 1982; SPIEGEL-ROY & VARDI, 1984). A análise dos resultados do experimento realizado permite incluir, didaticamente, a solução enzimática de GROSSER & CHANDLER (1987) no grupo das soluções fortes. Durante as contagens de protoplastos deste experimento, observou-se a presença de microcalos compactos que só liberavam protoplastos em suas extremidades, locais onde ocorria contato efetivo com a solução enzimática. Verificou-se, também, que após 10 horas de tratamento enzimático restavam, ainda, alguns aglomerados de células que não apresentavam a parede celular digerida pela solução enzimática. Por essa razão, foi realizado um segundo experimento com o objetivo de avaliar o efeito de um pré-tratamento enzimático na separação das células dos microcalos, visando aumentar a eficiência no isolamento de protoplastos. Para o pré-tratamento dos calos foi utilizada solução de macerozyme a l % por l e 2 horas, que promoveu uma pré-individualização das células dos calos. De acordo com BUTTON & BOTHA (1975), o uso de macerozyme, mesmo em concentrações mais elevadas de 2 a 3%, aparentemente não afeta a viabilidade das células, que permanecem intactas após o tratamento. Pela Figura l verifica-se, ainda, que os calos subcultivados em meio de cultura com menor teor de sacarose (4%) apresentam maior eficiência na liberação de protoplastos quando comparados aos calos cultivados em meio contendo reguladores de crescimento e/ou 5% de sacarose. RESULTADOS E DISCUSSÃO Na literatura existem vários protocolos de isolamento de protoplastos de citros como os de SPIEGEL-ROY & VARDI (1984); KOBAYASHI et al. (1985); GOLDMAN (1988) e GROSSER & GMITTER Jr. (1990). O protocolo de GROSSER & GMITTER Jr. (1990), que utiliza a solução enzimática de GROSSER & CHANDLER (1987), foi escolhido para a avaliação do experimento de isolamento de protoplastos obtidos a partir de calo da variedade tangerina Cleópatra por ser o mais recente e por se aplicar a diversas espécies. Para o isolamento de protoplas- tos macerou-se com um bastão de vidro l g de calo de cada tratamento em 3 ml de meio BH3 (GROSSER & GMITTER Jr., 1990) 0.7 M, em placa de Petri (45x20 mm); adicionando-se em seguida, l ml da solução enzimática. As placas de Petri foram seladas com parafilme e incubadas a 25 ± 2°C, sob condições de escuro a 50 rpm (GROSSER & GMITTER Jr., 1990). A avaliação do experimento foi feita em Câmara de Newbauer através da contagem do número de protoplastos intactos isolados, a intervalos de l hora, sendo utilizadas 2 repetições por tratamento. Inicialmente, foram estudados os efeitos da composição do meio de cultivo utilizado na multiplicação de calos e do tempo de digestão enzimática sobre a eficiência de isolamento de protoplastos. No presente estudo, (1977) e SIMMONDS et al. (1979), os quais verificaram aumento na eficiência de isolamento de protoplastos com a redução na concentração de sacarose dos subcultivos anteriores ao processo de isolamento. WALLIN et al. (1977), atribuíram o aumento na eficiência de isolamento de protoplastos à mudança na composição química da parede celular ocasionada pelo menor teor de sacarose. Este procedimento exige menor quantidade de enzima para o isolamento de protoplastos, reduzindo possíveis efeitos de toxicidade. Neste experimento observou-se aumento pronunciado no crescimento dos calos no meio MT-S. Esses calos eram mais friáveis e, provavelmente, apresentavam maior número de células na fase de crescimento logarítmico, visto que nessa fase as células dos calos são mais uniformes e apresentam parede celular mais fina, possibilitando maior eficiência no isolamento de protoplastos (POWER & CHAPMAN, 1985). notou-se, para todos os meios de cultivo estudados, que o maior número de protoplastos intactos isolados ocorreu após 4 horas de tratamento enzimático dos calos subcultivados (Figura 1). RESULTADOS E DISCUSSÃO Segundo SHAHIN (1985), embora generalizações possam ser feitas, as condições necessárias ao isolamento e cultivo de protoplastos podem variar consideravelmente de espécie para espécie e de genótipo para genótipo. O mesmo fato foi observado por VARDI et al. (1982), que verificaram grande variabilidade nos requisitos metodológicos exigidos por diferentes variedades e espécies, principalmente no que se refere às enzimas de maceração e aos açúcares utilizados como estabilizadores osmóticos. material biológico disponível. Segundo SHAHIN (1985), embora generalizações possam ser feitas, as condições necessárias ao isolamento e cultivo de protoplastos podem variar consideravelmente de espécie para espécie e de genótipo para genótipo. O mesmo fato foi observado por VARDI et al. (1982), que verificaram grande variabilidade nos requisitos metodológicos exigidos por diferentes variedades e espécies, principalmente no que se refere às enzimas de maceração e aos açúcares utilizados como estabilizadores osmóticos. CONCLUSÕES Os resultados relativos à eficiência de isolamento de protoplastos de calos de tangerina Cleópatra foram superiores aos obtidos por GOLDMAN (1988) e CRISTOFANI (1991), as quais ao trabalharem com a variedade laranja Pera isolaram em média 1.37 x 106 e 1.43 X 106 proto- plastos/g de peso fresco de calo, respectivamente. O método descrito por GROSSER & GMITTER Jr. (1990) permite o isolamento de protoplastos de tangerina Cleópatra em quantidades satisfatórias para posteriores trabalhos de hibridação somática via fusão de protoplastos. No entanto, alterações na metodologia, relacionadas a redução do nível de sacarose e ausência de reguladores de crescimento no meio de cultivo dos calos e a adoção de um pré- tratamento enzimático dos mesmos com macerozyme, permitem aumento da eficiência de isolamento de protoplastos viáveis. RESULTADOS E DISCUSSÃO Por essa razão, o número médio de protoplastos isolados por grama de peso fresco de calos de tangerina Cleópatra subcultivados nos meios de cultura MT-S, MT e MT + BAP foi de 2.2 x 106, 1.7 x 106 e 1.4 x 106, respectivamente. Na Figura 2, pode-se verificar o efeito benéfico do pré-tratamento enzimático, com solução contendo macerozyme a 1%, sobre a eficiência no isolamento de protoplastos. Esse tratamento promoveu a duplicação no número de protoplastos intactos isolados por grama de peso fresco de calos de tangerina Cleópatra (4.7 x 106), demonstrando que, muitas vezes, devem ser feitas alterações em protocolos consagrados na literatura visando ajustá-los às condições do laboratório e do Os resultados obtidos no presente estudo estão de acordo com as citações de SHEPARD & TOTTEN (1977); WALLIN et al. material biológico disponível. Segundo SHA (1985), embora generalizações possam ser feit condições necessárias ao isolamento e culti protoplastos podem variar consideravelmen espécie para espécie e de genótipo para gen O mesmo fato foi observado por VARDI (1982), que verificaram grande variabilidad requisitos metodológicos exigidos por difer variedades e espécies, principalmente no q refere às enzimas de maceração e aos açú utilizados como estabilizadores osmóticos. Os resultados relativo eficiência de isolamento de protoplastos de ca tangerina Cleópatra foram superiores aos ob por GOLDMAN (1988) e CRISTOFANI (199 quais ao trabalharem com a variedade laranja isolaram em média 1.37 x 106 e 1.43 X 106 p plastos/g de peso fresco de calo, respectivam Embora o tratamento com m rozyme a l % por 2 horas tenha proporcionado subcultivados no meio de cultura MT-S sendo realizado o pré-tratamento enzimático com macerozyme 1% por l hora nas condições propostas variou de 81 a 89%. Esses resultados indicam a eficiência do método e se aproximam dos obtidos por GOLDMAN (1988) e CRISTOFANI (1991) que trabalhando com protoplastos isolados da variedade laranja Pera obtiveram de 79.5 a 89.5% e de 80.7 a 84.0% de protoplastos viáveis, respectivamente. subcultivados no meio de cultura MT-S sendo realizado o pré-tratamento enzimático com macerozyme 1% por l hora nas condições propostas variou de 81 a 89%. Esses resultados indicam a eficiência do método e se aproximam dos obtidos por GOLDMAN (1988) e CRISTOFANI (1991) que trabalhando com protoplastos isolados da variedade laranja Pera obtiveram de 79.5 a 89.5% e de 80.7 a 84.0% de protoplastos viáveis, respectivamente. material biológico disponível. REFERÊNCIAS BIBLIOGRÁFICAS BUTTON, J.; BOTHA, C.E.J. Enzymic maceration of Citrus callus and the regeneration of plants from single cells. Journal of Experimental Botany, Oxon, v.26, n.94, p.723-729, 1975. CRISTOFANI, M. Adaptação de metodologias de cultura de tecidos visando o melhoramento através de indução de mutações em Citrus sinensis (L) Osbeck cv. Pera. Piracicaba, 1991. 185p. Dissertação (Mestrado) - Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo. Embora o tratamento com mace- rozyme a l % por 2 horas tenha proporcionado uma eficiência de isolamento de protoplastos semelhante ao tratamento pré-enzimático por l hora, este último deve ser recomendado devido a presença de menor quantidade de células danificadas, o que facilita a etapa de purificação. FAO QUARTELY BULLETIN OF STATISTICS. Rome, 1992. v.5, 117p. GOLDMAN, M.H.S. Cultura de tecidos nucelares, isolamento e radiossensitividade de protoplastos de Citrus sinensis (L) Osbeck cv. Pera. Piracicaba, 1988. 127p. Dissertação (Mestrado) - Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo. Nos experimentos realizados observou-se que, de uma forma geral, calos altamente friáveis, esbranquiçados (sem oxidação) e de rápido crescimento são os que promovem maior eficiência de isolamento de protoplastos. GROSSER, J.W.; CHANDLER, J.L. Aseptic isolation of leaf protoplasts from Citrus, Poncirus, Poncirus X Citrus hybrids and Severinia for use in somatic hybridization experiments. Scientia Horticulturae, Amsterdam, v.31, n.253-257, 1987. A viabilidade dos protoplastos isolados a partir de calos de tangerina Cleópatra SHEPARD, J.F.; TOTTEN, R.E. Mesophyll cell protoplasts of potato. Plant Pysiology, Rockville, v.60, p.313-316, 1977. GROSSER, J.W.; GMITTER JÚNIOR, F.G. Protoplast fusion and citrus improvement. Plant Breeding Reviews, New York, v.8, p.339-374, 1990. SIMMONDS, J.A.; SIMMONDS, D.A.; CUMMING, B.G. Isolation and cultivation of protoplasts from morphogenetic callus cultures of Lilium. Canadian Journal of Botany, Ottawa, v.57, p.512-519, 1979. HOOLEY, R.; McCARTHY, D. Extracts from virus infected hypersensitive tobacco leaves are detrimental to protoplast survival. Physiological and Molecular Plant Pathology, London, v.16, p.25-38, 1980. KOBAYASHI, S.; IKEDA, I.; UCHIMIYA, H. Conditions for high frequency embryogenesis from orange (Citrus sinensis Osb.) rotoplasts. Plant Cell, Tissue and Organ Culture, Dordrecht, v.4, p.149- 159, 1985. SPIEGEL-ROY, P.; VARDI, A. Citrus. In: AMMIRATO, P.V.; EVANS, D.A.; SHARP, W.R.; YAMADA, Y., ed. Handbook of plant cell culture; crop species. New York, MacMillan, 1984. v.3, p.355-72. MURASHIGE, T.; TUCKER, D.P.H. Growth factor requirements of citrus tissue culture. INTERNATIONAL CITRUS SYMPOSIUM, 1., Riverside, 1969. Proceedings... v.3, p. 1155-1161. VARDI, A.; SPIEGEL-ROY, P.; GALUN, E. Entregue para publicação em 30.05.94 Aceito para publicação em 03.08.94 REFERÊNCIAS BIBLIOGRÁFICAS Plant regeneration from Citrus protoplasts: variability in methodological requirements among cultivars and species. Theoretical and Applied Genetics, Berlin, v.62, p.171-176, 1982. OHGAWARA, T.; KOBAYASHI, S.; OHGAWARA, E.; UCHIMIYA, H.; ISHII, S. Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata. Theoretical and Applied Genetics, Berlin, v.71, p.1-4, 1985. WALLIN, A.; GLIMELIUS, K.; ERIKSON, T. Pretreatment of cell suspension as a method to increase the protoplast yield of Aplopappus gracilus. Physiologia Plantarum, Copenhagen, v.40, p .307- 311, 1977. POWER, J.B.; CHAPMAN, J.V. Isolation, culture and genetic manipulation of plant protoplasts. In: DIXON, R.A.,ed. Plant cell culture. Oxford, 1985. p.37-66. SHAHIN, E.A. Totipotency of tomato protoplasts. Theoretical and Applied Genetics, Berlin, v.69, p.235-240, 1985. Entregue para publicação em 30.05.94 Aceito para publicação em 03.08.94
https://openalex.org/W4254673647	https://bmgn-lchr.nl/article/download/URN%3ANBN%3ANL%3AUI%3A10-1-109767/4	Dutch	null	Redactioneel	Bijdragen en mededelingen betreffende de geschiedenis der Nederlanden	2,012	cc-by	873	bmgn - Low Countries Historical Review \| Volume 127-1 (2012) \| pp. 3-4 bmgn - Low Countries Historical Review \| Volume 127-1 (2012) \| pp. 3-4 Redactioneel ‘The minimal ambition of any history of masculinity is to demonstrate that masculinity has a history’. Met die zin begint gastredacteur Stefan Dudink zijn inleidende artikel in dit themanummer Low Countries Histories of Masculinity van bmgn - Low Countries Historical Review. Dudink en zijn co-auteurs gaan echter veel verder dan deze minimale doelstelling. Historici met een andere specialisatie zouden kunnen denken dat ‘mannelijkheid’ een tamelijk statisch concept is, dat vooral besproken wordt binnen genderstudies. Dudink en zijn collega’s laten echter zien hoe het concept mannelijkheid zich manifesteerde op een groot aantal terreinen. Matthijs Lok en Natalie Scholz beschrijven hoe mannelijkheid op verschillende manieren werd ingezet tijdens de Restauratie na de ondergang van Napoleon. Dudink werkt dit thema verder uit door erop te wijzen hoe de beeldvorming rond koning Willem I, die werd afgeschilderd volgens de conventies van geïdealiseerde mannelijkheid, het nieuwe regime legitimeerde na een lange crisistijd. En ook al maakte de mannelijkheid van de Belgische opstandelingen weinig indruk op de Nederlandse dichter Immerzeel, Josephine Hoegaerts laat zien dat mannelijkheid de machtsrelaties bepaalde binnen de Belgische legermacht tijdens de negentiende eeuw. Mannelijkheid werd niet alleen geassocieerd met de hang naar macht, maar ook met deugd, zoals Gemma Blok laat zien in haar bijdrage over de beweging rond drankbestrijding in de negentiende eeuw. En ondanks onze veronderstelling dat religie in de moderne tijd aan het feminiseren is, toont Tine Van Osselaer in haar studie over Belgische mannen die verschijningen zagen ons dat mannelijkheid op nieuwe manieren naar voren kwam in de katholieke kerk. De redactie van bmgn dankt Dudink als gastredacteur, en hem en zijn co-auteurs voor deze stimulerende bijdragen. Naast de inhoud zijn voor lezers en auteurs van een wetenschappelijk tijdschrift zoals bmgn - Low Countries Historical Review de status, de vindbaarheid en de toegang van het tijdschrift van groot belang. Het tijdschrift heeft een int1 rating van de European Science Foundation (voorheen bekend als A-status) en is opgenomen in Thomson Reuter’s Arts and Humanities Citation Index. Artikelen worden bibliografisch verwerkt in ebsco Publishing’s research databases, waaronder Historical Abstracts, en in Scopus (Elsevier). Vanaf deze jaargang verschijnt de bmgn - Low Countries Historical Review als tweetalig tijdschrift (Nederlands en Engels) in Open Access (oa). Redactioneel Doel van oa is het gratis beschikbaar stellen © 2012 Royal Netherlands Historical Society \| knhg Creative Commons Attribution 3.0 Unported License urn:nbn:nl:ui:10-1-109767 \| www.bmgn-lchr.nl \| e-issn 2211-2898 \| print issn 0615-0505 van wetenschappelijke publicaties via internet. Het maakt de verspreiding van onderzoeksresultaten vele malen groter en de toegang gemakkelijker. Met de overgang naar oa sluiten redactie en uitgever aan bij het beleid van nwo en knaw. Financieel kan het tijdschrift gebruik maken van een driejarige subsidie uit het Stimuleringsfonds voor Open Access van nwo, in de uitvoering werken we samen met Igitur publishing (Universiteitsbibliotheek Utrecht) waardoor de digitale toegang ook in de toekomst gegarandeerd is. Naast de digitale versie op www.bmgn-lchr.nl blijft de gedrukte versie van het tijdschrift bestaan. Beide versies zullen wel verschillen. Online publiceren biedt meer mogelijkheden om dicht bij de actualiteit te blijven: lezers en auteurs worden beter geïnformeerd en kunnen actief deelnemen aan het wetenschappelijke debat. Alle recensies verschijnen vanaf dit nummer doorlopend op het web. De recensies worden ook gepubliceerd op de homepage van www.historici.nl (rond 60.000 bezoeken per maand). Dit is de site die knhg en Huygens ing in juni 2011 lanceerden, en die in 2012 zal uitgroeien tot dé digitale ontmoetingsplaats voor professionele historici in Nederland. Potentiële recensenten kunnen op www.bmgn-lchr.nl kiezen uit het aanbod te recenseren boeken en zich aanmelden voor het schrijven van een bespreking. Voor lezers en auteurs bestaat de mogelijk om online commentaren te schrijven. De bibliografische gegevens van de recensies worden toegevoegd aan het eerst volgende nummer dat verschijnt (in druk zowel als online), zodat auteurs er gemakkelijk naar kunnen verwijzen en alle lezers goed op de hoogte blijven. Wij vragen u om u bij het eerste bezoek aan de website van het tijdschrift, die op 30 maart 2012 wordt gelanceerd, te registreren als gebruiker (reader), zodat wij u op de hoogte kunnen houden van nieuw verschenen nummers, recensies en discussies. Gebruik hiervoor de knop <log in> in de balk bovenaan de homepage van het tijdschrift en klik op <Not a user? Register with this site>. Na registratie ontvangt u per e-mail de inhoudsopgave van elk nieuw nummer, een lijst van de gepubliceerde recensies en andere relevantie informatie over bmgn - Low Countries Historical Review. En een laatste mededeling over www.bmgn-lchr.nl: alle jaargangen vanaf 1970 zullen ook daar vindbaar en doorzoekbaar zijn. Redactioneel En tenslotte zullen wij u moeten melden dat na vele jaren van dienst Karel Davids uit de redactie treedt, waarvan hij de laatste jaren tevens voorzitter was. Hij speelde een belangrijke rol in de totstandkoming van bovengenoemde veranderingen en we zijn hem daarvoor zeer erkentelijk. In zijn plaats komt een andere sociaal-economische historicus, Joost Jonker, die zowel hoogleraar is aan de UvA als hoofddocent in Utrecht. Met zijn kennis op het gebied van financiële en bedrijfsgeschiedenis van de laatste eeuwen verrijkt hij de redactie. We heten hem van harte welkom. Namens de redactie, james kennedy
https://openalex.org/W4254761611	https://gmd.copernicus.org/articles/12/3863/2019/gmd-12-3863-2019.pdf	English	null	Improved tropospheric and stratospheric sulfur cycle in the aerosol-chemistry-climate model SOCOL-AERv2	null	2,019	cc-by	21,790	Aryeh Feinberg1,2,3, Timofei Sukhodolov1,4, Bei-Ping Luo1, Eugene Rozanov1,4, Lenny H. E. Winkel2,3 Thomas Peter1 and Andrea Stenke1 1Institute for Atmospheric and Climate Science, ETH Zurich, Zurich, Switzerland 2Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, Zurich, Switzerland 3Eawag, Swiss Federal Institute of Aquatic Science and Technology, Dübendorf, Switzerland 4Physikalisch-Meteorologisches Observatorium Davos and World Radiation Center, Davos, Switzerland Correspondence: Aryeh Feinberg (aryeh.feinberg@env.ethz.ch) Received: 14 May 2019 – Discussion started: 27 May 2019 Revised: 23 July 2019 – Accepted: 30 July 2019 – Published: 3 September 2019 Received: 14 May 2019 – Discussion started: 27 May 2019 Revised: 23 July 2019 – Accepted: 30 July 2019 – Published: 3 September 2019 not further improved. Model performance under volcanically perturbed conditions has also undergone some changes, re- sulting in a slightly shorter volcanic aerosol lifetime after the Pinatubo eruption. With the improved deposition schemes of SOCOL-AERv2, simulated sulfur wet deposition ﬂuxes are within a factor of 2 of measured deposition ﬂuxes at 78 % of the measurement stations globally, an agreement which is on par with previous model intercomparison studies. Because of these improvements, SOCOL-AERv2 will be better suited to studying changes in atmospheric sulfur deposition due to variations in climate and emissions. Abstract. SOCOL-AERv1 was developed as an aerosol– chemistry–climate model to study the stratospheric sulfur cycle and its inﬂuence on climate and the ozone layer. It includes a sectional aerosol model that tracks the sulfate particle size distribution in 40 size bins, between 0.39 nm and 3.2 µm. Sheng et al. (2015) showed that SOCOL- AERv1 successfully matched observable quantities related to stratospheric aerosol. In the meantime, SOCOL-AER has undergone signiﬁcant improvements and more observa- tional datasets have become available. In producing SOCOL- AERv2 we have implemented several updates to the model: adding interactive deposition schemes, improving the sul- fate mass and particle number conservation, and expand- ing the tropospheric chemistry scheme. We compare the two versions of the model with background stratospheric sulfate aerosol observations, stratospheric aerosol evolu- tion after Pinatubo, and ground-based sulfur deposition net- works. SOCOL-AERv2 shows similar levels of agreement as SOCOL-AERv1 with satellite-measured extinctions and in situ optical particle counter (OPC) balloon ﬂights. The vol- canically quiescent total stratospheric aerosol burden simu- lated in SOCOL-AERv2 has increased from 109 Gg of sulfur (S) to 160 Gg S, matching the newly available satellite esti- mate of 165 Gg S. However, SOCOL-AERv2 simulates too high cross-tropopause transport of tropospheric SO2 and/or sulfate aerosol, leading to an overestimation of lower strato- spheric aerosol. Aryeh Feinberg1,2,3, Timofei Sukhodolov1,4, Bei-Ping Luo1, Eugene Rozanov1,4, Lenny H. E. Winkel2,3 Thomas Peter1 and Andrea Stenke1 Due to the current lack of upper tropospheric SO2 measurements and the neglect of organic aerosol in the model, the lower stratospheric bias of SOCOL-AERv2 was Geosci. Model Dev., 12, 3863–3887, 2019 https://doi.org/10.5194/gmd-12-3863-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. Geosci. Model Dev., 12, 3863–3887, 2019 https://doi.org/10.5194/gmd-12-3863-2019 © Author(s) 2019. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction The atmospheric sulfur cycle is of signiﬁcance for climate, atmospheric chemistry, ecosystems, agriculture, and human health. Sulfate aerosol reﬂects incoming shortwave solar ra- diation, leading to a cooling effect at the Earth’s surface. Sul- fate aerosol also absorbs outgoing longwave radiation, lead- ing to a warming of the lower stratosphere. In addition to these direct radiative effects, sulfate particles act as cloud condensation nuclei, leading to cloud formation and an indi- rect radiative effect (Myhre et al., 2013). Sulfate particles af- fect air chemistry in the troposphere and stratosphere by cat- alyzing chemical reactions that deactivate nitrogen (Dentener and Crutzen, 1993). In the cold polar winter stratosphere, they affect ozone depletion by activating chlorine species and serving as condensation nuclei for polar stratospheric clouds A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3864 (Solomon, 1999). For decades, atmospheric sulfate deposi- tion has been a concern due to its role in acidiﬁcation of soils and surface waters (Vet et al., 2014). On the other hand, sul- fur is a macronutrient for plants and decreasing sulfur depo- sition has led to increased demand for sulfur fertilizers in cer- tain regions (Hinckley and Matson, 2011; Kovar and Grant, 2011). These wide-ranging impacts have motivated the de- velopment of atmospheric sulfur models. nonsulfate aerosol species are often not considered in strato- spheric modeling studies, despite their possible contribution to observed aerosol quantities. There are around 15 active models that include strato- spheric aerosol microphysics, which can be separated into models with sectional (∼1/3) or modal (∼2/3) size distri- butions (Kremser et al., 2016). SOCOL-AERv1 is a model with a sectional scheme that divides the sulfate aerosol size distribution into 40 bins (Sheng et al., 2015). The model succeeded in reproducing the observed background strato- spheric aerosol extinctions compared to the Stratospheric Aerosol and Gas Experiment II (SAGE II) and Halogen Oc- cultation Experiment (HALOE) measurements (Thomason, 2012), as well as the particle size distributions measured by optical particle counters (OPCs) in the midlatitudes (Deshler et al., 2003; Deshler, 2008). The SOCOL-AERv1-simulated aerosol burden of 109 Gg sulfur (S) also matched the strato- spheric burden calculated from SAGE-4λ data (112 Gg S). Sulfur is emitted to the atmosphere in various compounds through both natural and anthropogenic sources. Natural sources of sulfur include SO2 from eruptive and degassing volcanoes and dimethylsulﬁde (DMS) from marine phyto- plankton and in small amounts from the terrestrial biosphere. Anthropogenic activities such as fossil fuel combustion, metal smelting, and biomass burning release sulfur mainly as SO2 to the atmosphere (Smith et al., 2011). Short-lived sulfur compounds such as hydrogen sulﬁde (H2S), DMS, and carbon disulﬁde (CS2) are almost completely oxidized in the troposphere and thus do not enter the stratosphere in large amounts. Longer-lived sulfur compounds, such as car- bonyl sulﬁde (OCS) and to some extent sulfur dioxide (SO2), are transported to the stratosphere where they ultimately oxi- dize to gaseous sulfuric acid (H2SO4) (Thomason and Peter, 2006; Kremser et al., 2016). H2SO4 molecules nucleate to form new sulfate aerosol particles or condense on existing particles. In the stratosphere, this sustains a layer of binary H2SO4-H2O particles between 15 and 30 km, often called the “Junge layer” (Junge et al., 1961). A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER The Junge layer is intensi- ﬁed compared to background conditions by sporadically oc- curring volcanic eruptions whose emissions reach the strato- sphere. In the troposphere, H2SO4-containing particles are removed by wet and dry deposition, closing the atmospheric sulfur cycle (Kremser et al., 2016). p ( g ) Despite the good agreement of SOCOL-AERv1 with stratospheric aerosol observations, several aspects of the tro- pospheric sulfur cycle are treated in a coarse manner. For example, the wet and dry deposition schemes are not in- teractive, i.e., wet removal of precursor gases and aerosol does not depend on the grid cell precipitation and dry de- position does not depend on the land surface type or parti- cle size. The Model Intercomparison Project on the climatic response to volcanic forcing (VolMIP) (Zanchettin et al., 2016), which investigated the response of four chemistry– climate models (CCMs) to the 1815 eruption of Mt. Tambora in Indonesia, highlighted several concerns with the deposi- tion ﬂuxes simulated by SOCOL-AERv1. Compared to the other models with interactive deposition schemes, SOCOL- AERv1 displayed lower sulfate deposition in the midlatitude storm tracks since its wet deposition scheme is not linked with precipitation. As well, SOCOL-AERv1 overestimated sulfate deposition to polar ice sheets in both the preindustrial background and Tambora cases (Marshall et al., 2018). Im- provements to the deposition schemes in SOCOL-AER are expected to lead to better reconstructions of past volcanic ac- tivity from deposited sulfate. OCS was ﬁrst suggested to be an important contributor to the stratospheric aerosol layer by Crutzen (1976). Recent modeling studies have quantiﬁed the contribution of different sulfur compounds to the stratospheric aerosol burden. Brühl et al. (2012) attributed 70 % of the background, nonvolcanic stratospheric aerosol burden above 20 km to OCS oxidation. Sheng et al. (2015) suggested that 56 % of the stratospheric burden arises due to OCS and 32 % due to SO2 emissions. It must be noted that these studies calculated these contribu- tions by turning off emissions from all other sulfur species, and lower sulfur emissions can lead to smaller aerosol parti- cles, slower sedimentation, and longer aerosol lifetimes. Since Sheng et al. (2015) was published, there have also been substantial updates and changes to the strato- spheric aerosol observations. OPC measurements have un- dergone revision due to a correction in the counting ef- ﬁciency of the instrument (Kovilakam and Deshler, 2015; Deshler et al., 2019). Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER sulfate aerosol microphysics to the SOCOL model, based on the two-dimensional sulfate aerosol model AER (Weisen- stein et al., 1997). The model considers eight gaseous sul- fur species: OCS, CS2, H2S, DMS, methanesulfonic acid (MSA), SO2, sulfur trioxide (SO3), and H2SO4. Sulfate aerosol particles are resolved in 40 size bins, ranging in ra- dius from 0.39 nm to 3.2 µm, with sequential bins doubling in volume. Chemical reaction rate coefﬁcients and absorp- tion cross sections of all reactions, including sulfur reac- tions, follow the recommendations by Sander et al. (2011). In the aqueous phase, sulfur is described as S(IV) and S(VI) without further speciation. Aqueous oxidation of S(IV) by ozone (O3) and hydrogen peroxide (H2O2) is calculated by the model using the scheme by Jacob (1986). The aqueous production ﬂux of S(VI) is added to the atmospheric sulfate aerosol tracers with the ﬂux to each bin proportional to the bin volume. we use these newly available datasets to evaluate results from the updated SOCOL-AERv2 model. This paper outlines the changes that have been made from SOCOL-AERv1 to v2 through the implementation of the new interactive deposition scheme and other improvements. Section 2 summarizes the changes to the SOCOL-AER code and details the experimental setup of the simulations. Sec- tion 3 discusses the results of three types of simulations: year 2000 time-slice runs (Sect. 3.1–3.3), 2000–2010 transient runs (Sect. 3.4), and post-Pinatubo transient runs (Sect. 3.5). We compare the model with stratospheric aerosol observa- tions, from both nonvolcanic background and post-Pinatubo periods, as well as with surface measurements of wet and dry deposition ﬂuxes. Section 3.6 presents updated estimates for the year 2000 atmospheric sulfur budget. Section 4 draws the conclusions of this work. The microphysical scheme of SOCOL-AERv1 consid- ers the nucleation (Vehkamäki et al., 2002), composition (Tabazadeh et al., 1997), growth through H2SO4 conden- sation, evaporation (Ayers et al., 1980; Kulmala and Laak- sonen, 1990), coagulation (Fuchs, 1964; Jacobson and Se- infeld, 2004), and sedimentation of sulfate aerosol (Kas- ten, 1968; Walcek, 2000). SOCOL-AERv1 employs a crude altitude-varying lifetime approach for tropospheric wet re- moval of sulfur species, with H2SO4 and MSA having a mean column lifetime of 5 d and SO2 having a mean life- time of 2.5 d (Weisenstein et al., 1997). Dry deposition of SO2, MSA, H2SO4, and sulfate aerosol is modeled assuming a deposition velocity of 1 cm s−1 at the ground. 2.1 Year 2000 time-slice simulations The development of SOCOL-AERv2 consisted of correc- tions to the SOCOL-AERv1 code and implementations of new schemes. To compare directly with the reference simu- lation from Sheng et al. (2015) we run time-slice simulations for the year 2000 at each stage of the code changes. For each model run we simulate 10 years, taking the ﬁrst 5 years as a spin-up from the initial conditions and analyzing only the last 5 years. In this section, we describe changes in the code for each of the time-slice simulations (summarized in Table 1). A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER The model is run with operator splitting so that dynamical quantities are recalculated every 15 min, whereas the chemistry, aerosol microphysics, and radiation schemes are called every 2 h. Twenty sub-time-steps are used for the aerosol microphys- ical scheme, yielding an aerosol microphysical time step of 6 min. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER Updated extinction values are avail- able through the Global Space-based Stratospheric Aerosol Climatology (GloSSAC) project (Thomason et al., 2018). This dataset was used to construct the SAGE-3λ dataset of stratospheric aerosol burdens, which is an input for models in phase 6 of the Coupled Model Intercomparison Project (CMIP6). New in situ measurements of SO2 in the upper tro- posphere have raised a discussion about the magnitude of the cross-tropopause SO2 ﬂux (Rollins et al., 2017, 2018). Here, As reviewed by Kremser et al. (2016), other studies have emphasized the minor, yet nonnegligible, contribution of nonsulfate species to the stratospheric aerosol burden. Me- teoritic dust particles enter the atmosphere at a rate of 3– 300 t d−1 (Plane, 2012), which would correspond to 0.15 %– 15 % of the stratospheric aerosol mass ﬂux estimated by Sheng et al. (2015). Modeling (Yu et al., 2015) and measure- ments (Froyd et al., 2009; Friberg et al., 2014; Murphy et al., 2014) suggest that organic carbon is a signiﬁcant fraction of the aerosol mass in the lowest part of the stratosphere. The www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 3865 2.1.2 Dry radius binning scheme (DRYRAD) In the CONSERVE simulation, corrections were made to the aerosol microphysics scheme in SOCOL-AER, mainly to im- prove aerosol mass conservation. In the scheme calculating H2SO4 condensation and evaporation, the equation for the “effective” mean free path of H2SO4 molecules in air was corrected to agree with Eq. (6) from Hamill et al. (1977). An additional constraint was added in the H2SO4 condensa- tion scheme such that the ﬂux of H2SO4 from the gas phase must equal the ﬂux of H2SO4 into the particle phase. This im- proves mass conservation in cases when H2SO4 is depleted below the saturation vapor pressure within one time step. Furthermore, the aerosol sedimentation scheme in SOCOL- AERv1 was not applied within boundary layer levels and sedimenting aerosol from the model level above the bound- ary layer was artiﬁcially removed. In the CONSERVE simu- lation this is amended: sedimenting aerosol from the model level above the boundary layer is added to the layer below. Sheng et al. (2015) had implemented several forced mass conservation checks on the total (gas-phase and aerosol) H2SO4 burden in each grid cell. If the total burden had changed by more than 0.1 % during aerosol microphysics, H2SO4 aerosol and gas-phase mixing ratios would be scaled to agree with the total H2SO4 burden before microphysics In SOCOL-AERv1, the sulfate aerosol is resolved in wet ra- dius bins. Uptake and evaporation of H2O during transport- induced changes in relative humidity and temperature cause shifts in the sulfate mass distribution with respect to wet aerosol radius, the coordinate variable. In SOCOL-AERv1, this process was treated by rescaling the number of sulfate aerosol particles so that each bin would have the correct H2SO4 weight percent. Although this procedure guarantees the conservation of the total number of H2SO4 molecules, it does not conserve the aerosol number density. To amend this, SOCOL-AERv2 resolves the sulfate aerosol distribution in dry radius bins, similar to the approach of other sectional models (e.g., Kleinschmitt et al., 2017). Dry radius bins can also be described as aerosol H2SO4 mass bins. The new dry radius bins range from 0.39 nm to 3.2 µm, corresponding to 2.8 to 1.6 × 1012 molecules of H2SO4 per particle (assuming a H2SO4 density of 1.8 g cm−3), with molecule number doubling between bins. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3866 Table 1. Description of the year 2000 time-slice simulations that were produced during the development of SOCOL-AERv2. Each simulation builds on the previous one, adding new features or correcting a certain process in the model. Two additional sensitivity runs are also listed that reduce the cross-tropopause transport of SO2 and tropospheric sulfate aerosol. Simulation name Description Section SHENG31 Model version (in T31 resolution) used in Sheng et al. (2015) 2.1.1 SOCOL-AERv1 SHENG31 model code but using T42 resolution 2.1.1 DRYRAD Implementation of dry sulfate aerosol radius as the binning scheme instead of wet radius 2.1.2 CONSERVE Improvement of mass conservation in aerosol microphysical schemes 2.1.3 CCMI Expansion of tropospheric chemistry scheme (Revell et al., 2015, 2018) 2.1.4 BNDLAYER Limiting emission and deposition boundary conditions to the lowermost model layer 2.1.5 DRYDEP Addition of interactive dry deposition scheme (Kerkweg et al., 2006; Kerkweg et al., 2009) 2.1.6 WETDEP Addition of interactive wet deposition scheme (Tost et al., 2006) 2.1.7 AQCHEM Fixes to the aqueous chemistry scheme (time step, transfer to wet deposition ﬂux, cloud pH) 2.1.8 SOCOL-AERv2 Correcting the supercooled liquid fraction to CALIOP-observed values for aqueous chemistry 2.1.9 Additional runs based on SOCOL-AERv2 ICE-OX Oxidation of S(IV) to S(VI) occurs in cloud ice water in addition to liquid water 2.1.10 AER-SCAV Aerosol scavenging coefﬁcient on ice is increased by a factor of 20 2.1.10 tated several changes to the sulfate condensation and coagu- lation schemes to ensure that the transfer of aerosol from bin to bin is based on molecular ﬂuxes rather than aerosol vol- ume ﬂuxes. For calculation of aerosol radiative properties, a new look-up table was produced as a function of relative hu- midity and temperature. To ease interpretation of the output, the outputted aerosol bins of SOCOL-AER are rebinned to the previous wet volume binning approach. simulations in Sheng et al. (2015), we output the sulfur cy- cle burdens and ﬂuxes as accumulated rather than instanta- neous quantities to reduce the inﬂuence of diurnal cycling on the 12-hourly output of the model. To test the effect of hori- zontal resolution on the atmospheric sulfur cycle we ran one simulation at T31 resolution (∼3.75◦×3.75◦in latitude and longitude, SHENG31) and one simulation at T42 resolution (SOCOL-AERv1). 2.1.1 Rerunning v1 in T31 (simulation: SHENG31) and T42 (SOCOL-AERv1) A full description of SOCOL-AERv1 can be found in Sheng et al. (2015), so here we will only summarize the main as- pects of the model. The model originated from the SOCOLv3 chemistry–climate model (Stenke et al., 2013), which con- sists of the middle atmosphere version of the global cir- culation model (GCM) European Centre/Hamburg 5 (MA- ECHAM5) (Roeckner et al., 2003; Giorgetta et al., 2006) and the chemistry model MEZON (Rozanov et al., 1999; Egorova et al., 2003). SOCOLv3 includes 39 hybrid vertical levels ranging from the Earth surface up to 0.01 hPa (80 km). The model is an atmosphere-only model prescribing global sea surface temperatures and sea ice coverage with observed data from the Hadley Centre (Rayner et al., 2003). The quasi- biennial oscillation is produced in the model by relaxing the simulated zonal winds in the equatorial stratosphere to ob- served wind proﬁles (Stenke et al., 2013). The model’s boundary conditions that we use for the year 2000 time-slice simulations are identical to Sheng et al. (2015). SO2 is emitted from anthropogenic and biomass burning sources according to a gridded emission inventory for the year 2000 (Lamarque et al., 2010; Smith et al., 2011) and from continuous volcanic degassing (Andres and Kas- gnoc, 1998; Dentener et al., 2006b). DMS ﬂuxes are calcu- lated online using a wind-driven parametrization (Nightin- gale et al., 2000) and a climatology of sea surface DMS con- centrations (Kettle et al., 1999; Kettle and Andreae, 2000). As in Weisenstein et al. (1997), 1 Tg S yr−1 of CS2 is emitted between the latitudes of 52◦S and 52◦N. The mixing ratios of H2S and OCS are ﬁxed at the surface to 30 pptv (Weisen- stein et al., 1997) and 500 pptv (Chin and Davis, 1995; Kettle et al., 2002; Montzka et al., 2007; Commane et al., 2013), re- spectively. The chemistry module in SOCOL-AERv1 includes a com- prehensive range of stratospheric chemical reactions and a simpliﬁed set of tropospheric reactions: of the atmospheric hydrocarbons, only methane photochemistry is included. Sheng et al. (2015) introduced online sulfur chemistry and To ensure comparability of results with the new develop- ment runs for this paper, we have rerun the source code from Sheng et al. (2015) in two experiments. As opposed to the www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 2.1.5 Treatment of the boundary layer (BNDLAYER) calculations. These forced mass corrections were found to be unnecessary after the above improvements to the mi- crophysics scheme, and therefore they were removed from SOCOL-AERv2. In the previous simulations, to allow for rapid boundary layer mixing, emissions of chemical species were immedi- ately dispersed over the four lowermost model levels (∼1 km altitude), species with prescribed mixing ratios (including OCS and H2S) were dispersed over the six lowermost lev- els (∼2 km altitude), and dry deposition of species occurred out of the four lowermost model levels. While such a coarse approach was sufﬁcient for stratospheric applications, it is inadequate for deposition ﬂux and tropospheric lifetime cal- culations. Instead of emitting the species in multiple lower layers, SOCOL-AERv2 emits only in the ﬁrst model layer (∼70 m), from which the species are mixed via the model’s boundary layer parametrizations. The BNDLAYER simula- tion tests speciﬁcally the effect of using only one model layer for emission and prepares the model for revised dry deposi- tion boundary conditions. 2.1.6 Interactive dry deposition (DRYDEP) We implemented the interactive dry deposition scheme de- scribed in Revell et al. (2018) in SOCOL-AERv2, replac- ing the simple prescribed constant deposition velocities of SOCOL-AERv1. The new treatment is based on the DRY- DEP scheme in the EMAC model (Kerkweg et al., 2006; Kerkweg et al., 2009). Dry deposition velocities are calcu- lated using an interactive resistance-based approach which considers surface properties, the solubility and reactivity of each gas tracer, and the radius and density of aerosol trac- ers (Wesely, 1989). Effective Henry’s law constants for near- neutral pH and reactivity of gas tracers are taken from We- sely (1989). These improvements are tested in the DRYDEP simulation. 2.1.4 Merging CCMI additions with SOCOL-AER (CCMI) Since the publication of Sheng et al. (2015), improvements have been made to the SOCOL model in preparation of the coordinated simulations within the Chemistry Climate Model Initiative (CCMI), mainly related to the improve- ment in tropospheric chemistry processes (Revell et al., 2015, 2018). Many of these improvements have been merged into SOCOL-AERv2 and have upgraded the representation of chemistry in our model, in particular in the troposphere. y p p p In SOCOL-AERv1, as well as SOCOLv3 (Stenke et al., 2013), ozone-depleting substances (ODSs) were transported in three families (short-lived Cl, long-lived Cl, and Br) to save computational cost. With modern supercomputers this treatment is no longer necessary and the ODS species are transported individually. The individual treatment of ODS species avoids a repartitioning of the family members, based on simpliﬁed age-of-air estimates, after each transport step. The chemistry scheme was expanded in the CCMI simu- lation, as described in Revell et al. (2015). We included the Mainz Isoprene Mechanism (MIM-1) in SOCOL-AER. This scheme considers the degradation of isoprene and ne- cessitates the addition of 14 organic species and 44 chem- ical reactions to SOCOL-AER (Pöschl et al., 2000). Addi- tional CO emissions were added to the model to account for the effect of oxidation of nonmethane volatile organic com- pounds (NMVOC) emitted from anthropogenic, biogenic, and biomass burning sources. Lightning NOx is now cal- culated interactively based on cloud-top height (Price and Rind, 1992) and grid cell scaling factors from satellite ob- servations (Christian et al., 2003). A cloud modiﬁcation fac- tor approach (Chang et al., 1987) was implemented to ac- count for the effect of clouds on photolysis rates. We de- rived a new look-up table of photolysis rates averaged over two solar cycles (22 years) from a comprehensive recon- struction of total and spectral solar irradiance (NRLSSI) by Lean et al. (2005), which was used in the CCMI REF-C1 ex- periment. Additional heating through Hartley and Huggins bands of ozone has also been implemented into SOCOL- AER. As documented by Revell et al. (2018), N2O5 hydrol- ysis on tropospheric aerosols is now included in SOCOL- AER. Methanesulfonic acid (MSA) chemistry is solved in the explicit scheme instead of the implicit Newton–Raphson scheme since otherwise the chemical solver does not prop- erly converge. Reaction rates have been updated or added from the NASA JPL data evaluation no. 18 (Burkholder et al., 2015). 2.1.2 Dry radius binning scheme (DRYRAD) Since aerosol mi- crophysics schemes and heterogeneous chemistry on sul- fate aerosol require wet aerosol volume and H2SO4 weight percent, we calculate these quantities for each bin online, based on the grid cell temperature and relative humidity. This change in the dimension variable of SOCOL-AER necessi- www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 3867 2.1.8 Improvement in aqueous-phase chemistry (AQCHEM) In the SO2 aqueous chemistry subroutine of SOCOL- AERv1, the pH of clouds is prescribed vertically according to Walcek and Taylor (1986) so that pH equals 3 from the sur- face to 600 hPa and 4.5 above 600 hPa. However, this paper reported modeled pH within a single cumulus cloud, where liquid water content increased with height. This pH distribu- tion is therefore not applicable to the whole atmosphere. Al- though in the interactive wet deposition scheme a constant cloud pH of 5 is used (Sect. 2.1.7), aqueous-phase sulfur chemistry is more sensitive to the choice of pH and therefore a more detailed pH distribution was applied. We use an ap- proximation of the modeled cloud pH from Tost et al. (2007) for the revised aqueous chemistry routine. Between the sur- face and 600 hPa, north of 20◦N a cloud pH of 5.2 is used and south of 20◦N a cloud pH of 4.2 is used. Above the 600 hPa level, a uniform pH of 3.5 is used. SLFHu = 1 + exp(−f (T )) −1, (1) f (T ) = 5.3608 + 0.4025T + 0.08387T 2 + 0.007182T 3 + 2.39 × 10−4T 4 + 2.87 × 10−6T 5, (2) (1) (2) where T is the air temperature in degrees Celsius (◦C). The determined SLF can is used to correct LWC in the aqueous chemistry subroutine, i.e., LWC = SLFHu × TWC. where T is the air temperature in degrees Celsius (◦C). The determined SLF can is used to correct LWC in the aqueous chemistry subroutine, i.e., LWC = SLFHu × TWC. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3868 et al., 2014; Cesana et al., 2015; Tan et al., 2016). The mod- eled liquid fraction in SOCOL-AERv1 underestimates the ﬁtted CALIOP satellite measurements from Hu et al. (2010) throughout most of the mixed-phase cloud temperature range (Fig. S1 in the Supplement). In the SOCOL-AERv2 run, we correct for the inﬂuence of the underestimated supercooled liquid fraction on SO2 aqueous chemistry. The ECHAM5 calculated liquid water content (LWC) and ice water con- tent (IWC) are added together and inputted into the aqueous- phase chemistry subroutine as total water content (TWC). If the grid cell temperature (T ) is in the mixed-phase cloud regime (−38 to 0 ◦C), we calculate the observed supercooled liquid fraction (SLF) from the ﬁtted sigmoid function from Hu et al. (2010): leased to the atmosphere in their original species, whereas evaporating scavenged sulfate aerosol species are transferred to the largest aerosol size bin. The wet deposition scheme is applied to SO2, gaseous H2SO4, and sulfate aerosol, as well as other gas chemical tracers such as O3, HNO3, N2O5, H2O2, etc. The WETDEP simulation includes this new inter- active wet deposition scheme instead of the ﬁxed wet depo- sition lifetimes used in SOCOL-AERv1. 2.1.10 Additional sensitivity runs (ICE-OX, AER-SCAV) We ran two additional simulations to probe whether the re- maining disagreement between observations and SOCOL- AERv2 could be caused by overestimated cross-tropopause ﬂuxes of SO2 and sulfate aerosol. In ICE-OX, the aqueous- phase oxidation of SO2 was allowed to occur in ice water as well as liquid water. Increased oxidation of SO2 in the up- per troposphere reduces its cross-tropopause ﬂux. In AER- SCAV, the scavenging coefﬁcient of aerosol particles on ice clouds was increased by a factor of 20 from 0.05 to 1. This enhances the removal of sulfate aerosol in the upper tropo- sphere. In SOCOL-AERv1, the SO2 oxidized in the aqueous cloud phase is released as aerosol. With the new interactive wet deposition scheme, it is possible to transfer the oxidized SO2 directly to the scavenged aerosol ﬂux in cloud water. The wet deposition routine is called at each dynamical time step (15 min), while the aqueous-phase chemistry was called at each chemical time step in SOCOL-AERv1 (2 h). To transfer the oxidized SO2 ﬂux to the wet deposition scheme directly, it was both logical and technically simpler to synchronize these two processes. In the AQCHEM simulation, the above changes were added to SOCOL-AER and the aqueous-phase chemistry is called at each dynamical time step. 2.2 Years 2000–2010 transient simulations In order to compare simulated deposition with observations, the model codes from SOCOL-AERv1 and v2 were used to run two sets of transient simulations from 2000 to 2010. Five ensemble members were simulated for both versions of SOCOL-AER and plotted results show ensemble means and standard deviations. For the transient simulations we made several updates to the boundary conditions used in Sheng et al. (2015). Anthropogenic emissions were taken from the Community Emissions Data Systems (CEDS), which will be used for CMIP6 simulations (Hoesly et al., 2018). Lana et al. (2011) updated the marine DMS dataset to include three times as many DMS measurements as the previous dataset (Kettle et al., 1999; Kettle and Andreae, 2000) used by Sheng et al. (2015). Transient degassing volcanic SO2 emissions were taken from Diehl et al. (2012). To represent eruptive 2.1.7 Interactive wet deposition (WETDEP) An interactive wet deposition scheme was added to SOCOL- AER, based on the SCAV submodule in the EMAC model (Tost et al., 2006). Grid scale variables from ECHAM5 such as liquid and ice water contents, cloud cover, convective and large-scale rain and ice formation and precipitation ﬂuxes, and the convective upward mass ﬂux are used by the wet de- position scheme. Since our model does not include a com- prehensive cloud aqueous chemistry mechanism, we imple- mented the EASY2 version of the SCAV submodule (Tost et al., 2007) with a constant pH of 5 for cloud water and rain water. The constant pH of 5 is within the wide range of pH values (3.6–7) measured by several hill cap cloud ﬁeld campaigns (Sellegri et al., 2003; Marinoni et al., 2004; van Pinxteren et al., 2016). Scavenging coefﬁcients for gas-phase species are calculated based on Henry’s law equilibrium con- stants. Scavenging of aerosol is based on a radius-dependent calculation of nucleation and impaction scavenging. During cloud evaporation, all scavenged gas-phase species are re- www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 3.1.4 CCMI chemistry changes (CCMI) Including the expanded chemistry set and updates from the CCMI version of SOCOLv3 in SOCOL-AER leads to altered distributions of gas-phase species in the troposphere. The rel- evant change for the tropospheric sulfur cycle is increased mixing ratios of H2O2, causing increased aqueous conver- sion of SO2 to S(VI). For this reason, the CCMI simula- tion shows a 19 Gg S lower SO2 burden and a 10 Gg S larger sulfate aerosol burden in the troposphere than the CON- SERVE simulation. Larger OH mixing ratios in the upper troposphere and lower stratosphere (UTLS) reduce the SO2 lifetime, causing 5 Gg lower SO2 and 2 Gg higher sulfate aerosol burdens in the stratosphere. These chemical changes also lead to differences in the Pinatubo simulation, to be dis- cussed in Sect. 3.5. 3.1 Impacts of performed changes in the development of SOCOL-AERv2 In the following section, we discuss the relevant impacts of each stage of code changes on the atmospheric sulfur cycle. Table 2 lists the stratospheric and tropospheric burdens of SO2 and sulfate aerosol and total deposition ﬂuxes for each time-slice simulation. 3.1.1 Rerunning SOCOL-AERv1 in T31 and T42 resolutions Since the SHENG31 simulation was rerun for this study, several quantities differ slightly in SHENG31 compared to Sheng et al. (2015) (e.g., 114 vs. 109 Gg S of stratospheric sulfate aerosol). The differences in the quantities could be caused by the switch in output format from instantaneous 12-hourly values to mean 12-hourly values. However, the changes are minor and the overall picture for the sulfur cy- cle remains unchanged. Reﬁning the horizontal resolution to T42 in SOCOL-AERv1 does not result in substantial changes for the tropospheric and stratospheric aerosol burdens. 3.1.5 Boundary layer levels (BNDLAYER) In BNDLAYER the boundary layer conditions are only im- plemented for the lowest level of the model. The conﬁnement of the boundary layer conditions to one model level reduces the burdens of SO2 and sulfate aerosol in the troposphere and stratosphere. This is a strong effect with reductions of the tropospheric aerosol burden by 40 % and the stratospheric A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER the effective aerosol radius, leading to a longer stratospheric lifetime. The decrease in tropospheric aerosol burden occurs mainly around the Northern Hemisphere midlatitudes, where anthropogenic emissions of SO2 are high and thus sulfate particles can grow through condensation. In the DRYRAD version of the model the wet particle radius is no longer re- stricted to 3.2 µm; accounting for the uptake of water the maximum radius can reach well above 10 µm. The possibility of larger particle formation can lead to enhanced sedimenta- tion velocities and therefore reduced aerosol lifetimes. emissions, we applied a satellite-derived dataset from Carn et al. (2016). The other data sources for the boundary con- ditions remained the same as in the time-slice simulations; however, transient boundary conditions were included rather than applying repeating year 2000 values. 2.3 Pinatubo transient simulations To verify the updated model’s performance under volcani- cally perturbed conditions, we have repeated two experi- ments from Sukhodolov et al. (2018), modeling the Mt. Pinatubo eruption with 7 and 6 Tg S emitted as SO2. As in Sukhodolov et al. (2018), we simulated ﬁve ensemble mem- bers with sulfur mass released from 14 to 15 June 1991 and spread between 16 and 30 km. We performed two additional runs with SOCOL-AERv1 and SOCOL-AERv2, including the Pinatubo eruption magnitude of 7 Tg S but with all other sulfur sources switched off, to check the sulfur mass con- servation by analyzing the integrated deposition ﬂuxes. To compare with modeled burdens, we used observational esti- mates from SAGE-4λ and SAGE-3λ datasets and from the High-Resolution Infrared Radiation Sounder (HIRS) mea- surements (Baran and Foot, 1994). 3.1.3 Mass conservation ﬁxes (CONSERVE) The corrections in the sedimentation and H2SO4 condensa- tion schemes do improve the mass conservation of sulfur species. The tropospheric sulfate aerosol burden increases by 4 %, mainly due to the correction of the artiﬁcial removal of particles sedimenting from the model level above the bound- ary layer level. This artiﬁcial loss due to sedimentation rep- resents a ∼3 Tg S yr−1 sink since the outputted total sulfur deposition increases by this amount from the DRYRAD to the CONSERVE simulations. Furthermore, in the DRYRAD simulation the total sum of tropospheric aerosol inﬂuxes and outﬂuxes result in an imbalance of 3339 Gg S yr−1, which corresponds to about 8 % of the source ﬂux of tropospheric sulfate aerosol. In the CONSERVE simulation, this imbal- ance is reduced to 63 Gg S yr−1, i.e., around 0.1 % of the aerosol source ﬂux. These improvements to the model pro- vide more conﬁdence to the outputted sulfur cycle ﬂuxes, which will be used to study the sulfur budget in Sect. 3.6. 2.1.9 Final development run for SOCOL-AERv2 Section 2.1.1–2.1.8 complete the description of improve- ments in developing SOCOL-AERv2 with one exception, namely how SO2 oxidation is calculated in clouds in the mixed-phase temperature regime. This is important because the S(IV) to S(VI) conversion only occurs in the liquid phase in the model. Therefore, in the ﬁnal development simula- tion (SOCOL-AERv2) we wanted to investigate whether the aqueous SO2 reaction was hampered by the model’s repre- sentation of the liquid fraction in mixed-phase clouds. It has recently been discussed in the literature that many general circulation models (GCMs) underpredict the supercooled liq- uid fraction (SLF) observed by satellite products (Komurcu Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ 3869 3.1.6 Interactive dry deposition (DRYDEP) aerosol burden by 20 % in BNDLAYER. The ﬁrst cause is the reduction in effective S emissions into the atmosphere since H2S is prescribed to be 30 ppt in only one model level instead of six model levels. Assuming steady-state condi- tions over the 5-year averaging period, the 8.5 Tg S yr−1 de- crease in total sulfur deposition (Table 2) corresponds to an 8.5 Tg S yr−1 decrease in the sulfur emissions. Another cause for the SO2 and aerosol burden decrease is that SO2 is emit- ted close to the surface in BNDLAYER, leading to less dis- persion of SO2 in the atmosphere and enhanced dry deposi- tion close to emission regions. The shorter SO2 lifetime re- duces its atmospheric burden, as well as reducing the conver- sion of SO2 to H2SO4 and subsequent sulfate aerosol forma- tion. The implementation of interactively calculated dry deposi- tion velocities, compared to the previously included constant dry deposition velocities, results in much longer dry deposi- tion lifetimes for both SO2 and sulfate aerosol. SO2 dry depo- sition velocities decrease more drastically over land than over ocean in the DRYDEP simulation (Fig. 1). Over land, the SO2 dry deposition velocity is smaller than 1 cm s−1, which was the original value set in SOCOL-AERv1. The only lo- cations where SO2 dry deposition velocities are greater than 1 cm s−1 are above certain parts of the ocean, due to the high solubility of SO2 in waters at near-neutral pH. The dry de- position velocities of SO2 agree well with the distribution simulated by the EMAC model (Kerkweg et al., 2006). The resultant longer SO2 dry deposition lifetime increases the tro- pospheric SO2 burden, the conversion of SO2 to aerosol, and consequently the tropospheric aerosol burden. In addition, dry deposition velocities of sulfate aerosol decrease glob- ally compared to the assumed constant deposition velocity in SOCOL-AERv1 (1 cm s−1), leading to longer aerosol life- times with respect to dry deposition. Due to augmented trans- port of tropospheric SO2 and primary sulfate aerosol from the troposphere, the stratospheric aerosol burden increases by 22 The correct treatment of the lowermost model levels re- mains difﬁcult and is model dependent. Owing to their coarse resolution, CCMs cannot resolve the transport of chemi- cal species by rapid boundary layer convection and turbu- lence. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3870 Table 2. Global annual mean sulfate aerosol and SO2 burdens in the troposphere and stratosphere for all volcanically quiescent time-slice (year 2000) simulations in the development of SOCOL-AERv2. Total sulfur deposition (last column) is listed as a check of whether the mass balance of the model has changed. Model results are compared to two datasets, SAGE-3λ and SAGE-4λ, which derived stratospheric aerosol burdens from satellite extinction measurements. The model tropopause is used to separate tropospheric and stratospheric burdens, whereas for the SAGE-3λ and SAGE-4λ calculations the MERRA tropopause is used. The WMO assessment (Vet et al., 2014) calculates a multimodel mean total sulfur deposition ﬂux of 84.8 ± 6.1 Tg yr−1 (±σ) and total emissions of 91.0 ± 7.3 Tg yr−1 for 2001. Simulated interannual variability in one simulation is on the order of 1 %–2 % for burdens and 0.3 % for total deposition. Simulation Tropospheric Stratospheric Tropospheric Stratospheric Total sulfur aerosol burden aerosol burden SO2 burden SO2 burden deposition (Gg S) (Gg S) (Gg S) (Gg S) (Tg S yr−1) SHENG31 395 114 259 12.4 99.8 SOCOL-AERv1 397 116 261 11.9 99.8 DRYRAD 367 125 261 12.0 97.9 CONSERVE 382 128 261 12.3 101.1 CCMI 392 130 242 7.1 101.0 BNDLAYER 236 106 137 5.8 92.5 DRYDEP 508 128 218 6.6 92.8 WETDEP 769 202 351 8.2 93.6 AQCHEM 667 166 245 6.6 94.5 SOCOL-AERv2 640 160 217 6.3 94.4 Additional sensitivity tests ICE-OX 613 92 188 3.9 94.2 AER-SCAV 579 133 215 6.4 94.5 Observational datasets SAGE-4λ 117 ± 8 SAGE-3λ 165 ± 11 WMO assessment 84.8 ± 6.1 3.1.2 Dry radius binning (DRYRAD) The change from wet radius to dry radius binning reduces the tropospheric aerosol burden and increases the stratospheric aerosol burden in DRYRAD (Table 2). The increased strato- spheric aerosol burden can be explained by a decrease in www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3871 Figure 1. Annual mean dry deposition velocities for SO2 (a) and sulfate aerosol (b) simulated by SOCOL-AERv2. The mean dry deposition velocity for aerosol particles is calculated by weighting the dry deposition velocity for each size bin with the bin’s mass concentration at the surface. The color bar highlights differences between the newly simulated deposition velocities and the former homogeneous deposition velocity of 1 cm s−1, which is shown in whitish colors on both plots. Figure 1. Annual mean dry deposition velocities for SO2 (a) and sulfate aerosol (b) simulated by SOCOL-AERv2. The mean dry deposition velocity for aerosol particles is calculated by weighting the dry deposition velocity for each size bin with the bin’s mass concentration at the surface. The color bar highlights differences between the newly simulated deposition velocities and the former homogeneous deposition velocity of 1 cm s−1, which is shown in whitish colors on both plots. Figure 1. Annual mean dry deposition velocities for SO2 (a) and sulfate aerosol (b) simulated by SOCOL-AERv2. The mean dry deposition velocity for aerosol particles is calculated by weighting the dry deposition velocity for each size bin with the bin’s mass concentration at the surface. The color bar highlights differences between the newly simulated deposition velocities and the former homogeneous deposition velocity of 1 cm s−1, which is shown in whitish colors on both plots. to 128 Gg S. The changes in DRYDEP largely compensate the changes in BNDLAYER, for which emissions were con- ﬁned to a single model level. tions (Marshall et al., 2018). Calculating a global aerosol wet deposition lifetime with respect to wet deposition (lifetime is tropospheric aerosol burden divided by aerosol wet deposi- tion ﬂux), DRYDEP has an aerosol wet deposition lifetime of 4.9 d and WETDEP has a lifetime of 5.1 d. Therefore, there is not a large change in the global aerosol wet deposition life- time; however, the spatial distribution of the wet deposition sink has shifted. In WETDEP the tropospheric column wet deposition lifetime of sulfate aerosol varies from 2 d in the northern midlatitude storm tracks to more than 3 years over the southwestern United States. The introduction of interac- tive wet deposition to SOCOL-AER has the largest impact of any step on the stratospheric sulfate burden. Driven by the longer SO2 wet deposition lifetime, the stratospheric sulfate aerosol burden climbs by around 60 % to 202 Gg S. 3.1.7 Interactive wet deposition (WETDEP) When the constant wet deposition lifetimes for sulfur species are replaced with interactively calculated wet removal in the WETDEP simulation, the SO2 wet deposition ﬂux is reduced from 20.1 to 0.3 Tg yr−1, revealing an overestimation in the approach of SOCOL-AERv1. With the elimination of the wet deposition sink for SO2, the tropospheric SO2 burden in- creases by around 60 % and the total (aqueous + gas phase) conversion ﬂux of SO2 to S(VI) increases by around 40 %. In Sheng et al. (2015), the mean wet deposition lifetime for SO2 was selected as 2.5 d following the two-dimensional AER model. However, the AER model includes the 2.5 d lifetime for SO2 to account for aqueous oxidation of SO2, which is not explicitly modeled by AER (Weisenstein et al., 1997). As SOCOL-AERv1 already includes a mechanism for aqueous oxidation of SO2 by H2O2 and O3 in clouds, this resulted in double counting of the loss of SO2 by aqueous oxidation in previous simulations. The aerosol wet deposition maps and the relative difference between the DRYDEP and WETDEP simulations are shown in Fig. 2. The inclusion of an inter- active wet deposition enhances sulfate aerosol deposition in areas with high precipitation and suppresses it in drier re- gions. Sulfate deposition is reduced over polar regions, the eastern part of ocean basins, and the Sahara (lower precipita- tion regions), and is enhanced in the tropics and midlatitude storm tracks (higher precipitation regions). The reductions in sulfate deposition ﬂuxes above Greenland and Antarctica are notable since SOCOL-AERv1 overestimated the magnitude of polar sulfate deposition ﬂuxes compared to ice core mea- surements, which are used as proxies for past volcanic erup- A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER As will be discussed in Sect. 3.2.1, this value is much higher than the inferred stratospheric burden from SAGE II data for back- ground nonvolcanic conditions. To improve the agreement with observations, we focus on a possible underestimation of the sulfate aqueous chemistry ﬂux since the unintended dou- ble counting of this ﬂux led to good agreement of SOCOL- AERv1 with stratospheric observations (Sheng et al., 2015). www.geosci-model-dev.net/12/3863/2019/ 3.1.6 Interactive dry deposition (DRYDEP) This leaves the boundary layer parametrizations in SOCOL-AER imperfect and the number of model levels that should be included in the emission boundary conditions un- certain. For the subsequent simulations we use the single- level boundary layer treatment. Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3.2.1 Comparison with SAGE II-derived burdens Sheng et al. (2015) compared the modeled stratospheric sul- fate aerosol burden to the value calculated by the SAGE-4λ method (Arfeuille et al., 2013). In this method, extinctions measured by the SAGE II satellite product are used to esti- mate the stratospheric aerosol size distribution, which can then be used to determine the aerosol burden. The back- ground stratospheric aerosol burden derived from SAGE-4λ almost exactly matched the burden simulated by SOCOL- AERv1. Since that time, a new SAGE II retrieval has been published as part of the GloSSAC database (Thomason et al., 2018). A new method (SAGE-3λ) has been used to calculate the aerosol size distribution from the GloSSAC database. In this method, the surface area density and mass density of very small particles, which are invisible to the satellite extinction measurements, are added to the lognormal size distributions derived from the GloSSAC data. The stratospheric aerosol burden derived from SAGE-3λ for the volcanically quiescent period 2000–2004 is 165 ± 11 (±σ) Gg S. This aerosol bur- den is about 40 % larger than the stratospheric burden calcu- lated from SAGE-4λ, 117±8 Gg S. (Note that this value dif- fers slightly from the value reported in Sheng et al. (2015), 112.5 Gg S, possibly due to different assumptions about the tropopause height.) The addition of small aerosol particles derived from OPC measurements contributes 18 Gg S to the SAGE-3λ. The rest of the increase from SAGE-4λ to SAGE- 3λ can be attributed to the new retrieval methods. 3.1.9 SOCOL-AERv2 and aqueous chemistry in the supercooled liquid fraction Because of the underprediction of the SLF (Fig. S1) and ox- idation of SO2 occurring only in liquid water in SOCOL- AER, the oxidation of SO2 is likely underestimated in the upper troposphere, leading to a too intensive transport of SO2 to the stratosphere. Therefore, in the SOCOL-AERv2 simu- lation we increased the supercooled liquid fraction to agree with the SLF–temperature relationship observed by CALIOP (Hu et al., 2010). This increase in SLF enhances the SO2 oxi- dation rate in the middle and upper tropospheric mixed-phase clouds, reducing the cross-tropopause SO2 ﬂux by around 10 %. However, the impact on the stratospheric aerosol bur- den is minor, with only a reduction of 6 Gg S (−4 %) com- pared to the AQCHEM simulation. Therefore, the underesti- mation of the SLF does not play a major role in SOCOL- AER’s stratospheric sulfur cycle. However, the amount of SO2 oxidation in the upper troposphere may be affected by other processes, e.g. by oxidation on ice surfaces. This will be discussed further in Sect. 3.3. The stratospheric aerosol burden simulated by SOCOL- AERv2, 160 Gg S, agrees well with the SAGE-3λ-derived burden of 165 Gg S. However, evaluating a model’s perfor- mance with the stratospheric aerosol burden is not straight- forward since both SAGE-3λ and SAGE-4λ are themselves derived products and not direct measurements. The retrieval of size distributions from measured SAGE II wavelengths is A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3872 Figure 2. Maps of sulfate wet deposition simulated by SOCOL-AER for the year 2000. Three plots are shown: wet deposition in the DRYDEP simulation, which uses the old wet deposition scheme (a); wet deposition in the WETDEP simulation, which uses the interactive wet deposition scheme (b); and relative percent differences in deposition, which is WETDEP minus DRYDEP (c). Figure 2. Maps of sulfate wet deposition simulated by SOCOL-AER for the year 2000. Three plots are shown: wet deposition in the DRYDEP simulation, which uses the old wet deposition scheme (a); wet deposition in the WETDEP simulation, which uses the interactive wet deposition scheme (b); and relative percent differences in deposition, which is WETDEP minus DRYDEP (c). results change in the new version and where deﬁciencies re- main. tropospheric S to the stratosphere. The stratospheric aerosol burden decreases by 18 % from 202 to 165 Gg S. From sep- arate sensitivity studies (not shown), we ﬁnd that the shorter aqueous chemistry time step is the main cause of the in- creased aqueous ﬂux. Goto et al. (2011) investigated the sen- sitivity of sulfate aqueous chemistry to different settings and also found that reducing the aqueous chemistry time step in- creases the conversion of S(IV) to S(VI). This is because the Henry’s law equilibration rate and aqueous oxidation is fast so with shorter time steps more SO2 can be dissolved in cloud droplets and converted to S(VI). 3.1.8 Aqueous chemistry changes (AQCHEM) In the AQCHEM simulation we amended the cloud pH dis- tribution for aqueous chemistry, reduced the aqueous chem- istry time step to 15 min, and directly transferred oxidized S(IV) to the scavenged sulfate aerosol in the wet deposition scheme. This increases the aqueous oxidation ﬂux of S(IV) to S(VI) by around 50 %. The enhanced aqueous conversion of SO2 to sulfate aerosol leads to increased aerosol formation in the lowermost troposphere, where deposition is efﬁcient. This results in smaller tropospheric burdens of both SO2 and sulfate aerosol, meaning that there is also less transport of www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 3.2.2 Comparison with SAGE II extinctions Figure 3 shows the comparison of annual mean SOCOL- AERv1 and v2 extinctions with SAGE II at the Equator and 45◦N for 525 and 1020 nm. Below 20 km at the Equa- tor, SOCOL-AERv2 shows higher extinctions at both wave- lengths, which match better with the SAGE II observations. However, in the lowest 1–3 km of the stratosphere, organ- ics are a nonnegligible fraction of the overall aerosol bur- den (Murphy et al., 2014) and therefore can contribute to the aerosol extinction observed by SAGE II. If anything, SOCOL-AER as a sulfate aerosol-only model should under- estimate the extinction at these altitudes, although to what extent is unknown. Since SOCOL-AERv2 matches or over- estimates the SAGE II extinctions in the lowermost strato- sphere, SOCOL-AERv2 may have too high sulfate aerosol concentrations in the lower stratosphere. Between 20 and 25 km SOCOL-AERv2 overestimates the SAGE II extinc- tions, while SOCOL-AERv1 matches observations. Between 25 and 30 km both model versions overestimate the SAGE II extinctions. The model versions are within observed variabil- ity between 30 and 35 km; however, above 35 km they tend to underestimate the extinction, possibly because of meteoritic dust, which is the major contributor to extinction in the upper stratosphere (Neely et al., 2011). The comparison at 45◦N is similar to the equatorial comparison with SOCOL-AERv2 overestimating the observed aerosol extinctions below 20 km and otherwise showing similar behavior to SOCOL-AERv1. Si th ti ti f SOCOL AER 2 i th l Figure 3. Comparison between annual mean model extinctions at 525 and 1020 nm and SAGE II measurements from the GloSSAC project (Thomason et al., 2018) at the Equator (a) and 45◦N (b). Observations are averaged between 2000 and 2004, representing the volcanically quiescent part of the record. Model results are av- eraged over 5 years of the year 2000 time slice for SOCOL-AERv1 and SOCOL-AERv2. Horizontal bars represent the modeled or ob- served standard deviation. The highlighted region in the upper plot corresponds to the altitudes where nonsulfate aerosols may play a role. sues remain in SOCOL-AERv2’s representation of sulfate aerosol extinction below 20 km at 45◦N and between 20 and 30 km at the Equator. 3.2.2 Comparison with SAGE II extinctions Since the overestimation of SOCOL-AERv2 in the lower stratosphere originates from the introduction of interactive deposition schemes, it possibly stems from too fast cross- tropopause transport of primary sulfate aerosol and/or SO2, whereas the better agreement of SOCOL-AERv1 may be for- tuitous due to the double counting of the SO2 oxidation ﬂux in the wet-deposition scheme. SOCOL-AERv2 is the version that is more physically consistent in its representation of the tropospheric sulfur cycle. However, several outstanding is- A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3873 Figure 3. Comparison between annual mean model extinctions at 525 and 1020 nm and SAGE II measurements from the GloSSAC project (Thomason et al., 2018) at the Equator (a) and 45◦N (b). Observations are averaged between 2000 and 2004, representing the volcanically quiescent part of the record. Model results are av- eraged over 5 years of the year 2000 time slice for SOCOL-AERv1 and SOCOL-AERv2. Horizontal bars represent the modeled or ob- served standard deviation. The highlighted region in the upper plot corresponds to the altitudes where nonsulfate aerosols may play a role. uncertain, as can be seen when comparing the change be- tween SAGE-3λ and SAGE-4λ stratospheric burdens. In ad- dition, the MERRA climatological tropopause height (Rie- necker et al., 2011) was used to calculate the stratospheric burden for the SAGE-3λ and SAGE-4λ products. However, for SOCOL-AER’s burden, the WMO-deﬁned (World Me- teorological Organization) tropopause height from the model was used (WMO, 1957). Differences between the tropopause heights used in different calculations can play a big role in the derived burden since the majority of the aerosol bur- den is located in the lower stratosphere. For example, if the tropopause height from SOCOL-AER instead of MERRA is used, the stratospheric burdens derived from SAGE-3λ and SAGE-4λ are around 7 % smaller. For these reasons we will evaluate SOCOL-AER with the extinctions measured di- rectly by SAGE II of version GloSSACv1.0, in addition to the derived burdens. 3.2 Comparison of SOCOL-AER versions with stratospheric observations In this section, we will compare the SOCOL-AERv1 and v2 simulations with observations to understand how the model Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ www.geosci-model-dev.net/12/3863/2019/ A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3874 Deshler, 2015; Deshler et al., 2019). In Fig. 4, we apply the measured counting efﬁciencies for the channels r > 0.15, r > 0.25, and r > 0.30 µm from Deshler et al. (2019) to the SOCOL-AER size bins (counting efﬁciencies were not mea- sured for other channels). In this manner, we can calculate the number density that an OPC instrument would measure given a simulated size distribution. SO2 is around 24 pptv at 17 km, substantially higher than the other observations. SO2 is around 24 pptv at 17 km, substantially higher than the other observations. The annual means of SOCOL-AERv1 and v2 agree with MIPAS-observed SO2 and overestimate the three other ob- servation sets at the tropopause, simulating SO2 mixing ra- tios between 20 and 30 pptv at 17 km. MIPAS satellite obser- vations of SO2 under nonvolcanic conditions are uncertain, which may explain the systematic offset between MIPAS SO2 measurements and the other observation sets (Höpfner et al., 2015; Rollins et al., 2017). On the other hand, in situ measurements lack the spatial and temporal coverage of satellites, which reduces their comparability with global models. More aircraft campaigns will be invaluable for de- termining the background level of UTLS SO2. If anything, the currently available observations suggest that SOCOL- AER’s cross-tropopause SO2 transport might be too high. In Sect. 3.3 we will investigate the consequences of an overes- timated UTLS SO2. g SOCOL-AERv2 simulates higher number densities of condensation nuclei (CN, r > 0.01 µm) above 25 km, match- ing the shape of the observed curve better than SOCOL- AERv1. The transport of polar H2SO4-rich air to midlati- tudes during the breakup of the polar vortex may lead to the high number densities of CN above 25 km in the mea- surements (Campbell and Deshler, 2014; Sheng et al., 2015). The improved agreement in SOCOL-AERv2 is due to the implementation of dry radius binning and the improvement in sulfur mass conservation, which enable the model to cap- ture the increased transport of CN to midlatitudes during late winter and spring. SOCOL-AERv2 also displays a kink at the tropopause for particle channels larger than r > 0.15 µm, which appeared after the addition of interactive wet deposi- tion. In the interactive wet deposition scheme, the scaveng- ing efﬁciency of particles depends on radius, which leads to stronger removal of larger aerosol particles in the tropo- sphere. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER Lauder OPC measurements may also show a similar kink at the tropopause for the larger particle channels; how- ever, it is difﬁcult to verify this given the large variability in the measurements (Fig. 4). 3.2.3 Comparison with OPC size distributions We also compare the SOCOL-AER simulations with in situ OPC measurements from Laramie, USA, and Lauder, New Zealand (Fig. 4). Since the publication of Sheng et al. (2015), the counting efﬁciencies of OPC channels as a function of radius have undergone important revisions (Kovilakam and www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 3.3 Observational disagreements with SOCOL-AERv2 To summarize, SOCOL-AERv2 shows similar levels of agreement with stratospheric sulfur observations as SOCOL- AERv1. The stratospheric aerosol burden simulated by SOCOL-AERv2, 160 Gg S, agrees very well with the SAGE- 3λ retrieved burden, 165 Gg S. SOCOL-AERv2 slightly overestimates the SAGE II aerosol extinction in the low- ermost stratosphere at the Equator and in the lowermost stratosphere at 45◦N, namely by up to 25 % at wavelength 1020 nm and by up to 40 % at 525 nm. Since organic particles may contribute to the aerosol burden in the lowest 1–3 km of the stratosphere, we think that SOCOL-AERv2 is actu- ally overestimating the cross-tropopause transport of sulfur. OPC measurements also show that large particle channels (r > 0.25 µm) are overestimated in the UTLS at midlatitudes (by up to a factor of 3). A second region where SAGE II ex- tinctions diverge from the simulated values is between 25 and 30 km at the Equator, where SOCOL-AERv2 overestimates extinctions. Above 35 km at the Equator and above 30 km at 45◦N, SOCOL-AERv2 underestimates aerosol extinctions; however, this is likely caused by a lack of meteoritic material in the model. Otherwise, both model versions show similar levels of agreement with the OPC measurements. All four channels larger than r > 0.25 µm have too high number densities com- pared to observations at Laramie, with the agreement becom- ing even worse with altitude. At Lauder the agreement of the largest three channels (r > 0.30 µm) is better. Sheng et al. (2015) attributed the worsening agreement of larger aerosol particles with altitude to either numerical diffusion in the sedimentation scheme or an overestimate in the speed of the Brewer-Dobson circulation, a known artifact in the SOCOL model (Stenke et al., 2013). A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER It is important to mention that although the agreement in the UTLS was improved by AER-SCAV and ICE-OX, there may be other reasons behind the too high cross-tropopause transport in SOCOL-AERv2. Convective transport of SO2 and aerosol to the upper troposphere may be too strong in SOCOL-AERv2, which is a common problem with other GCMs (Allen and Landuyt, 2014). In this case, chemical ox- idation of SO2 on ice or increased aerosol scavenging, rather than being a missing feature in itself, would be compensating for the strong convective transport. Numerical diffusion may further enhance the SO2 cross-tropopause transport due to the strong vertical gradient at the tropopause. The inﬂuence of convection could be further investigated by testing the sen- sitivity of SOCOL-AER’s sulfur cycle to different convec- tion schemes, as has been done for other models (Tost et al., 2010). The choice of the convective scheme and the order in OX now shows too low number densities of CN in the UTLS compared to OPC measurements, suggesting either that too much SO2 is removed or that other aerosol types contribute to CN at these altitudes. The available SO2 measurements also imply that too much SO2 is removed in ICE-OX since the simulated SO2 concentration at 17 km (∼1 pptv) is lower than the in situ and ACE-FTS values of 5 to 10 pptv (Fig. 5). abilities and surface poisoning during the reaction, make it difﬁcult to extrapolate the measurements to atmospheric conditions (Clegg and Abbatt, 2001). Furthermore, Rotstayn and Lohmann (2002) found improved model agreement with Arctic sulfate measurements when they included SO2 oxida- tion also in ice water. In addition, physical uptake of SO2 without conversion to S(VI) on ice has been observed in the laboratory (Huthwelker et al., 2001) and may lead to grav- itational settling; uptake of SO2 on ice is not considered in either SOCOL-AERv1 or v2. Assuming that SO2 oxidation occurs in cloud ice water at the same rate as cloud liquid wa- ter is likely an upper limit estimate for the scavenging of SO2 on ice. It is important to mention that although the agreement in the UTLS was improved by AER-SCAV and ICE-OX, there may be other reasons behind the too high cross-tropopause transport in SOCOL-AERv2. 3.2.4 Comparison with UTLS SO2 measurements There has been an ongoing debate in the literature regard- ing the magnitude of the cross-tropopause SO2 ﬂux and its relative importance in establishing the Junge layer (Rollins et al., 2018). The debate has been fueled by a lack of in situ measurements in the UTLS region and the high tempo- ral and spatial variability in UTLS SO2. Figure 5 compares the tropical UTLS SO2 measured by two aircraft campaigns (Rollins et al., 2017, 2018) with two annual mean satellite products, MIPAS (Höpfner et al., 2015), and ACE-FTS (Do- eringer et al., 2012), averaged during volcanically quiescent periods. The two in situ measurements and the ACE-FTS satellite product all show SO2 mixing ratios of 5 to 10 pptv around the tropical tropopause (∼17 km). MIPAS-observed To address the possible overestimation of sulfur transport to the stratosphere, we ran two additional simulations, AER- SCAV and ICE-OX. In AER-SCAV, we increased the scav- enging coefﬁcient of aerosol on ice clouds by a factor of 20, from 0.05 to 1, maximizing the effect of upper tropospheric sulfate aerosol removal. In ICE-OX, the ice water content was added to the liquid water content before the aqueous- phase chemistry routine so that SO2 oxidation occurs as well in middle and upper tropospheric ice clouds, maximizing the effect of condensed phase S(IV) to S(VI) oxidation. One lab- oratory study has identiﬁed the SO2 + H2O2 reaction on the surface of ice as a possible sink for SO2, although compli- cating factors, like partial-pressure-dependent reaction prob- www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3875 Figure 4. Number densities of particle size bins measured by OPC (Deshler et al., 2003; Deshler, 2008) and modeled by SOCOL-AERv1 and v2 over Laramie, Wyoming, USA (41◦N, 105◦W), and Lauder, New Zealand (45◦S, 170◦W). Measured number densities are shown as box plots (minimum excluding outliers below the 0.4th percentile, 25th percentile, median, 75th percentile, maximum excluding outliers above the 99.6th percentile) and modeled number densities as solid lines. For the Laramie plots (a), OPC measurements are used from the period 1999–2008 and model results are averaged over the 5 years of the time slice. For the Lauder plots (b), OPC measurements are used from January to April 1998–2001 and zonal mean model results are averaged from January to April over 5 years of the time slice. Model results are weighted with the counting efﬁciencies for OPC channels from Deshler et al. (2019) for direct comparability with the measurements. Figure 4. Number densities of particle size bins measured by OPC (Deshler et al., 2003; Deshler, 2008) and modeled by SOCOL-AERv1 and v2 over Laramie, Wyoming, USA (41◦N, 105◦W), and Lauder, New Zealand (45◦S, 170◦W). Measured number densities are shown as box plots (minimum excluding outliers below the 0.4th percentile, 25th percentile, median, 75th percentile, maximum excluding outliers above the 99.6th percentile) and modeled number densities as solid lines. For the Laramie plots (a), OPC measurements are used from the period 1999–2008 and model results are averaged over the 5 years of the time slice. For the Lauder plots (b), OPC measurements are used from January to April 1998–2001 and zonal mean model results are averaged from January to April over 5 years of the time slice. Model results are weighted with the counting efﬁciencies for OPC channels from Deshler et al. (2019) for direct comparability with the measurements. OX now shows too low number densities of CN in the UTLS compared to OPC measurements, suggesting either that too much SO2 is removed or that other aerosol types contribute to CN at these altitudes. The available SO2 measurements also imply that too much SO2 is removed in ICE-OX since the simulated SO2 concentration at 17 km (∼1 pptv) is lower than the in situ and ACE-FTS values of 5 to 10 pptv (Fig. 5). 3.4.1 Wet deposition The wet deposition scheme in SOCOL-AERv2 is coupled to the climate model’s cloud and precipitation ﬁelds, whereas in SOCOL-AERv1 constant wet deposition lifetimes are ap- plied. SOCOL-AERv1 therefore simulates too large deposi- tion ﬂuxes in dry regions and too small deposition ﬂuxes in wet regions compared to SOCOL-AERv2 (Sect. 3.1.7). To verify the SOCOL-AERv2 wet deposition ﬂuxes in both dry and wet regions, we compare simulated and observed sulfate deposition ﬂuxes over 3 orders of magnitude in Fig. 6. The presentation is logarithmic since using linear axes would give too high a weight to large deposition ﬂuxes, obscuring biases at the lower range of deposition ﬂuxes. Figure 5. Comparing modeled and measured SO2 mixing ratios in the tropical UTLS region between 10 and 25◦N. Modeled re- sults from three simulations are averaged over 5 years of the time slice and are shown as colored dashed lines. Horizontal error bars indicate the interquartile range of monthly means. Observational datasets are shown as solid lines with interquartile ranges, extracted from data in Fig. 3 of Rollins et al. (2017) and Fig. 11 of Rollins et al. (2018). The satellite datasets (triangles), MIPAS and ACE- FTS, show mean SO2 values between 2002–2012 and 2004–2010, respectively, and have been ﬁltered to remove any data affected by major volcanic eruptions. Rollins et al. (2017) data (brown circles) represent in situ ﬂight data from October 2015 over the Gulf of Mexico and the tropical eastern Paciﬁc Ocean. Rollins et al. (2018) data (orange circles) were measured in a ﬂight campaign over the tropical western Paciﬁc Ocean in October 2016. Model and satellite data are averaged over all longitudes between 10 and 25◦N. g p SOCOL-AERv1 indeed overestimates low deposition ﬂuxes (< 1 kg S ha−1), corresponding to sites in drier areas (< 50 cm yr−1 precipitation). For both time periods (2000– 2002 and 2005–2007), SOCOL-AERv2 improves the agree- ment with observations compared to SOCOL-AERv1 (R2 = 0.61 and R2 = 0.69 for SOCOL-AERv2 vs. R2 = 0.51 and R2 = 0.58 for SOCOL-AERv1). The fraction of stations where the model is within a factor of 2 of observations (f2×) also improves slightly for both measurement periods in SOCOL-AERv2. The variability in simulated wet deposi- tion ﬂuxes, shown by the ensemble standard deviation bars in Fig. 6, increases in SOCOL-AERv2 because wet deposi- tion is coupled to modeled precipitation. 3.4.1 Wet deposition Due to the inter- nal variability in modeled precipitation in a free-running cli- mate model, multiple-ensemble simulations as well as long- term deposition measurements are required when comparing models with observations. Overall, SOCOL-AERv2 matches the measurements better than SOCOL-AERv1, especially for sites with low and high deposition ﬂuxes. which it is called relative to the wet deposition routine could be used to further tune SOCOL-AERv2 in the UTLS; how- ever, a clear challenge is the lack of in situ measurements at these altitudes. Further measurements of these species in the UTLS would be helpful to constrain the importance of SO2 and primary sulfate aerosol in establishing the stratospheric aerosol layer. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3876 WMO assessment corrected wet deposition measurements for sea salt contributions of sulfate at sites less than 100 km from coastlines and at all African measurement sites. For our study, we only use the sites where measurement methodol- ogy and temporal data coverage were assessed as “satisfac- tory” or “conditional” in the WMO database. We interpo- lated modeled annual mean deposition to the coordinates of the measurements stations. Several previous model intercom- parison projects that simulated deposition (Dentener et al., 2006a; Lamarque et al., 2013; Vet et al., 2014; Tan et al., 2018) will be used as benchmarks for the performance of SOCOL-AER compared with observations. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER Convective transport of SO2 and aerosol to the upper troposphere may be too strong in SOCOL-AERv2, which is a common problem with other GCMs (Allen and Landuyt, 2014). In this case, chemical ox- idation of SO2 on ice or increased aerosol scavenging, rather than being a missing feature in itself, would be compensating for the strong convective transport. Numerical diffusion may further enhance the SO2 cross-tropopause transport due to the strong vertical gradient at the tropopause. The inﬂuence of convection could be further investigated by testing the sen- sitivity of SOCOL-AER’s sulfur cycle to different convec- tion schemes, as has been done for other models (Tost et al., 2010). The choice of the convective scheme and the order in These extreme simulations succeed in reducing the cross- tropopause sulfur transport, leading to strongly reduced stratospheric aerosol burdens, namely 133 Gg S in AER- SCAV and 92 Gg S in ICE-OX. In these two simulations, ex- tinctions at 45◦N now match observations in the lowermost stratosphere, while equatorial extinctions underestimate ob- servations, which may be reasonable since organic aerosol particles play a role in this level (Fig. S2). Similarly, the mod- eled OPC channels are reduced in number density in AER- SCAV and ICE-OX at midlatitudes (Fig. S3). However, ICE- www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 3876 Figure 5. Comparing modeled and measured SO2 mixing ratios in the tropical UTLS region between 10 and 25◦N. Modeled re- sults from three simulations are averaged over 5 years of the time slice and are shown as colored dashed lines. Horizontal error bars indicate the interquartile range of monthly means. Observational datasets are shown as solid lines with interquartile ranges, extracted from data in Fig. 3 of Rollins et al. (2017) and Fig. 11 of Rollins et al. (2018). The satellite datasets (triangles), MIPAS and ACE- FTS, show mean SO2 values between 2002–2012 and 2004–2010, respectively, and have been ﬁltered to remove any data affected by major volcanic eruptions. Rollins et al. (2017) data (brown circles) represent in situ ﬂight data from October 2015 over the Gulf of Mexico and the tropical eastern Paciﬁc Ocean. Rollins et al. (2018) data (orange circles) were measured in a ﬂight campaign over the tropical western Paciﬁc Ocean in October 2016. Model and satellite data are averaged over all longitudes between 10 and 25◦N. 3876 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3877 Figure 6. Evaluation of modeled total sulfur wet (a, b, e, f) and dry (c, d, g, h) deposition ﬂuxes against measurement sites from the WMO database (Vet et al., 2014). SOCOL-AERv1 and SOCOL-AERv2 are compared with measurements averaged in two different time periods, 2000–2002 and 2005–2007. The ensemble standard deviation for the model results is shown as vertical bars. A power regression between the simulation results and measurements is shown in blue and can be compared to the one-to-one line shown in black. Two model evaluation metrics are listed on the plots: the goodness of ﬁt of the power regression between model and measurements (R2) and the fraction of stations where the model is within a factor of 2 of measurements (f2×). Points are colored according to the region (for the wet deposition plots) or the measurement network (for the dry deposition plots) of the measurement stations. Figure 6. Evaluation of modeled total sulfur wet (a, b, e, f) and dry (c, d, g, h) deposition ﬂuxes against measurement sites from the WMO database (Vet et al., 2014). SOCOL-AERv1 and SOCOL-AERv2 are compared with measurements averaged in two different time periods, 2000–2002 and 2005–2007. The ensemble standard deviation for the model results is shown as vertical bars. A power regression between the simulation results and measurements is shown in blue and can be compared to the one-to-one line shown in black. Two model evaluation metrics are listed on the plots: the goodness of ﬁt of the power regression between model and measurements (R2) and the fraction of stations where the model is within a factor of 2 of measurements (f2×). Points are colored according to the region (for the wet deposition plots) or the measurement network (for the dry deposition plots) of the measurement stations. results. However, the analysis periods for these intercompar- ison projects differed from this study for both the simula- tions and observations (Table S1), which can contribute to the differences in the results. SOCOL-AERv2 shows similar levels of agreement with observations as the previous model intercomparison studies in the European and East Asian re- gions. The model biases, correlation coefﬁcients, and frac- tion of values within ±50 % of measurements fall within or very close to the range of the intercomparison projects. Geosci. Model Dev., 12, 3863–3887, 2019 3.4 Evaluation of SOCOL-AER deposition in transient simulations In order to evaluate the performance of SOCOL-AER ver- sions in the troposphere, we will compare simulated annual mean deposition ﬂuxes with the database compiled for the World Meteorological Organization (WMO) assessment of precipitation chemistry and deposition (Vet et al., 2014). The WMO assessment only included regionally representative sites, e.g., excluding measurements within 50 km of indus- trial or urban areas, which should be comparable to the sim- ulated values in a global model with coarse resolution. The deposition ﬂuxes reported in the WMO assessment were av- eraged in 3-year periods, 2000–2002 and 2005–2007. The Nevertheless, there are remaining biases in the deposition ﬁelds of SOCOL-AERv2, e.g. high biases in many North American sites compared to the WMO observations (Fig. 6 and Table 3). Since the model’s deposition scheme is cou- pled to precipitation ﬁelds, inaccuracies in the modeled pre- cipitation distribution can lead to incorrect deposition ﬂuxes. We calculated the model’s precipitation biases compared to the WMO database for each station. The bias in precipitation depth in SOCOL-AERv2 correlates with the bias that we ﬁnd www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3878 Table 3. Metrics comparing the agreement of simulated sulfate wet deposition with the WMO database, separated by measurement region. Results from SOCOL-AERv2 from two time periods are compared with the range of values from past model intercomparison projects (MIP), including Photocomp (Dentener et al., 2006b), HTAP I (Vet et al., 2014), ACCMIP (Lamarque et al., 2013), and HTAP II (Tan et al., 2018). Note that the observational and simulation time periods covered by the other model intercomparison projects differ from SOCOL-AER in this study (see Table S1 in the Supplement). The metrics are calculated in linear space to conform with past MIPs: linear ﬁt slopes differ from the power regression in Fig. 6 and fractions of sites where the model is within ±50 % of observations differ from the fraction within a factor of 2 listed in Fig. 6. Metric North America Europe East Asia Range of MIPs SOCOL-AERv2 Range of MIPs SOCOL-AERv2 Range of MIPs SOCOL-AERv2 2000 to 2005 to 2000 to 2005 to 2000 to 2005 to 2002 2007 2002 2007 2002 2007 Mean observations 2.5–3.1 3.3 3.1 2.3–4.0 3.8 2.9 6.5–6.9 8.3 7.8 (kg S ha−1) Mean model 2.8–3.6 4.4 4.0 2.0–4.6 3.7 3.5 3.9–5.0 5.8 5.9 (kg S ha−1) Mean bias −0.2 to 0.5 1.1 0.9 −1.3 to 0.5 −0.1 0.6 −2.9 to −1.6 −2.5 −1.9 (kg S ha−1) Linear ﬁt slope 0.6–1.0 1.2 1.1 0.3–0.6 0.5 0.6 0.3–0.5 0.4 0.4 (observations vs. model) Pearson correlation 0.8–0.9 0.8 0.8 0.6–0.7 0.6 0.5 0.6–0.9 0.8 0.7 coefﬁcient, R Fraction of sites 70–77 48 60 53–86 72 61 69–88 66 73 within ±50 % (%) ulations (Tan et al., 2018) and SOCOL-AER was run at a relatively coarse resolution (2.8◦× 2.8◦) compared to many of the models used in the intercomparison project. Finally, three of the intercomparison projects (Photocomp, HTAP I, and HTAP II) were based on ofﬂine chemistry-transport models that are run with observed meteorology. On the other hand, SOCOL-AER is used in free-running mode, pro- ducing ﬁve ensemble members that are subsequently aver- aged. SOCOL-AER, and its parent climate model ECHAM5, include precipitation biases that impact the simulation of deposition. Considering these aspects, the performance of SOCOL-AERv2 compares well with state-of-the-art sulfate wet deposition models. 50 % higher than those of the CASTNET network (Schwede et al., 2011). A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER The inferred dry deposition ﬂuxes are therefore less reliable for comparisons with models than the measured wet deposition ﬂuxes. p Figure 6 displays the comparison between SOCOL-AER versions and the total sulfur (sum of SO2 and aerosol) in- ferred dry deposition ﬂuxes from North American measure- ment networks. Despite the systematic bias between the CAPMoN and CASTNET networks, we calculate the power regression, f2×, and the R2 value based on the combined dataset of both networks. We take this approach as it is un- clear which network’s dry deposition velocity calculation is more accurate (Wu et al., 2018). SOCOL-AERv2, with its new interactive dry deposition scheme, shows a similar correlation with the observations as SOCOL-AERv1. How- ever, SOCOL-AERv2’s improved deposition scheme simu- lates much more realistic slopes of the correlation lines and higher fractions of model results within a factor of 2 of ob- servations. The improved agreement of SOCOL-AERv2 is likely caused by the better spatial representation of SO2 dry deposition velocities in the interactive scheme. The modeled dry deposition ﬂuxes can also be affected by the new wet deposition scheme since enhanced sulfur removal through wet deposition leaves less sulfur available for dry deposi- tion, and vice versa. Similar to the wet deposition compar- A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER In North America, SOCOL-AERv2 correlates similarly with observations (Pearson correlation coefﬁcient, R = 0.8); how- ever, biases and the fraction within ±50 % are worse than the past intercomparison projects. As mentioned above, the North American deposition ﬂuxes may be affected by inac- curacies in our model’s precipitation ﬁelds and/or errors in the anthropogenic SO2 emission inventory. In addition, there are several factors that advantage the model-intercomparison projects compared to the SOCOL-AER runs. Firstly, we have compared ranges of the multimodel means and not the in- dividual model values from the intercomparison projects, which likely have a wider spread and individually worse per- formance. Secondly, model resolution can play an impor- tant role in the comparison between observations and sim- for sulfate deposition ﬂuxes (Spearman’s correlation coefﬁ- cient, ρ = 0.5, Fig. S4). This ﬁnding suggests that some of the model biases can be explained by errors in precipitation ﬁelds rather than errors in the wet deposition scheme. Both versions of SOCOL-AER match the observations better in the period 2005–2007 rather than 2000–2002. Since there are no large differences in the precipitation biases between these periods, this improvement could be related to more accurate SO2 emission maps for the 2005–2007 period. One known issue with the CEDS anthropogenic SO2 inventory is that the emissions from the western United States are overestimated compared to the eastern United States (Hoesly et al., 2018). SOCOL-AERv2 also shows higher deposition biases in the western United States. Since errors in emission inventories and model precipitation ﬁelds impact the evaluation of the deposition ﬁeld, it is difﬁcult to attribute errors to the depo- sition scheme itself. Table 3 compares the performance of SOCOL-AERv2 to past model intercomparison studies, including Photocomp (Dentener et al., 2006a), ACCMIP (Lamarque et al., 2013), HTAP I (Vet et al., 2014), and HTAP II (Tan et al., 2018). These intercomparison projects used observational data from the same networks as in the WMO database to evaluate their www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER sions switched off (orange and grey lines in Fig. 7). This experiment shows that by the end of 1995, when almost all the volcanic aerosol is already removed from the atmosphere, SOCOL-AERv1 deposited only 6.4 Tg S, while in SOCOL- AERv2 this is now improved to 6.7 Tg S, i.e., the volcanic sulfur mass loss decreased from 8.6 % to 4.2 %. The remain- ing part of the mass loss is likely due to limitations of the transport scheme (Stenke et al., 2013). Overall, in SOCOL- AERv2 the Mt. Pinatubo emission estimate required for the model to reproduce the observed burden peak is still between 6 and 7 Tg S, given the large observational uncertainty. ison, the model performs better in 2005–2007 compared to 2000–2002, suggesting that the North American emission inventories may be biased in the earlier time period. Addi- tional comparisons for the SO2 and aerosol dry deposition ﬂuxes with observations show better agreement for SOCOL- AERv2 than SOCOL-AERv1 (Fig. S5 and S6). g Compared to past model-intercomparison projects for to- tal sulfur dry deposition, SOCOL-AERv2 simulates a lower bias and a higher fraction of model values within ±50 % of the observation sites (Table 4). The agreement nevertheless remains poor with only 19 % to 28 % of the modeled total dry deposition ﬂuxes within ±50 % of the observations. Aerosol dry deposition biases are larger in SOCOL-AER compared to past model intercomparison projects. However, since SO2 dominates the dry deposition ﬂux (compare Figs. S5 and S6), the reduced biases in the SO2 deposition ﬂuxes in SOCOL- AERv2 leads to overall lower total sulfur dry deposition biases. It is difﬁcult to conclude whether the observations should actually be the target for the model in this case since dry deposition ﬂuxes are inferred values with inher- ent uncertainties. As observational networks improve their parametrizations for deriving the dry deposition ﬂux, they will become more reliable standards with which to compare modeled results. However, the similar, if not better, agree- ment of SOCOL-AERv2 compared to sulfur dry deposition from model-intercomparison studies adds conﬁdence to the implemented dry deposition scheme. 3.6 Updated nonvolcanic sulfur budget for the year 2000 Figure 8 shows an update from SOCOL-AERv2 for the at- mospheric sulfur budget under volcanically quiescent back- ground conditions. The model was run with repeating bound- ary conditions for the year 2000, with the updated sulfur emission datasets (Sect. 2.2). Due to higher emissions in the updated sulfur emission datasets, the stratospheric aerosol burden shown in Fig. 8 (167 Gg S) is slightly larger than the burden for the SOCOL-AERv2 run in Table 2 (160 Gg S). We have added diagnostics to track tracer ﬂuxes within the model’s planetary boundary layer (PBL), expanding from the budget ﬁgure from SOCOL-AERv1 (Fig. 3 in Sheng et al., 2015). For these calculations, we extracted the mod- eled height of the PBL from the ECHAM5 vertical diffusion routines. In Fig. 8, italicized ﬂuxes are calculated from the other outputted ﬂuxes assuming steady-state conditions, i.e., that the ﬂuxes into/from a species add up to zero. The ﬂuxes in the PBL do not fully balance due to failures in the steady- state assumption, difﬁculties in extracting chemical ﬂuxes from the iterative chemical solver, or remaining mass con- servation errors in the model. However, the imbalances in the PBL are at most 4 % of the total input ﬂuxes in the PBL for each species. Therefore, the outputted ﬂuxes shown in Fig. 8 can be considered reliable. 3.4.2 Dry deposition Dry deposition ﬂuxes compiled by the WMO assessment are based on North American stations from the Canadian Air and Precipitation Monitoring Network (CAPMoN) and the US Clean Air Status and Trends Network (CASTNET). Dry deposition ﬂuxes are not measured directly but are inferred through surface-based measurements of gas and particle con- centrations and estimates of their dry deposition velocities (Wesely and Hicks, 2000; Vet et al., 2014). The estimation of dry deposition velocities is uncertain; the SO2 dry deposition velocities calculated by the CAPMoN network are around www.geosci-model-dev.net/12/3863/2019/ www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 3879 3.5 Pinatubo simulation with SOCOL-AERv2 The eruption of Mt. Pinatubo in 1991 remains the strongest directly observed volcanic event, which makes it a valuable test for models. Sukhodolov et al. (2018) analyzed the per- formance of SOCOL-AERv1 for this case in a series of sensitivity experiments, demonstrating reasonable agreement with observations of different aerosol parameters. Figure 7 shows the SOCOL-AERv1 and v2 global total stratospheric aerosol burden for emission estimates of 6 and 7 Tg S com- pared to HIRS, SAGE-3λ (v4), and SAGE-4λ. The same Mt. Pinatubo emission estimates modeled with SOCOL-AERv2 show clear differences compared to SOCOL-AERv1, ex- pressed in both the shape of the peak and its magnitude. SOCOL-AERv2 is ∼0.4 Tg S higher at the peak values in late 1991 but lower at the tail after mid-1992. The main rea- sons for a narrower and stronger peak in SOCOL-AERv2 are the changes to the chemistry scheme for the CCMI simu- lation and the update of reaction coefﬁcients to Burkholder et al. (2015) recommendations (Sect. 2.1.4), which led to higher OH concentrations in the UTLS. In SOCOL-AERv2, the oxidation of SO2 is therefore faster, causing faster aerosol formation and its earlier removal from the stratosphere. An- other change that contributed to the differences is the im- proved sulfur mass conservation. This effect is illustrated by the integrated total sulfur deposition in the two model ver- sions, after the emission of 7 Tg S with all other sulfur emis- Figure 8 reveals differences between the tropospheric sul- fur cycle in the PBL and the free troposphere. OCS, MSA, and sulfate aerosol all show higher burdens in the free tro- posphere compared to the PBL since these species are pro- duced chemically in the atmosphere and/or they are long- lived species. On the other hand, several sulfur species (CS2, DMS, H2S, and SO2) have higher burdens in the boundary layer compared to the free troposphere, despite the free tro- posphere having more volume. Emission from the surface is the most important atmospheric source for these species. Still, free tropospheric SO2 is supplied not only by cross- PBL transport (67 %) but also through transport and oxi- dation of short-lived species (mainly DMS and H2S). This result supports another modeling study that highlighted the transport pathway of DMS to the upper troposphere by deep convection over the ocean (Marandino et al., 2013). Aqueous-phase SO2 oxidation dominates oxidation of SO2 www.geosci-model-dev.net/12/3863/2019/ Geosci. www.geosci-model-dev.net/12/3863/2019/ A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3880 Table 4. Metrics comparing the agreement of simulated dry deposition with the WMO database in North America, separated by measurement quantity (total sulfur, SO2, and sulfate aerosol). Results from SOCOL-AERv2 from two time periods are compared with the range of values from past model intercomparison projects (MIP), including HTAP I (Vet et al., 2014), ACCMIP (Lamarque et al., 2013), and HTAP II (Tan et al., 2018). Note that the observational and simulation time periods covered by the other model intercomparison projects differ from SOCOL-AER in this study (see Table S1). The metrics are calculated in linear space to conform with past MIPs: linear ﬁt slopes differ from the power regression in Fig. 6 and fractions of sites where the model is within ±50 % of observations differ from the fraction within a factor of 2 of observations listed in Fig. 6. ( , ) p y p p j SOCOL-AER in this study (see Table S1). The metrics are calculated in linear space to conform with past MIPs: linear ﬁt slopes differ from the power regression in Fig. 6 and fractions of sites where the model is within ±50 % of observations differ from the fraction within a factor of 2 of observations listed in Fig. 6. Metric Total sulfur dry deposition SO2 dry deposition Sulfate aerosol dry deposition Range of MIPs SOCOL-AERv2 Range of MIPs SOCOL-AERv2 Range of MIPs SOCOL-AERv2 2000 to 2005 to 2000 to 2005 to 2000 to 2005 to 2002 2007 2002 2007 2002 2007 Mean observations 1.1–2.3 2.5 2.0 0.8–1.9 2.0 1.6 0.2–0.4 0.4 0.4 (kg S ha−1) Mean model 3.6–5.1 4.9 3.8 3.2–4.6 3.8 2.9 0.4–0.5 1.1 0.9 (kg S ha−1) Mean bias 2.5–3.7 2.4 1.7 2.4–2.6 1.7 1.2 0.1–0.2 0.7 0.5 (kg S ha−1) Linear ﬁt slope 1.0–2.7 1.3 1.1 1.0–2.7 1.1 0.9 1.0–1.6 2.1 2.0 (observations vs. model) Pearson correlation 0.8 0.8 0.8 0.8 0.8 0.8 0.8–0.9 0.6 0.6 coefﬁcient, R Fraction of sites 6–13 19 28 5–6 22 31 47–48 19 29 within ±50 % (%) Figure 7. Time evolution of the globally averaged stratospheric aerosol burden calculated by SOCOL-AERv1 (Fig. 1a from Sukhodolov et al., 2018) and SOCOL-AERv2 compared with the HIRS and SAGE II-derived data (SAGE-4λ and SAGE-3λv4). Light blue shaded area: uncertainties in HIRS. Black shaded area: 2σ ﬁve-member ensemble spread of one of the model experiments; others are shown as ensemble means. 3.5 Pinatubo simulation with SOCOL-AERv2 Model Dev., 12, 3863–3887, 2019 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER Model experiments are performed with two emission estimates: 6 and 7 Tg S. Orange and grey lines represent the accumulated (acc) globally integrated deposition of sulfur emitted from Pinatubo. The accumulated deposition from Pinatubo is calculated from simulations where all sulfur sources other than Pinatubo are turned off. Metric Total sulfur dry deposition SO2 dry deposition Sulfate aerosol dry deposition Range of MIPs SOCOL-AERv2 Range of MIPs SOCOL-AERv2 Range of MIPs SOCOL-AERv2 2000 to 2005 to 2000 to 2005 to 2000 to 2005 to 2002 2007 2002 2007 2002 2007 Mean observations 1.1–2.3 2.5 2.0 0.8–1.9 2.0 1.6 0.2–0.4 0.4 0.4 (kg S ha−1) Mean model 3.6–5.1 4.9 3.8 3.2–4.6 3.8 2.9 0.4–0.5 1.1 0.9 (kg S ha−1) Mean bias 2.5–3.7 2.4 1.7 2.4–2.6 1.7 1.2 0.1–0.2 0.7 0.5 (kg S ha−1) Linear ﬁt slope 1.0–2.7 1.3 1.1 1.0–2.7 1.1 0.9 1.0–1.6 2.1 2.0 (observations vs. model) Pearson correlation 0.8 0.8 0.8 0.8 0.8 0.8 0.8–0.9 0.6 0.6 coefﬁcient, R Fraction of sites 6–13 19 28 5–6 22 31 47–48 19 29 within ±50 % (%) Figure 7. Time evolution of the globally averaged stratospheric aerosol burden calculated by SOCOL-AERv1 (Fig. 1a from Sukhodolov et al., 2018) and SOCOL-AERv2 compared with the HIRS and SAGE II-derived data (SAGE-4λ and SAGE-3λv4). Light blue shaded area: uncertainties in HIRS. Black shaded area: 2σ ﬁve-member ensemble spread of one of the model experiments; others are shown as ensemble means. Model experiments are performed with two emission estimates: 6 and 7 Tg S. Orange and grey lines represent the accumulated (acc) globally integrated deposition of sulfur emitted from Pinatubo. The accumulated deposition from Pinatubo is calculated from simulations where all sulfur sources other than Pinatubo are turned off. www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3881 Figure 8. Atmospheric sulfur budget from SOCOL-AERv2 under volcanically quiescent conditions for the year 2000. The ﬁgure is produced using output diagnostics that track sulfur ﬂuxes and burdens within the planetary boundary layer (PBL), free troposphere, and stratosphere. Solid arrows show net ﬂuxes and dashed arrows show one-way ﬂuxes, all in gigagrams of sulfur per year (Gg S yr−1). Simulated burdens of sulfur species are given within the boxes, in units of gigagrams of sulfur (Gg S). Italicized numbers represent ﬂuxes that are derived by balancing other ﬂuxes, assuming steady state of the upper layers. For example, net cross-tropopause ﬂuxes are calculated by balancing the stratospheric chemical ﬂuxes and net cross-PBL ﬂuxes are calculated by balancing the free tropospheric chemical ﬂuxes. Upward OCS and sulfate aerosol cross-tropopause ﬂuxes are calculated based on the residual meridional and vertical air velocities (v∗and w∗) and concentrations at the tropopause. Black numbers: SOCOL-AERv2 results. Red numbers: (a) SAGE-3λ stratospheric aerosol burden (b) DMS emissions estimated by (Lana et al., 2011) (c) dry deposition ﬂuxes from NCAR-CAM3.5 model (Lamarque et al., 2012) for the year 2000, which participated in ACCMIP (d) multimodel mean wet deposition from ACCMIP models for the year 2000 (Lamarque et al., 2013). Figure 8. Atmospheric sulfur budget from SOCOL-AERv2 under volcanically quiescent conditions for the year 2000. The ﬁgure is produced using output diagnostics that track sulfur ﬂuxes and burdens within the planetary boundary layer (PBL), free troposphere, and stratosphere. Solid arrows show net ﬂuxes and dashed arrows show one-way ﬂuxes, all in gigagrams of sulfur per year (Gg S yr−1). Simulated burdens of sulfur species are given within the boxes, in units of gigagrams of sulfur (Gg S). Italicized numbers represent ﬂuxes that are derived by balancing other ﬂuxes, assuming steady state of the upper layers. For example, net cross-tropopause ﬂuxes are calculated by balancing the stratospheric chemical ﬂuxes and net cross-PBL ﬂuxes are calculated by balancing the free tropospheric chemical ﬂuxes. Upward OCS and sulfate aerosol cross-tropopause ﬂuxes are calculated based on the residual meridional and vertical air velocities (v∗and w∗) and concentrations at the tropopause. Black numbers: SOCOL-AERv2 results. www.geosci-model-dev.net/12/3863/2019/ Red numbers: (a) SAGE-3λ stratospheric aerosol burden (b) DMS emissions estimated by (Lana et al., 2011) (c) dry deposition ﬂuxes from NCAR-CAM3.5 model (Lamarque et al., 2012) for the year 2000, which participated in ACCMIP (d) multimodel mean wet deposition from ACCMIP models for the year 2000 (Lamarque et al., 2013). 2015). With the modiﬁcations that were made in this pa- per, SOCOL-AERv2 now simulates a ﬂux of 228 Gg S yr−1 into stratospheric aerosol. Of this ﬂux, 46 % is due to up- ward transport of primary tropospheric aerosol, 27 % due to SO2, 21 % due to OCS, 4 % due to DMS, and 1 % due to H2S. These contributions are very similar to the contribu- tions reported for SOCOL-AERv1 (Sheng et al., 2015). As discussed in Sect. 3.3, the AER-SCAV and ICE-OX runs sug- gest that too much SO2 and tropospheric aerosol are trans- ported across the tropopause in SOCOL-AERv2. Future im- provements of convection schemes and increased availability of reliable observational data will further constrain the ac- curacy of the SO2 contribution to the stratospheric aerosol layer. in both parts of the troposphere in SOCOL-AERv2 (74 % of total SO2 oxidation in the PBL and 68 % in the free tro- posphere). Sulfate aerosol is produced in the PBL through condensation of H2SO4 on existing particles and aqueous- phase oxidation of SO2, with negligible nucleation ﬂuxes. In the upper troposphere, nucleation of new H2SO4-H2O droplets becomes more important, although growth of ex- isting particles remains the largest mass ﬂux to the parti- cle phase. Figure 8 shows a large downward ﬂux of aerosol from the free troposphere to the PBL (18 Tg S yr−1). This balanced ﬂux does not represent only gravitational sedimen- tation to the PBL but rather mostly wet removal of free tro- pospheric aerosol to the surface. The PBL diagnostics in SOCOL-AERv2 are a useful tool for understanding transport and transformation of sulfur species in the troposphere. SOCOL-AERv1 calculated a total ﬂux to stratospheric aerosol of 181 Gg S yr−1 by summing the net cross- tropopause ﬂuxes of gaseous sulfur species and the upward cross-tropopause ﬂux of primary sulfate aerosol (Sheng et al., 4 Conclusions For SOCOL-AER to be used to study the tropospheric sulfur cycle, as well as the deposition response to vol- www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER The supplement related to this article is available on- line at: https://doi.org/10.5194/gmd-12-3863-2019-supplement. Author contributions. AF conducted most of the simulations, ana- lyzed the data, and drafted the paper. TS conducted and analyzed the simulations for the post-Pinatubo period. AF was supervised di- rectly by LW, TP, and AS during the model development work. All authors participated in the model development, discussions about the results, and revisions to the article. Competing interests. The authors declare that they have no conﬂict of interest. Acknowledgements. Thanks to Jianxiong Sheng for providing plot- ting codes to analyze SOCOL-AER results and for providing fur- ther information about SOCOL-AERv1. Thanks to Debra Weisen- stein for discussions about the original AER code. Thanks to Jean-François Lamarque for providing the sulfur deposition data for the NCAR-CAM3.5 model. We acknowledge the researchers behind the GloSSAC SAGE II data and the OPC measure- ments for use of their data. We also gratefully acknowledge the sources of precipitation chemistry and deposition data ac- knowledged on p. 92 of Vet et al. (2014), Atmos. Environ., 93, https://doi.org/10.1016/j.atmosenv.2013.10.060. Color tables from ColorBrewer 2.0 (http://colorbrewer2.org/#t) were used for the ﬁg- ures in this paper. The model developments presented here increase the ap- plicability of SOCOL-AER to scientiﬁc questions in both the troposphere and stratosphere. Namely, due to its im- proved deposition ﬂuxes, SOCOL-AERv2 is more suitable for comparison with ice-core-derived magnitudes of past vol- canic eruptions and their atmospheric impacts (e.g., Marshall et al., 2018); modeling the atmospheric budget of cosmo- genic isotopes, which attach to sulfate aerosols (Delaygue et al., 2015); and studying future changes to sulfur deposi- tion relevant to agriculture and ecosystems (e.g., Vet et al., 2014). With its updated chemistry and improved sulfur mass conservation compared to SOCOL-AERv1, SOCOL-AERv2 is more reliable for studying the impacts of volcanic erup- tions and stratospheric sulfate geoengineering. Financial support. This research has been supported by the ETH Zurich (grant no. ETH-39 15-2). Timofei Sukhodolov and Eugene Rozanov have been supported from the Swiss National Science Foundation (VEC (grant no. 200021-169241) and POLE (grant no. 200020_182239)). Review statement. This paper was edited by Samuel Remy and re- viewed by Claudia Timmreck and one anonymous referee. Geosci. Model Dev., 12, 3863–3887, 2019 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3882 canic eruptions, we implemented new features and ap- plied several corrections to the code. Compared to SOCOL- AERv1, the implemented interactive deposition schemes in SOCOL-AERv2 result in much improved agreement with measurements from sulfur deposition networks. Our tests for Pinatubo showed that SOCOL-AERv2 now gives more aerosol mass in 1991 due to faster SO2 oxidation. Better sulfur mass conservation allowed us to decrease the sulfur mass loss after Pinatubo from 8.6 % to 4.2 %. With the im- proved mass conservation in SOCOL-AERv2, we are also able to separate free tropospheric ﬂuxes from PBL ﬂuxes in the atmospheric sulfur budget, revealing that short-lived sul- fur species (DMS and H2S) contribute strongly to SO2 in the free troposphere. With respect to stratospheric aerosol observations, SOCOL-AERv2 shows similar levels of agree- ment as SOCOL-AERv1. The modeled estimate for the bur- den of background stratospheric aerosol has increased from 116 Gg S in SOCOL-AERv1 to 160 Gg S in SOCOL-AERv2. At the same time, the burden derived from SAGE extinc- tions and OPC measurements increased from the SAGE-4λ estimate of 117 Gg S by 40 % to the most recent estimate from the SAGE-3λ dataset of 165 Gg S. Given the uncer- tainty in the SAGE-3λ and SAGE-4λ estimates, this might be to some degree fortuitous. Therefore, it is more reliable to compare the model with the satellite extinction measure- ments directly. Aerosol extinctions in the lower stratosphere are overestimated by SOCOL-AERv2 more than SOCOL- AERv1. We speculate that this might be related to inaccura- cies in the model’s convective transport scheme, which were also present in SOCOL-AERv1 but compensated by double counting of the SO2 aqueous oxidation ﬂux. Nevertheless, uncertain processes related to SO2 and aerosol scavenging by ice clouds could also lead to overestimation of lower strato- spheric sulfate aerosol. Comparison with other models in the background experiments of the Interactive Stratospheric Aerosol Model Intercomparison Project (ISA-MIP) (Timm- reck et al., 2018) may help to identify the cause of SOCOL- AERv2’s overestimated lower stratospheric aerosol. canic eruptions, we implemented new features and ap- plied several corrections to the code. Compared to SOCOL- AERv1, the implemented interactive deposition schemes in SOCOL-AERv2 result in much improved agreement with measurements from sulfur deposition networks. Our tests for Pinatubo showed that SOCOL-AERv2 now gives more aerosol mass in 1991 due to faster SO2 oxidation. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER Better sulfur mass conservation allowed us to decrease the sulfur mass loss after Pinatubo from 8.6 % to 4.2 %. With the im- proved mass conservation in SOCOL-AERv2, we are also able to separate free tropospheric ﬂuxes from PBL ﬂuxes in the atmospheric sulfur budget, revealing that short-lived sul- fur species (DMS and H2S) contribute strongly to SO2 in the free troposphere. With respect to stratospheric aerosol observations, SOCOL-AERv2 shows similar levels of agree- ment as SOCOL-AERv1. The modeled estimate for the bur- den of background stratospheric aerosol has increased from 116 Gg S in SOCOL-AERv1 to 160 Gg S in SOCOL-AERv2. At the same time, the burden derived from SAGE extinc- tions and OPC measurements increased from the SAGE-4λ estimate of 117 Gg S by 40 % to the most recent estimate from the SAGE-3λ dataset of 165 Gg S. Given the uncer- tainty in the SAGE-3λ and SAGE-4λ estimates, this might be to some degree fortuitous. Therefore, it is more reliable to compare the model with the satellite extinction measure- ments directly. Aerosol extinctions in the lower stratosphere are overestimated by SOCOL-AERv2 more than SOCOL- AERv1. We speculate that this might be related to inaccura- cies in the model’s convective transport scheme, which were also present in SOCOL-AERv1 but compensated by double counting of the SO2 aqueous oxidation ﬂux. Nevertheless, uncertain processes related to SO2 and aerosol scavenging by ice clouds could also lead to overestimation of lower strato- spheric sulfate aerosol. Comparison with other models in the background experiments of the Interactive Stratospheric Aerosol Model Intercomparison Project (ISA-MIP) (Timm- reck et al., 2018) may help to identify the cause of SOCOL- AERv2’s overestimated lower stratospheric aerosol. Code and data availability. Since the SOCOL-AER code is based on ECHAM5, users must ﬁrst sign the ECHAM5 license agree- ment before accessing the SOCOL-AER code (http://www.mpimet. mpg.de/en/science/models/license/, last access: 22 August 2019). SOCOL-AER code is then freely available upon request from the authors. The simulation data presented in this paper are avail- able at https://doi.org/10.3929/ethz-b-000342078 (Feinberg et al., 2019). SAGE II data from the GloSSAC database can be found on- line at https://doi.org/10.5067/GloSSAC-L3-V1.0. The OPC mea- surements from the University of Wyoming were obtained at ftp: //cat.uwyo.edu/pub/permanent/balloon/Aerosol_InSitu_Meas/ (last access: 22 August 2019). Deposition ﬂux measurements can be downloaded online from the World Data Centre for Precipitation Chemistry at http://www.wdcpc.org/global-assessment-data (last access: 22 August 2019). Supplement. Geosci. Model Dev., 12, 3863–3887, 2019 References J., and Tost, H.: The role of carbonyl sulphide as a source of stratospheric sulphate aerosol and its impact on climate, Atmos. Chem. Phys., 12, 1239–1253, https://doi.org/10.5194/acp-12-1239-2012, 2012. Burkholder, J., Sander, S., Abbatt, J., Barker, J., Huie, R., Kolb, C., Kurylo, M., Orkin, V., Wilmouth, D., and Wine, P.: Chem- ical Kinetics and Photochemical Data for Use in Atmospheric Studies, Evaluation No. 18, Tech. rep., Jet Propulsion Labora- tory, Pasadena, available at: https://jpldataeval.jpl.nasa.gov/pdf/ JPL_Publication_15-10.pdf (last access: 9 July 2019), 2015. Dentener, F. J. and Crutzen, P. J.: Reaction of N2O5 on tropospheric aerosols: Impact on the global distributions of NOx, O3, and OH, J. Geophys. Res., 98, 7149–7163, https://doi.org/10.1029/92JD02979, 1993. Deshler, T.: A review of global stratospheric aerosol: Measurements, importance, life cycle, and local stratospheric aerosol, Atmos. Res., 90, 223–232, https://doi.org/10.1016/j.atmosres.2008.03.016, 2008. Campbell, P. and Deshler, T.: Condensation nuclei measurements in the midlatitude (1982–2012) and Antarctic (1986–2010) strato- sphere between 20 and 35 km, J. Geophys. Res.-Atmos., 119, 137–152, https://doi.org/10.1002/2013JD019710, 2014. Deshler, T., Hervig, M., Hofmann, D., Rosen, J., and Liley, J.: Thirty years of in situ stratospheric aerosol size distri- bution measurements from Laramie, Wyoming (41◦N), us- ing balloon-borne instruments, J. Geophys. Res., 108, 4167, https://doi.org/10.1029/2002JD002514, 2003. Carn, S., Clarisse, L., and Prata, A.: Multi-decadal satellite mea- surements of global volcanic degassing, J. Volcanol. Geoth. Res., 311, 99–134, https://doi.org/10.1016/j.jvolgeores.2016.01.002, 2016. Cesana, G., Waliser, D., Jiang, X., and Li, J.: Multimodel evaluation of cloud phase transition using satellite and re- analysis data, J. Geophys. Res.-Atmos., 120, 7871–7892, https://doi.org/10.1002/2014JD022932, 2015. Deshler, T., Luo, B., Kovilakam, M., Peter, T., and Kalnajs, L. E.: Retrieval of aerosol size distributions from in situ particle counter measurements: instrument counting efﬁciency and comparisons with satellite measurements, J. Geophys. Res.-Atmos., 124, 5058–5087, https://doi.org/10.1029/2018JD029558, 2019. Chang, J., Brost, R., Isaksen, I., Madronich, S., Middleton, P., Stockwell, W., and Walcek, C.: A three-dimensional Eulerian acid deposition model: Physical concepts and formulation, J. Geophys. Res., 92, 14681–14700, https://doi.org/10.1029/jd092id12p14681, 1987. Diehl, T., Heil, A., Chin, M., Pan, X., Streets, D., Schultz, M., and Kinne, S.: Anthropogenic, biomass burning, and volcanic emis- sions of black carbon, organic carbon, and SO2 from 1980 to 2010 for hindcast model experiments, Atmos. Chem. Phys. Dis- cuss., 12, 24895–24954, https://doi.org/10.5194/acpd-12-24895- 2012, 2012. Chin, M. and Davis, D.: A reanalysis of carbonyl sulﬁde as a source of stratospheric background sulfur aerosol, J. Geophys. Res., 100, 8993–9005, https://doi.org/10.1029/95jd00275, 1995. References Clegg, S. M. and Abbatt, J. P. D.: Oxidation of SO2 by H2O2 on ice surfaces at 228 K: a sink for SO2 in ice clouds, Atmos. Chem. Phys., 1, 73–78, https://doi.org/10.5194/acp-1-73-2001, 2001. Allen, R. J. and Landuyt, W.: The vertical distribution of black car- bon in CMIP5 models: Comparison to observations and the im- portance of convective transport, J. Geophys. Res.-Atmos., 119, 4808–4835, https://doi.org/10.1002/2014jd021595, 2014. Commane, R., Herndon, S., Zahniser, M., Lerner, B., Mc- Manus, J., Munger, J., Nelson, D., and Wofsy, S.: Carbonyl sulﬁde in the planetary boundary layer: Coastal and conti- nental inﬂuences, J. Geophys. Res.-Atmos., 118, 8001–8009, https://doi.org/10.1002/jgrd.50581, 2013. Andres, R. and Kasgnoc, A.: A time-averaged inventory of subaerial volcanic sulfur emissions, J. Geophys. Res., 103, 25251–25261, https://doi.org/10.1029/98jd02091, 1998. Crutzen, P. J.: The possible importance of CSO for the sul- fate layer of the stratosphere, Geophys. Res. Lett., 3, 73–76, https://doi.org/10.1029/GL003i002p00073, 1976. Arfeuille, F., Luo, B. P., Heckendorn, P., Weisenstein, D., Sheng, J. X., Rozanov, E., Schraner, M., Brönnimann, S., Thoma- son, L. W., and Peter, T.: Modeling the stratospheric warm- ing following the Mt. Pinatubo eruption: uncertainties in aerosol extinctions, Atmos. Chem. Phys., 13, 11221–11234, https://doi.org/10.5194/acp-13-11221-2013, 2013. Delaygue, G., Bekki, S., and Bard, E.: Modelling the strato- spheric budget of beryllium isotopes, Tellus B, 67, 28582, https://doi.org/10.3402/tellusb.v67.28582, 2015. Dentener, F., Drevet, J., Lamarque, J.-F., Bey, I., Eickhout, B., Fiore, A. M., Hauglustaine, D., Horowitz, L. W., Krol, M., and Kulshrestha, U.: Nitrogen and sulfur deposition on regional and global scales: A multimodel evaluation, Global Biogeochem. Cy., 20, GB4003, https://doi.org/10.1029/2005GB002672, 2006a. Ayers, G., Gillett, R., and Gras, J.: On the vapor pres- sure of sulfuric acid, Geophys. Res. Lett., 7, 433–436, https://doi.org/10.1029/GL007i006p00433, 1980. Baran, A. and Foot, J.: New application of the operational sounder HIRS in determining a climatology of sulphuric acid aerosol from the Pinatubo eruption, J. Geophys. Res., 99, 25673–25679, https://doi.org/10.1029/94jd02044, 1994. Dentener, F., Kinne, S., Bond, T., Boucher, O., Cofala, J., Gen- eroso, S., Ginoux, P., Gong, S., Hoelzemann, J. J., Ito, A., Marelli, L., Penner, J. E., Putaud, J.-P., Textor, C., Schulz, M., van der Werf, G. R., and Wilson, J.: Emissions of primary aerosol and precursor gases in the years 2000 and 1750 pre- scribed data-sets for AeroCom, Atmos. Chem. Phys., 6, 4321– 4344, https://doi.org/10.5194/acp-6-4321-2006, 2006b. Brühl, C., Lelieveld, J., Crutzen, P. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3883 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER At- mos. Sci., 34, 1104–1119, https://doi.org/10.1175/1520- 0469(1977)034<1104:mpasap>2.0.co;2, 1977. Kettle, A., Andreae, M., Amouroux, D., Andreae, T., Bates, T., Berresheim, H., Bingemer, H., Boniforti, R., Curran, M., and DiTullio, G.: A global database of sea surface dimethylsulﬁde (DMS) measurements and a procedure to predict sea surface DMS as a function of latitude, lon- gitude, and month, Global Biogeochem. Cy., 13, 399–444, https://doi.org/10.1029/1999gb900004, 1999. Hinckley, E.-L. S. and Matson, P. A.: Transformations, transport, and potential unintended consequences of high sulfur inputs to Napa Valley vineyards, P. Natl. Acad. Sci. USA, 108, 14005– 14010, https://doi.org/10.1073/pnas.1110741108, 2011. Hoesly, R. M., Smith, S. J., Feng, L., Klimont, Z., Janssens- Maenhout, G., Pitkanen, T., Seibert, J. J., Vu, L., Andres, R. J., Bolt, R. M., Bond, T. C., Dawidowski, L., Kholod, N., Kurokawa, J.-I., Li, M., Liu, L., Lu, Z., Moura, M. C. P., O’Rourke, P. R., and Zhang, Q.: Historical (1750–2014) anthro- pogenic emissions of reactive gases and aerosols from the Com- munity Emissions Data System (CEDS), Geosci. Model Dev., 11, 369–408, https://doi.org/10.5194/gmd-11-369-2018, 2018. Kettle, A., Kuhn, U., Von Hobe, M., Kesselmeier, J., and Andreae, M.: Global budget of atmospheric carbonyl sulﬁde: Temporal and spatial variations of the dominant sources and sinks, J. Geo- phys. Res., 107, 4658, https://doi.org/10.1029/2002jd002187, 2002. Kleinschmitt, C., Boucher, O., Bekki, S., Lott, F., and Platt, U.: The Sectional Stratospheric Sulfate Aerosol module (S3A-v1) within the LMDZ general circulation model: description and evaluation against stratospheric aerosol observations, Geosci. Model Dev., 10, 3359–3378, https://doi.org/10.5194/gmd-10- 3359-2017, 2017. Höpfner, M., Boone, C. D., Funke, B., Glatthor, N., Grabowski, U., Günther, A., Kellmann, S., Kiefer, M., Linden, A., Los- sow, S., Pumphrey, H. C., Read, W. G., Roiger, A., Stiller, G., Schlager, H., von Clarmann, T., and Wissmüller, K.: Sulfur diox- ide (SO2) from MIPAS in the upper troposphere and lower stratosphere 2002–2012, Atmos. Chem. Phys., 15, 7017–7037, https://doi.org/10.5194/acp-15-7017-2015, 2015. Komurcu, M., Storelvmo, T., Tan, I., Lohmann, U., Yun, Y., Penner, J. E., Wang, Y., Liu, X., and Takemura, T.: In- tercomparison of the cloud water phase among global cli- mate models, J. Geophys. Res.-Atmos., 119, 3372–3400, https://doi.org/10.1002/2013JD021119, 2014. Hu, Y., Rodier, S., Xu, K., Sun, W., Huang, J., Lin, B., Zhai, P., and Josset, D.: Occurrence, liquid water content, and fraction of supercooled water clouds from combined CALIOP/IIR/MODIS measurements, J. Geophys. Res., 115, D00H34, https://doi.org/10.1029/2009JD012384, 2010. Kovar, J. L. and Grant, C. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3884 Feinberg, A., Sukhodolov, T., Luo, B.-P., Rozanov, E., Winkel, L., Stenke, A., and Peter, T.: Simulation data in the development of SOCOL-AERv2, ETH Zurich, https://doi.org/10.3929/ethz-b- 000342078, 2019. oxymonosulfate, J. Geophys. Res., 91, 9807–9826, https://doi.org/10.1029/JD091iD09p09807, 1986. oxymonosulfate, J. Geophys. Res., 91, 9807–9826, https://doi.org/10.1029/JD091iD09p09807, 1986. Jacobson, M. Z. and Seinfeld, J. H.: Evolution of nanoparticle size and mixing state near the point of emission, Atmos. Environ., 38, 1839–1850, https://doi.org/10.1016/j.atmosenv.2004.01.014, 2004. Friberg, J., Martinsson, B. G., Andersson, S. M., Brenninkmei- jer, C. A., Hermann, M., Van Velthoven, P. F., and Zahn, A.: Sources of increase in lowermost stratospheric sulphurous and carbonaceous aerosol background concentrations during 1999– 2008 derived from CARIBIC ﬂights, Tellus B, 66, 23428, https://doi.org/10.3402/tellusb.v66.23428, 2014. Junge, C. E., Chagnon, C. W., and Manson, J. E.: Stratospheric aerosols, J. Meteorol., 18, 81–108, https://doi.org/10.1175/1520- 0469(1961)018<0081:SA>2.0.CO;2, 1961. Kasten, F.: Falling speed of aerosol particles, J. Appl. Meteorol., 7, 944–947, https://doi.org/10.1175/1520- 0450(1968)007<0944:FSOAP>2.0.CO;2, 1968. Froyd, K. D., Murphy, D. M., Sanford, T. J., Thomson, D. S., Wil- son, J. C., Pﬁster, L., and Lait, L.: Aerosol composition of the tropical upper troposphere, Atmos. Chem. Phys., 9, 4363–4385, https://doi.org/10.5194/acp-9-4363-2009, 2009. Kerkweg, A., Buchholz, J., Ganzeveld, L., Pozzer, A., Tost, H., and Jöckel, P.: Technical Note: An implementation of the dry removal processes DRY DEPosition and SEDImentation in the Modu- lar Earth Submodel System (MESSy), Atmos. Chem. Phys., 6, 4617–4632, https://doi.org/10.5194/acp-6-4617-2006, 2006. Fuchs, N.: The Mechanics of Aerosols, Pergamon Press, Oxford, 1964. Giorgetta, M., Manzini, E., Roeckner, E., Esch, M., and Bengts- son, L.: Climatology and forcing of the quasi-biennial oscil- lation in the MAECHAM5 model, J. Climate, 19, 3882–3901, https://doi.org/10.1175/JCLI3830.1, 2006. Kerkweg, A., Buchholz, J., Ganzeveld, L., Pozzer, A., Tost, H., and Jöckel, P.: Corrigendum to “Technical Note: An implemen- tation of the dry removal processes DRY DEPosition and SED- Imentation in the Modular Earth Submodel System (MESSy)” published in Atmos. Chem. Phys., 6, 4617–4632, 2006, Atmos. Chem. Phys., 9, 9569–9569, https://doi.org/10.5194/acp-9-9569- 2009, 2009. Goto, D., Nakajima, T., Takemura, T., and Sudo, K.: A study of un- certainties in the sulfate distribution and its radiative forcing as- sociated with sulfur chemistry in a global aerosol model, Atmos. Chem. Phys., 11, 10889–10910, https://doi.org/10.5194/acp-11- 10889-2011, 2011. Kettle, A. and Andreae, M.: Flux of dimethylsulﬁde from the oceans: A comparison of updated data sets and ﬂux models, J. Geophys. Res., 105, 26793–26808, https://doi.org/10.1029/2000jd900252, 2000. Hamill, P., Toon, O., and Kiang, C.: Microphysical pro- cesses affecting stratospheric aerosol particles, J. References Doeringer, D., Eldering, A., Boone, C., González Abad, G., and Bernath, P.: Observation of sulfate aerosols and SO2 from the Sarychev volcanic eruption using data from the Atmospheric Chemistry Experiment (ACE), J. Geophys. Res., 117, D03203, https://doi.org/10.1029/2011JD016556, 2012. Christian, H. J., Blakeslee, R. J., Boccippio, D. J., Boeck, W. L., Buechler, D. E., Driscoll, K. T., Goodman, S. J., Hall, J. M., Koshak, W. J., and Mach, D. M.: Global fre- quency and distribution of lightning as observed from space by the Optical Transient Detector, J. Geophys. Res., 108, 4005, https://doi.org/10.1029/2002JD002347, 2003. Egorova, T., Rozanov, E., Zubov, V., and Karol, I.: Model for inves- tigating ozone trends (MEZON), Izv. Atmos. Ocean. Phy., 39, 277–292, 2003. Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER Murphy, D., Froyd, K., Schwarz, J., and Wilson, J.: Ob- servations of the chemical composition of stratospheric aerosol particles, Q. J. Roy. Meteor. Soc., 140, 1269–1278, https://doi.org/10.1002/qj.2213, 2014. Myhre, G., Shindell, D., Bréon, F.-M., Collins, W., Fuglestvedt, J., Huang, J., Koch, D., Lamarque, J.-F., Lee, D., Men- doza, B., Nakajima, T., Robock, A., Stephens, G., Takemura, T., and Zhang, H.: Anthropogenic and natural radiative forc- ing, Cambridge University Press, Cambridge, UK, 659–740, https://doi.org/10.1017/CBO9781107415324.018, 2013. Lamarque, J.-F., Emmons, L. K., Hess, P. G., Kinnison, D. E., Tilmes, S., Vitt, F., Heald, C. L., Holland, E. A., Lauritzen, P. H., Neu, J., Orlando, J. J., Rasch, P. J., and Tyndall, G. K.: CAM-chem: description and evaluation of interactive at- mospheric chemistry in the Community Earth System Model, Geosci. Model Dev., 5, 369–411, https://doi.org/10.5194/gmd-5- 369-2012, 2012. Neely, R. R., English, J. M., Toon, O. B., Solomon, S., Mills, M., and Thayer, J. P.: Implications of extinction due to mete- oritic smoke in the upper stratosphere, Geophys. Res. Lett., 38, L24808, https://doi.org/10.1029/2011gl049865, 2011. Nightingale, P. D., Malin, G., Law, C. S., Watson, A. J., Liss, P. S., Liddicoat, M. I., Boutin, J., and Upstill-Goddard, R. C.: In situ evaluation of air-sea gas exchange parameterizations using novel conservative and volatile tracers, Global Biogeochem. Cy., 14, 373–387, https://doi.org/10.1029/1999GB900091, 2000. Lamarque, J.-F., Dentener, F., McConnell, J., Ro, C.-U., Shaw, M., Vet, R., Bergmann, D., Cameron-Smith, P., Dalsoren, S., Doherty, R., Faluvegi, G., Ghan, S. J., Josse, B., Lee, Y. H., MacKenzie, I. A., Plummer, D., Shindell, D. T., Skeie, R. B., Stevenson, D. S., Strode, S., Zeng, G., Curran, M., Dahl-Jensen, D., Das, S., Fritzsche, D., and Nolan, M.: Multi-model mean nitrogen and sulfur deposition from the Atmospheric Chem- istry and Climate Model Intercomparison Project (ACCMIP): evaluation of historical and projected future changes, Atmos. Chem. Phys., 13, 7997–8018, https://doi.org/10.5194/acp-13- 7997-2013, 2013. Plane, J. M.: Cosmic dust in the Earth’s atmosphere, Chem. Soc. Rev., 41, 6507–6518, https://doi.org/10.1039/c2cs35132c, 2012. Pöschl, U., Von Kuhlmann, R., Poisson, N., and Crutzen, P. J.: De- velopment and intercomparison of condensed isoprene oxidation mechanisms for global atmospheric modeling, J. Atmos. Chem., 37, 29–52, https://doi.org/10.1023/A:1006391009798, 2000. Price, C. and Rind, D.: A simple lightning parameterization for calculating global lightning distributions, J. Geophys. Res., 97, 9919–9933, https://doi.org/10.1029/92JD00719, 1992. Lana, A., Bell, T., Simó, R., Vallina, S., Ballabrera-Poy, J., Ket- tle, A., Dachs, J., Bopp, L., Saltzman, E., Stefels, J., John- son, J. E., and Liss, P. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3885 and HALOE extinction measurements, J. Geophys. Res.-Atmos., 120, 8426–8447, https://doi.org/10.1002/2015JD023303, 2015. Bekki, S., Brooke, J. S. A., Dhomse, S., Johnson, C., Lamarque, J.-F., LeGrande, A. N., Mills, M. J., Niemeier, U., Pope, J. O., Poulain, V., Robock, A., Rozanov, E., Stenke, A., Sukhodolov, T., Tilmes, S., Tsigaridis, K., and Tummon, F.: Multi-model comparison of the volcanic sulfate deposition from the 1815 eruption of Mt. Tambora, Atmos. Chem. Phys., 18, 2307–2328, https://doi.org/10.5194/acp-18-2307-2018, 2018. Bekki, S., Brooke, J. S. A., Dhomse, S., Johnson, C., Lamarque, J.-F., LeGrande, A. N., Mills, M. J., Niemeier, U., Pope, J. O., Poulain, V., Robock, A., Rozanov, E., Stenke, A., Sukhodolov, T., Tilmes, S., Tsigaridis, K., and Tummon, F.: Multi-model comparison of the volcanic sulfate deposition from the 1815 eruption of Mt. Tambora, Atmos. Chem. Phys., 18, 2307–2328, https://doi.org/10.5194/acp-18-2307-2018, 2018. and HALOE extinction measurements, J. Geophys. Res.-Atmos., 120, 8426–8447, https://doi.org/10.1002/2015JD023303, 2015. Bekki, S., Brooke, J. S. A., Dhomse, S., Johnson, C., Lamarque, J.-F., LeGrande, A. N., Mills, M. J., Niemeier, U., Pope, J. O., Kremser, S., Thomason, L. W., Hobe, M., Hermann, M., Desh- ler, T., Timmreck, C., Toohey, M., Stenke, A., Schwarz, J. P., and Weigel, R.: Stratospheric aerosol–Observations, pro- cesses, and impact on climate, Rev. Geophys., 54, 278–335, https://doi.org/10.1002/2015RG000511, 2016. Poulain, V., Robock, A., Rozanov, E., Stenke, A., Sukhodolov, T., Tilmes, S., Tsigaridis, K., and Tummon, F.: Multi-model comparison of the volcanic sulfate deposition from the 1815 eruption of Mt. Tambora, Atmos. Chem. Phys., 18, 2307–2328, https://doi.org/10.5194/acp-18-2307-2018, 2018. Montzka, S., Calvert, P., Hall, B., Elkins, J., Conway, T., Tans, P., and Sweeney, C.: On the global distribution, seasonal- ity, and budget of atmospheric carbonyl sulﬁde (COS) and some similarities to CO2, J. Geophys. Res., 112, D09302, https://doi.org/10.1029/2006jd007665, 2007. Kulmala, M. and Laaksonen, A.: Binary nucleation of water– sulfuric acid system: Comparison of classical theories with dif- ferent H2SO4 saturation vapor pressures, J. Chem. Phys., 93, 696–701, https://doi.org/10.1063/1.459519, 1990. , p g , Lamarque, J.-F., Bond, T. C., Eyring, V., Granier, C., Heil, A., Klimont, Z., Lee, D., Liousse, C., Mieville, A., Owen, B., Schultz, M. G., Shindell, D., Smith, S. J., Stehfest, E., Van Aardenne, J., Cooper, O. R., Kainuma, M., Mahowald, N., McConnell, J. R., Naik, V., Riahi, K., and van Vuuren, D. P.: Historical (1850–2000) gridded anthropogenic and biomass burning emissions of reactive gases and aerosols: methodol- ogy and application, Atmos. Chem. Phys., 10, 7017–7039, https://doi.org/10.5194/acp-10-7017-2010, 2010. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER A.: Nutrient cycling in soils: Sulfur, in: Soil Management: Building a Stable Base for Agriculture, edited by: Hatﬁeld, J. L. and Sauer, T. J., American Society of Agron- omy and Soil Science Society of America, Madison, Wisconsin, 2011. Huthwelker, T., Lamb, D., Baker, M., Swanson, B., and Peter, T.: Uptake of SO2 by polycrystalline water ice, J. Colloid In- terf. Sci., 238, 147–159, https://doi.org/10.1006/jcis.2001.7507, 2001. Kovilakam, M. and Deshler, T.: On the accuracy of stratospheric aerosol extinction derived from in situ size distribution measure- ments and surface area density derived from remote SAGE II Jacob, D. J.: Chemistry of OH in remote clouds and its role in the production of formic acid and per- Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3886 Phys., 18, 16155–16172, https://doi.org/10.5194/acp-18-16155- 2018, 2018. ide emissions: 1850–2005, Atmos. Chem. Phys., 11, 1101–1116, https://doi.org/10.5194/acp-11-1101-2011, 2011. ide emissions: 1850–2005, Atmos. Chem. Phys., 11, 1101–1116, https://doi.org/10.5194/acp-11-1101-2011, 2011. Solomon, S.: Stratospheric ozone depletion: A review of concepts and history, Rev. Geophys., 37, 275–316, https://doi.org/10.1029/1999RG900008, 1999. Rienecker, M. M., Suarez, M. J., Gelaro, R., Todling, R., Bacmeis- ter, J., Liu, E., Bosilovich, M. G., Schubert, S. D., Takacs, L., and Kim, G.-K.: MERRA: NASA’s modern-era retrospective anal- ysis for research and applications, J. Climate, 24, 3624–3648, https://doi.org/10.1175/JCLI-D-11-00015.1, 2011. Stenke, A., Schraner, M., Rozanov, E., Egorova, T., Luo, B., and Peter, T.: The SOCOL version 3.0 chemistry–climate model: description, evaluation, and implications from an ad- vanced transport algorithm, Geosci. Model Dev., 6, 1407–1427, https://doi.org/10.5194/gmd-6-1407-2013, 2013. Roeckner, E., Bäuml, G., Bonaventura, L., Brokopf, R., Esch, M., Giorgetta, M., Hagemann, S., Kirchner, I., Kornblueh, L., Manzini, E., Rhodin, A., Schlese, U., Schulzweida, U., and Tompkins, A.: The atmospheric general circulation model ECHAM 5. PART I: Model description, Tech. Rep. 349, Max-Planck-Institut für Meteorologie, Hamburg, available at: http://www.mpimet.mpg.de/ﬁleadmin/publikationen/Reports/ max_scirep_349.pdf (last access: 9 July 2019), 2003. Sukhodolov, T., Sheng, J.-X., Feinberg, A., Luo, B.-P., Pe- ter, T., Revell, L., Stenke, A., Weisenstein, D. K., and Rozanov, E.: Stratospheric aerosol evolution after Pinatubo sim- ulated with a coupled size-resolved aerosol–chemistry–climate model, SOCOL-AERv1.0, Geosci. Model Dev., 11, 2633–2647, https://doi.org/10.5194/gmd-11-2633-2018, 2018. Rollins, A., Thornberry, T., Watts, L., Yu, P., Rosenlof, K., Mills, M., Baumann, E., Giorgetta, F., Bui, T., and Höpfner, M.: The role of sulfur dioxide in stratospheric aerosol for- mation evaluated by using in situ measurements in the trop- ical lower stratosphere, Geophys. Res. Lett., 44, 4280–4286, https://doi.org/10.1002/2017GL072754, 2017. Tabazadeh, A., Toon, O. B., Clegg, S. L., and Hamill, P.: A new parameterization of H2SO4/H2O aerosol composition: At- mospheric implications, Geophys. Res. Lett., 24, 1931–1934, https://doi.org/10.1029/97GL01879, 1997. Tan, I., Storelvmo, T., and Zelinka, M. D.: Observational constraints on mixed-phase clouds imply higher climate sensitivity, Science, 352, 224–227, https://doi.org/10.1126/science.aad5300, 2016. Rollins, A., Thornberry, T. D., Atlas, E., Navarro, M., Schaufﬂer, S., Moore, F., Elkins, J. W., Ray, E., Rosenlof, K., and Aquila, V.: SO2 observations and sources in the western Paciﬁc tropical tropopause region, J. Geophys. Res.-Atmos., 123, 13549–13559, https://doi.org/10.1029/2018JD029635, 2018. Tan, J., Fu, J. S., Dentener, F., Sun, J., Emmons, L., Tilmes, S., Sudo, K., Flemming, J., Jonson, J. E., Gravel, S., Bian, H., Davila, Y., Henze, D. K., Lund, M. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER T., Kucsera, T., Takemura, T., and Keating, T.: Multi-model study of HTAP II on sulfur and nitrogen deposition, Atmos. Chem. Phys., 18, 6847–6866, https://doi.org/10.5194/acp-18-6847-2018, 2018. Rotstayn, L. D. and Lohmann, U.: Simulation of the tro- pospheric sulfur cycle in a global model with a physi- cally based cloud scheme, J. Geophys. Res., 107, 4592, https://doi.org/10.1029/2002JD002128, 2002. Thomason, L. W.: Toward a combined SAGE II-HALOE aerosol climatology: an evaluation of HALOE version 19 strato- spheric aerosol extinction coefﬁcient observations, Atmos. Chem. Phys., 12, 8177–8188, https://doi.org/10.5194/acp-12- 8177-2012, 2012. Rozanov, E. V., Zubov, V. A., Schlesinger, M. E., Yang, F., and Andronova, N. G.: The UIUC three-dimensional stratospheric chemical transport model: Description and evaluation of the sim- ulated source gases and ozone, J. Geophys. Res., 104, 11755– 11781, https://doi.org/10.1029/1999JD900138, 1999. Thomason, L. and Peter, T.: SPARC Assessment of Stratospheric Aerosol Properties (ASAP), Tech. Rep. No. 4, SPARC, available at: https://www.sparc-climate.org/publications/sparc-reports/ (last access: 9 July 2019), 2006. Sander, S., Abbatt, J., Barker, J., Burkholder, J., Friedl, R., Golden, D., Huie, R., Kolb, C., Kurylo, M., Moortgat, G., Orkin, V., and Wine, P.: Chemical Kinetics and Photochemical Data for Use in Atmospheric Studies, Evaluation No. 17, Tech. Rep. No. 10- 6, Jet Propulsion Laboratory, Pasadena, available at: https: //jpldataeval.jpl.nasa.gov/pdf/JPL10-6Final15June2011.pdf (last access: 9 July 2019), 2011. Thomason, L. W., Ernest, N., Millán, L., Rieger, L., Bourassa, A., Vernier, J.-P., Manney, G., Luo, B., Arfeuille, F., and Peter, T.: A global space-based stratospheric aerosol cli- matology: 1979–2016, Earth Syst. Sci. Data, 10, 469–492, https://doi.org/10.5194/essd-10-469-2018, 2018. Schwede, D., Zhang, L., Vet, R., and Lear, G.: An intercom- parison of the deposition models used in the CASTNET and CAPMoN networks, Atmos. Environ., 45, 1337–1346, https://doi.org/10.1016/j.atmosenv.2010.11.050, 2011. Timmreck, C., Mann, G. W., Aquila, V., Hommel, R., Lee, L. A., Schmidt, A., Brühl, C., Carn, S., Chin, M., Dhomse, S. S., Diehl, T., English, J. M., Mills, M. J., Neely, R., Sheng, J., Toohey, M., and Weisenstein, D.: The Interactive Stratospheric Aerosol Model Intercomparison Project (ISA-MIP): motivation and experimental design, Geosci. Model Dev., 11, 2581–2608, https://doi.org/10.5194/gmd-11-2581-2018, 2018. Sellegri, K., Laj, P., Marinoni, A., Dupuy, R., Legrand, M., and Preunkert, S.: Contribution of gaseous and particulate species to droplet solute composition at the Puy de Dôme, France, Atmos. Chem. Phys., 3, 1509–1522, https://doi.org/10.5194/acp-3-1509- 2003, 2003. Tost, H., Jöckel, P., Kerkweg, A., Sander, R., and Lelieveld, J.: Tech- nical note: A new comprehensive SCAVenging submodel for global atmospheric chemistry modelling, Atmos. Chem. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER S.: An updated climatology of sur- face dimethlysulﬁde concentrations and emission ﬂuxes in the global ocean, Global Biogeochem. Cy., 25, GB1004, https://doi.org/10.1029/2010GB003850, 2011. Rayner, N., Parker, D. E., Horton, E., Folland, C., Alexander, L., Rowell, D., Kent, E., and Kaplan, A.: Global analyses of sea surface temperature, sea ice, and night marine air temperature since the late nineteenth century, J. Geophys. Res., 108, 4407, https://doi.org/10.1029/2002JD002670, 2003. Lean, J., Rottman, G., Harder, J., and Kopp, G.: SORCE contribu- tions to new understanding of global change and solar variabil- ity, in: The Solar Radiation and Climate Experiment (SORCE), Springer, New York, NY, 27–53, 2005 Revell, L. E., Tummon, F., Stenke, A., Sukhodolov, T., Coulon, A., Rozanov, E., Garny, H., Grewe, V., and Peter, T.: Drivers of the tropospheric ozone budget throughout the 21st cen- tury under the medium-high climate scenario RCP 6.0, Atmos. Chem. Phys., 15, 5887–5902, https://doi.org/10.5194/acp-15- 5887-2015, 2015. Marandino, C. A., Tegtmeier, S., Krüger, K., Zindler, C., Atlas, E. L., Moore, F., and Bange, H. W.: Dimethylsulphide (DMS) emis- sions from the western Paciﬁc Ocean: a potential marine source for stratospheric sulphur?, Atmos. Chem. Phys., 13, 8427–8437, https://doi.org/10.5194/acp-13-8427-2013, 2013. Revell, L. E., Stenke, A., Tummon, F., Feinberg, A., Rozanov, E., Peter, T., Abraham, N. L., Akiyoshi, H., Archibald, A. T., Butchart, N., Deushi, M., Jöckel, P., Kinnison, D., Michou, M., Morgenstern, O., O’Connor, F. M., Oman, L. D., Pitari, G., Plummer, D. A., Schoﬁeld, R., Stone, K., Tilmes, S., Vi- sioni, D., Yamashita, Y., and Zeng, G.: Tropospheric ozone in CCMI models and Gaussian process emulation to understand bi- ases in the SOCOLv3 chemistry–climate model, Atmos. Chem. Marinoni, A., Laj, P., Sellegri, K., and Mailhot, G.: Cloud chemistry at the Puy de Dôme: variability and relationships with environmental factors, Atmos. Chem. Phys., 4, 715–728, https://doi.org/10.5194/acp-4-715-2004, 2004. Marshall, L., Schmidt, A., Toohey, M., Carslaw, K. S., Mann, G. W., Sigl, M., Khodri, M., Timmreck, C., Zanchettin, D., Ball, W. T., Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER 3887 Wesely, M.: Parameterization of surface resistances to gaseous dry deposition in regional-scale numerical models, Atmos. Environ., 23, 1293–1304, https://doi.org/10.1016/0004-6981(89)90153-4, 1989. Tost, H., Lawrence, M. G., Brühl, C., Jöckel, P., The GABRIEL Team, and The SCOUT-O3-DARWIN/ACTIVE Team: Un- certainties in atmospheric chemistry modelling due to con- vection parameterisations and subsequent scavenging, Atmos. Chem. Phys., 10, 1931–1951, https://doi.org/10.5194/acp-10- 1931-2010, 2010. Wesely, M. and Hicks, B.: A review of the current status of knowledge on dry deposition, Atmos. Environ., 34, 2261–2282, https://doi.org/10.1016/S1352-2310(99)00467-7, 2000. van Pinxteren, D., Fomba, K. W., Mertes, S., Müller, K., Spindler, G., Schneider, J., Lee, T., Collett, J. L., and Herrmann, H.: Cloud water composition during HCCT-2010: Scavenging efﬁciencies, solute concentrations, and droplet size dependence of inorganic ions and dissolved organic carbon, Atmos. Chem. Phys., 16, 3185–3205, https://doi.org/10.5194/acp-16-3185-2016, 2016. WMO, M.: A three-dimensional science, WMO Bulletin, 6, 134– 138, 1957. Wu, Z., Schwede, D. B., Vet, R., Walker, J. T., Shaw, M., Staebler, R., and Zhang, L.: Evaluation and intercompari- son of ﬁve North American dry deposition algorithms at a mixed forest site, J. Adv. Model. Earth Sy., 10, 1571–1586, https://doi.org/10.1029/2017MS001231, 2018. Vehkamäki, H., Kulmala, M., Napari, I., Lehtinen, K. E., Timm- reck, C., Noppel, M., and Laaksonen, A.: An improved pa- rameterization for sulfuric acid-water nucleation rates for tro- pospheric and stratospheric conditions, J. Geophys. Res., 107, 4622, https://doi.org/10.1029/2002jd002184, 2002. Yu, P., Toon, O. B., Neely, R. R., Martinsson, B. G., and Bren- ninkmeijer, C. A.: Composition and physical properties of the Asian tropopause aerosol layer and the North American tro- pospheric aerosol layer, Geophys. Res. Lett., 42, 2540–2546, https://doi.org/10.1002/2015GL063181, 2015. Vet, R., Artz, R. S., Carou, S., Shaw, M., Ro, C.-U., Aas, W., Baker, A., Bowersox, V. C., Dentener, F., and Galy-Lacaux, C.: A global assessment of precipitation chemistry and depo- sition of sulfur, nitrogen, sea salt, base cations, organic acids, acidity and pH, and phosphorus, Atmos. Environ., 93, 3–100, https://doi.org/10.1016/j.atmosenv.2013.10.060, 2014. Zanchettin, D., Khodri, M., Timmreck, C., Toohey, M., Schmidt, A., Gerber, E. P., Hegerl, G., Robock, A., Pausata, F. S. R., Ball, W. T., Bauer, S. E., Bekki, S., Dhomse, S. S., LeGrande, A. N., Mann, G. W., Marshall, L., Mills, M., Marchand, M., Niemeier, U., Poulain, V., Rozanov, E., Rubino, A., Stenke, A., Tsigaridis, K., and Tummon, F.: The Model Intercomparison Project on the climatic response to Volcanic forcing (VolMIP): experimental design and forcing input data for CMIP6, Geosci. A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER Phys., 6, 565–574, https://doi.org/10.5194/acp-6-565-2006, 2006. Sheng, J., Weisenstein, D. K., Luo, B., Rozanov, E., Stenke, A., Anet, J., Bingemer, H., and Peter, T.: Global atmospheric sulfur budget under volcanically quiescent conditions: Aerosol-chem- istry-climate model predictions and validation, J. Geophys. Res.- Atmos., 120, 256–276, https://doi.org/10.1002/2014JD021985, 2015. Tost, H., Jöckel, P., Kerkweg, A., Pozzer, A., Sander, R., and Lelieveld, J.: Global cloud and precipitation chem- istry and wet deposition: tropospheric model simulations with ECHAM5/MESSy1, Atmos. Chem. Phys., 7, 2733–2757, https://doi.org/10.5194/acp-7-2733-2007, 2007. Smith, S. J., van Aardenne, J., Klimont, Z., Andres, R. J., Volke, A., and Delgado Arias, S.: Anthropogenic sulfur diox- Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ Geosci. Model Dev., 12, 3863–3887, 2019 A. Feinberg et al.: Improved sulfur cycle in SOCOL-AER Model Dev., 9, 2701–2719, https://doi.org/10.5194/gmd-9-2701-2016, 2016. Walcek, C. J.: Minor ﬂux adjustment near mixing ratio ex- tremes for simpliﬁed yet highly accurate monotonic calcula- tion of tracer advection, J. Geophys. Res., 105, 9335–9348, https://doi.org/10.1029/1999JD901142, 2000. Walcek, C. J. and Taylor, G. R.: A theoretical method for computing vertical distributions of acidity and sulfate production within cumulus clouds, J. At- mos. Sci., 43, 339–355, https://doi.org/10.1175/1520- 0469(1986)043<0339:ATMFCV>2.0.CO;2, 1986. Weisenstein, D. K., Yue, G. K., Ko, M. K., Sze, N., Rodriguez, J. M., and Scott, C. J.: A two-dimensional model of sulfur species and aerosols, J. Geophys. Res., 102, 13019–13035, https://doi.org/10.1029/97JD00901, 1997. Geosci. Model Dev., 12, 3863–3887, 2019 www.geosci-model-dev.net/12/3863/2019/ www.geosci-model-dev.net/12/3863/2019/ www.geosci-model-dev.net/12/3863/2019/ www.geosci-model-dev.net/12/3863/2019/
https://openalex.org/W2189256394	https://europepmc.org/articles/pmc4594164?pdf=render	English	null	Mobility impairment and bad feet … who'd of guessed? The Foot Disease in Inpatients Study (FDIS)	Journal of foot and ankle research	2,015	cc-by	879	* Correspondence: Peter.Lazzarini@health.qld.gov.au 1School of Clinical Sciences, Queensland University of Technology, Brisbane, Queensland, 4059, Australia Full list of author information is available at the end of the article © 2015 Lazzarini et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/ zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background analysis, and controlling for other risk factors, those people with mobility impairment were independently associated with increasing years of age (OR = 1.04 (95% CI) (1.02-1.05)), male gender (OR = 1.7 (1.2-2.5)), being born in Australia (OR = 1.7 (1.1-2.8), vision impairment (2.0 (1.2-3.1)), peripheral neuropathy (OR = 3.1 (2.0-4.6) and foot deformity (OR = 2.0 (1.3-3.0). Foot complications have been found to be predictors of mobility impairment and falls in community dwelling elderly patients. However, fewer studies have investi- gated the link between foot complications and mobility impairment in hospital in patient populations. The aim of this paper was to investigate the associations between mobility impairment and various foot complications in general inpatient populations. Conclusions These findings support the results of other large studies investigating community dwelling elderly patients that peripheral neuropathy and foot deformity are indepen- dently associated with mobility impairment and poten- tially falls. Furthermore the findings suggest routine clinical diagnosis of foot complications as defined by national diabetic foot guidelines were sufficient to deter- mine these associated foot complication risk factors for mobility impairment. Further research is required to establish if these foot complication risk factors for mobi- lity impairment are predictors of actual falls in the inpati- ent environment. Mobility impairment and bad feet … who’d of guessed? The Foot Disease in Inpatients Study (FDIS) Peter A Lazzarini1,2,3, Vanessa Ng2,3, Suzanne S Kuys2,4, Maarten C Kamp1,5, Michael C d’Emden5,6, Courtney Thomas7, Jude Wills8, Ewan M Kinnear2,3, Scott Jen9, Sheree E Hurn1, Lloyd Reed1 From Australasian Podiatry Conference 2015 Queensland, Australia. 6-8 May 2015 From Australasian Podiatry Conference 2015 Queensland, Australia. 6-8 May 2015 Methods Eligible participants were all adults admitted overnight, for any reason, into five diverse hospitals on one day; excluding maternity, mental health and cognitively impaired patients. Participants underwent a foot exami- nation to clinically diagnose different foot complications; including foot wounds, infections, deformity, peripheral arterial disease and peripheral neuropathy. They were also surveyed on social determinant, medical history, self-care, footwear, foot complication history risk factors, and, mobility impairment defined as requiring a mobility aid for mobilisation prior to hospitalisation. JOURNAL OF FOOT AND ANKLE RESEARCH JOURNAL OF FOOT AND ANKLE RESEARCH JOURNAL OF FOOT AND ANKLE RESEARCH Lazzarini et al. Journal of Foot and Ankle Research 2015, 8(Suppl 2):O26 http://www.jfootankleres.com/content/8/S2/O26 JOURNAL OF FOOT AND ANKLE RESEARCH Open Access Results 1School of Clinical Sciences, Queensland University of Technology, Brisbane, Queensland, 4059, Australia. 2Allied Health Research Collaborative, Metro North Hospital & Health Service, Queensland Health, Brisbane, Queensland, 4032, Australia. 3Department of Podiatry, Metro North Hospital & Health Service, Queensland Health, Brisbane, Queensland, 4032, Australia. 4Musculoskeletal Research Program, Griffith Health Institute, Griffith University, Gold Coast, Queensland, 4222, Australia. 5Department of Endocrinology and Diabetes, Royal Brisbane and Womens Hospital, Brisbane, Queensland, 4029, Australia. 6School of Medicine, The University of Queensland, Brisbane, Queensland, 4072, Australia. 7Department of Podiatry, North West Hospital & Health Service, Mount Isa, Queensland, 4825, Australia. 8Department of Podiatry, Central Queensland Hospital & Health Service, Rockhampton, Queensland, 4700, Australia. 9Department of Podiatry, Overall, 733 participants consented; mean(±SD) age 62(±19) years, 408 (55.8%) male, 172 (23.5%) diabetes. Mobility impairment was present in 242 (33.2%) partici- pants; diabetes populations reported more mobility impairment than non-diabetes populations (40.7% vs 30.9%, p < 0.05). In a backwards stepwise multivariate Correspondence: Peter.Lazzarini@health.qld.gov.au 1School of Clinical Sciences, Queensland University of Technology, Brisbane, Queensland, 4059, Australia Full list of author information is available at the end of the article West Moreton Hospital & Health Service, Queensland Health, Ipswich, Queensland, 4305, Australia. Published: 22 September 2015 doi:10.1186/1757-1146-8-S2-O26 Cite this article as: Lazzarini et al.: Mobility impairment and bad feet … who’d of guessed? The Foot Disease in Inpatients Study (FDIS). Journal of Foot and Ankle Research 2015 8(Suppl 2):O26. Lazzarini et al. Journal of Foot and Ankle Research 2015, 8(Suppl 2):O26 http://www.jfootankleres.com/content/8/S2/O26 Page 2 of 2 Page 2 of 2 Lazzarini et al. Journal of Foot and Ankle Research 2015, 8(Suppl 2):O26 http://www.jfootankleres.com/content/8/S2/O26 West Moreton Hospital & Health Service, Queensland Health, Ipswich, Queensland, 4305, Australia. Published: 22 September 2015 doi:10.1186/1757-1146-8-S2-O26 Cite this article as: Lazzarini et al.: Mobility impairment and bad feet … who’d of guessed? The Foot Disease in Inpatients Study (FDIS). Journal of Foot and Ankle Research 2015 8(Suppl 2):O26. doi:10.1186/1757-1146-8-S2-O26 Cite this article as: Lazzarini et al.: Mobility impairment and bad feet … who’d of guessed? The Foot Disease in Inpatients Study (FDIS). Journal of Foot and Ankle Research 2015 8(Suppl 2):O26. 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https://openalex.org/W2539318458	https://www.e3s-conferences.org/articles/e3sconf/pdf/2016/02/e3sconf_flood2016_16004.pdf	English	null	Post-flood damage data: requirements for disaster forensic investigation	E3S web of conferences	2,016	cc-by	9,652	© 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/). Post-flood damage data: requirements for disaster forensic investigation Martin Dolan 1,a, Nicholas Walliman 2, Daniela Molinari3, Scira Menoni4, Raymond Ogden 5, Shahrzad Amouzad6 and Francesco Ballio 7 n 1,a, Nicholas Walliman 2, Daniela Molinari3, Scira Menoni4, Raymond Ogden 5, Shahrzad Amouzad6 and Ballio 7 1,2,5,6School of Architecture, Oxford Brookes University, Gipsy Lane, Oxford, UK 3,4,7Politecnico di Milano, Department of Civil and Environmental Engineering, Piazza Leonardo da Vinci, 32, Milano, Italy 1,2,5,6School of Architecture, Oxford Brookes University, Gipsy Lane, Oxford, UK 3,4,7Politecnico di Milano, Department of Civil and Environmental Engineering, Piazza Leonardo da Vinci, 32, Milano, Italy 1,2,5,6School of Architecture, Oxford Brookes University, Gipsy Lane, Oxford, UK 3,4,7Politecnico di Milano, Department of Civil and Environmental Engineering, Piazza Leonardo da Vinci, 32, Milano, Italy Abstract. Disaster forensic investigation analyses the unfolding of a disaster and attempts to identify its multiple causes of damage which can lead to (i) improved disaster prevention and management from lessons learnt, and (ii) more effective mitigation measures in the aftermath of a disaster. The way in which damage data are collected after a flood event as well as the types of collected data influences their usability within forensic investigations. In order to explore whether or not existing data can be used for disaster forensic analysis, the European Project IDEA (Improving Damage assessments to Enhance cost-benefit Analyses) is investigating existing gaps in damage information so as to identify possible paths towards improving data quality. This paper focuses in detail on a forensic analysis of the interlinked damage to economic activities and infrastructure in the Severn floods of 2007 in the UK. Besides investigating the usability of existing data, this research investigated: (i) the relative weight of direct and indirect costs to business and infrastructure companies; (ii) to what extent damage to infrastructure has impacted on indirect damage to businesses. Finally recommendations for improving the data for use in forensic investigation are offered. a Corresponding author: Martin Dolan: mdolan@brookes.ac.uk , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) DOI: 10.1051/ 6 e3sconf/201 0716004 1 Introduction major policy decisions and smaller incremental social and cultural decisions that lead to accumulated vulnerabilities.1 Despite the increased understanding of the science of proved technology, the costs associated with these events are increasing at a rapid rate in both developed countries, where stronger technology and science have failed to stabilise losses, and in developing countries, where the increase in losses are growing much faster than wealth highlighting deficiencies in current research on disasters1. Another trait of forensic investigation is that the research must be carried out independently and free from influence from any external bodies while still maintaining the authority and support of all public and private bodies involved and affected. This allows the investigation to achieve unadulterated results and report on the multitude of actions and responsibilities which account for the damage caused2. The aim of this type of investigation is not to pin blame on any one stakeholder but to expose the myriad of contributing factors and aid in the identification of the most effective responses in order to avoid a reoccurrence of the damage caused. Many approaches to understanding disasters tend to be conducted within the frame of a narrow specialist field such as geological science, meteorology, technology, politics, economics or social sciences. Efforts rarely attempt to delve deeply into the underlying and cross- disciplinary causes with the focus often on reacting to the disaster in order to recover, while the concept of One of the principles of forensic investigations is the ability to be able to accumulate evidence of good practices by examining the different impact of similar events in different locations or the effects of a single event on different sectors in the same geographical location. In an attempt to address some of the deficiencies in current disaster research methodologies and improve the long-term recovery and prevention needed to reduce the impacts of such events, forensic investigation has been purposed as an alternative approach that offers a multidisciplinary framework. 2.1.1 Stakeholder identification and categorisation Assessing the reliability of damage data collection, storage and management for forensic investigations requires a deep understanding of the present state of art, as well as of limits to be overcome and useful requirements to be supplied by tools developed by the IDEA project. A review of event reports and literature allowed first to identify principle stakeholders. A questionnaire was then sent to individual stakeholders so that profiles were created for each stakeholder, based on questionnaires results. Preliminary information was further integrated with knowledge acquired by direct interviews arranged with engaged stakeholders. Interestingly, further stakeholders were identified through such discussions. This paper focuses in detail on the Severn flood in the UK in 2007. Following the severe flooding in Gloucestershire and elsewhere in 2007, the UK government commissioned Sir Michael Pitt to examine the response and to provide lessons to be learned from that event. The Pitt Review 2007 made 92 recommendations to the government including advice on improving data collection and recording. The report noted that effective information flows during the response and recovery periods are necessary. This was followed by engagement with stakeholders and the categorisation of these into data owners, users or collectors. Stakeholders were also categorised as public or private organisations, with public groups relating to policy level government bodies, county councils, district councils and emergency services. Private stakeholders included insurance companies, service providers such as water companies, electricity providers, gas and telecommunications companies, private businesses and voluntary organisations. The focus of this case study is on identifying the vulnerabilities that lead to damage to commercial and industrial activities. In detail, observed direct and indirect damages are analysed according to a forensic approach. Particular care is taken to analyse indirect damage due to direct damage, as well as damage to infrastructure, as it is often the case that business activities suffer because of disruption of essential services. It is through this process that the research aimed to understand the requirements for damage data in order to carry out a successful forensic analysis. As regard data owners, characterisation implied the identification of (i) which data they are responsible for, (ii) at which spatial and temporal scales, (iii) with what rights (e.g. private or public owners, voluntarily or by law, etc.) and (iv) tools used for the storage of data. 2 Methodology The methodological approach entailed firstly the collection and analysis of existing data obtained from reports, databases and directly from stakeholders. Reliability and completeness of data is crucial in being able to ascertain the reality of what occurred on the ground. When dealing with data that is collected during and in the aftermath of an event of high uncertainty, such as a natural disaster, this can often be difficult task to achieve. This was followed by carrying out a forensic analysis of the Severn floods of 2007 which was considered a suitable case study due to the extensive coverage and post-disaster analysis carried out at all levels. Finally the results from the forensic analysis were assessed for their accuracy and applicability which led to a judgement of the quality of the data used to carry out the forensic analysis. To explore whether or not existing data can be used for disaster forensic analyses, several case studies were analysed within the European Project IDEA (Improving Damage assessments to Enhance cost-benefit Analyses). The main objective was to investigate existing gaps in damage information so as to identify possible paths toward improvement of both data quality and procedures to collect and manage them. 1 Introduction Forensic investigation aims to identify not only the physical vulnerabilities such as poor building standards and planning policies but also the The participation of experts in a wide range of professional disciplines from natural sciences, social and economic studies, engineering, public health, emergency planners and humanitarian relief organisations is essential for an in-depth forensic investigation and input from as a Corresponding author: Martin Dolan: mdolan@brookes.ac.uk , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conferenc 7 16004 ( 2016) DOI: 10.1051/ 6 e3sconf/201 0716004 many stakeholders as possible will lead to improved understanding of the causes and effects of damage and suitable mitigation measures to be implemented. The methodological approach to the study is presented here before findings and results, with their implications for forensic analysis and data, are discussed. Finally a selected number of recovery interventions for the business sector and utilities were analysed to assess to what extent the forensic investigation has been used or may be used to guarantee effective investments. However, a critical challenge in forensic investigation is the way in which data are collected, analysed and shared among these stakeholders and how this data is also relayed to the forensic investigating team. A lack of data or inaccessibility can restrict the essential multi- disciplinary insight that defines the approach. Where certain principle stakeholders are unwilling or unable to cooperate in the investigation the completeness of the results can be compromised. 2.2 Forensic analysis In the process of forensic analysis it is important to understand the ultimate goal of carrying out such investigations. In the case of disaster vulnerability analysis we may, for example, aim to assess the impact of varying different factors on different sectors of the exposed community. In order to be able to carry out a variety of analyses, it is essential to quantify as many values and variants as possible. Following this step, an estimated weight was assigned to the direct impact of each factor on the main affected sector. In order to do this, firstly a judgement was made on which sectors were affected. For example, this could include businesses, residences, infrastructure companies, emergency operations, government agencies etc. Once the affected sectors are decided on, a decision is made on which sector was affected by each factor. This is done by Table 2 below. In this case it is clear that the hazard affected all sectors while the large amount of information to deal with affects mostly emergency services and the Environment Agency. In this particular case, it was extremely valuable to gain insight into how particular sectors were affected in different ways by varying vulnerability factors in order to establish the most beneficial actions to take in building resilience. This section explains how to arrive at the point where this is possible. The forensic analysis began with the creation of a timeline of events as they unfolded, beginning with the first evidence of heavy rainfall. The entire event unfolded over quite a long period and emergency operations and other actions took place outside of the research area and also unrelated to infrastructure and business. These events were considered to be outside the limits of this study. The next step in the process was to make a judgement of the weight of the impact on each sector for each factor. This is done on a scale of 1-3 with 1 being minimal, 2 being significant and 3 being major. A value of -1,-2 or -3 may be assigned to factors if it is believed that these factors contributed more to a reduction of vulnerability than to an increase. 2.1.2 Data gathering Following this a judgement was made as to whether these factors contributed to or reduced damage or both. This is relevant in that some factors were a combination of both. For example, the shutting down of a water treatment plant protected the machinery of the plant and sets and reduced long term damage but it had an impact on businesses and residences in the short term. 2.1.1 Stakeholder identification and categorisation With respect to data collectors, investigation concerned (i) reasons for data collection, (ii) type of collected data and spatial and temporal scales of interest (iii) tools and procedures presently implemented, and (iv) available human and economic resources. Data users were characterised in terms of (i) data of interest, (ii) scales of interest and (iii) uses of data. Besides investigating the usability of existing data, this process allowed the exploration of: (i) what is the relative weight of direct and indirect costs to business and infrastructure companies; Moreover links among the different stakeholders were identified as some data are collected or owned by certain stakeholders who shared them with other stakeholders for the different purposes related to risk management. (ii) to what extent damage to infrastructure has impacted on indirect damage to businesses. 2 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) the complexity of the infrastructure network and the interrelations within the system. the complexity of the infrastructure network and the interrelations within the system. the complexity of the infrastructure network and the interrelations within the system. Finally, stakeholders that are/could be in charge of coordinating the data analysis and representation were defined. Previous experience has shown that without such entity taking care of integrating the data no comprehensive picture of disaster impacts will be ever possible From the timeline an extensive list of the perceived contributing factors was created. This included such aspects as the suitability of emergency response plans in place before the flood and the times of flood warnings. Following this, the factors were categorised depending on whether they were considered to be related to the hazard or to physical, social, organisational or systemic vulnerability. Some factors fell into several of these categories simultaneously and the categorisation reflected this. This process is illustrated in Table 1 below. 2.1.3 Data analysis An assessment of the comprehensiveness of the collected data was performed. In detail, availability of data to depict the full range of likely impacts as well as their drivers (i.e. hazard, exposure and vulnerability factors) was assessed. Finally, a categorisation of main data sources was carried out based on format and level of detail in order to make the use of this data more suitable for the scale of forensic analysis desired. This exercise built up a strong picture of where the largest vulnerabilities lay. For example, the majority of contributing factors may lie in the organisational vulnerability of a company or agency. This is to say that emergency planning, management and decision making would be the primary contributing factors in causing damage. Additionally systemic issues may be prevalent in the causing of damage as the failure of a single element of an infrastructure system can result in large-scale impact. 2.2 Forensic analysis The importance of a negative value is revealed in the following step when values are accumulated and thus reflect on the overall weight of contributing factors whether they reduce or contribute to damage. The value of this is clear where one particular factor may both It was deemed necessary to treat each individual element and sector of the infrastructure network separately by carrying out individual forensic investigations for each in order to firstly establish the direct impact, and then take an overarching approach to how direct impact to each of these sectors indirectly impacted on businesses. This decision was taken due to 3 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences 7 16004 ( 2016) LOODrisk 2016 - 3rd European Conference on Flood Risk Management FLOODrisk 2016 - 3rd European Conference on Flood Risk Management Welfare and social systems such as food distribution centres and financial centres as well as emergency services are also of importance for the functioning of normal services during a flood event6. In order to quantify the impacts on society it is important to assess the duration of disruption, the area affected, the number of people affected and combinations of these, having first analysed the effect of element failure on the network and then the effect of this on other networks. In order to be able to analyse the real weight of factors of vulnerability and be able to comment on the varying degrees of relevance for different sectors it was essential to be able to apply values to the importance of each factor. In order to do this, informed judgements were made and there was an element of subjectivity. Statistically speaking, by combining the sums of various subjective values, any discrepancies are aggregated and cancel each other out. A cumulative approach is thus beneficial. So the next step is a return to the previous section where the above weighted values are added together and input into the vulnerabilities columns as shown in Table 4. This gives a cumulative number of the impact of each vulnerability factor based on comparable parameters.3 This then allowed for the calculation of the total impact of each vulnerability in order to establish which is the most critical as shown in Table 5. Similar problems are faced by businesses subjected to disruption of the road transport system due to the damage to primary roads which may be closed for some time. Organisations, businesses and infrastructure systems can be considered as networks consisting of various actors and information flow. Studies of networks demonstrate that networks consisting of a few key nodes, or hubs, and high connectivity between them, followed by an increasing number of other nodes with decreasing connectivity are less vulnerable to failure due to a deletion of nodes or breaking of links7. Because external shocks to the network can happen at random the likelihood that a peripheral hub would be affected is small and the network will not lose its connectedness. However, many networks can have a weakness in that they are vulnerable to a shock that affects the most highly connected hubs. FLOODrisk 2016 - 3rd European Conference on Flood Risk Management Table 1 Forensic analysis listing contributing factors assigned to vulnerability class Table 1 Forensic analysis listing contributing factors assigned to vulnerability class Table 2 Assigning of impact to affected sectors Table 2 Assigning of impact to affected sectors Table 2 Assigning of impact to affected sectors 4 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences 7 16004 ( 2016) FLOODrisk 2016 - 3rd European Conference on Flood Risk Management Table 3 Assigning of weight to impact on affected sectors. Total is calculated by summing weights across all sectors. Table 3 Assigning of weight to impact on affected sectors. Total is calculated by summing weights across all sector Table 4 Total weight of factor is substituted back into vulnerabilities columns in order to calculate total significance for each vulnerability. Table 4 Total weight of factor is substituted back into vulnerabilities columns in order to calculate total significance for each vulnerability. 5 , E3S Web of Conferences 7 16004 ( 2016) DOI: 10.1051/ 6 e3sconf/201 0716004 FLOODrisk 2016 - 3rd European Conference on Flood Risk Management ! " ! # $%& '% ( ) ! * + ! " !! ! !! # !$ Table 5 Total weight of each vulnerability calculated by summing accumulated impact. Table 5 Total weight of each vulnerability calculated by summing accumulated impact. 6 6 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences 7 16004 ( 2016) , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) , FLOODrisk 2016 - 3rd European Conference on Flood Risk Management ( ) reduce and contribute to damage for varying sectors. This is shown in Table 3. transportation, and communication systems. 3 Infrastructure and business As part of the IDEA project this paper focuses specifically on the indirect impact of infrastructure failure on businesses in the 2007 Severn Trent floods in the U.K.. In a report produced as part of the FloodProBE project,4 critical infrastructure is defined as that infrastructure which is essential for the functioning of society, whose failure would seriously affect many people. The report offers an assessment of the vulnerability to infrastructure which includes an analysis of the effects of element failure on a network. The - effects of infrastructure failure and the inter-dependency of infrastructures. In fact the study focuses more on these secondary impacts than on the direct impacts and offers a relevant analysis of the vulnerability of infrastructure and its impact on businesses. Table 6 Interdependancies within infrastructure system and with other sectors. Rapid recovery of businesses may be hampered more by the loss of critical infrastructure than by direct physical damage. Tierney5 found businesses were forced to close by disruption to utility services more often than by direct damage during 1993 flooding in the U.S. Midwest. 15% of Des Moines businesses experienced flood damage while 42% were forced to close due to lack of regular services such as electricity or water. If a business itself is not directly impacted by a disaster, damage to offsite lifelines will still have a negative impact on recovery. Table 6 Interdependancies within infrastructure system and with other sectors. FLOODrisk 2016 - 3rd European Conference on Flood Risk Management The Internet is one example of this in that internet traffic is rarely disrupted by disruptions to smaller servers but can be extremely disrupted by a failure of a few key hubs8. Such perspectives suggest that the more reliant a business is on the satisfactory operation of infrastructure, the more vulnerable it will be to the impacts of climate related incidents such as flooding. Table 6 below shows a diagramatic example of how infrastructures relate to each other and how other sectors rely on the functioning of the system. Having carried out this exercise for each individual element or sector of the infrastructure network, the factors that were judged to have had an indirect impact on businesses were then translated into the forensic analysis of the business sector and the same process then carried out again. This narrows the field of analysis and allows for the assessment of importance of factors based on their perceived impact. Similar analysis can then be carried out offering insight into the categories of vulnerabilities. 4.1 Infrastructure During the flooding of 2007, infrastructure in the Gloucestershire area was heavily impacted with the closure of the Mythe Water Treatment plant leaving 350,000 people without drinking water for up to 16 days, an estimated 10,000 people stranded on the M5 motorway and 42,000 people left without power when a controlled shutdown of the Castlemeads Power Station was carried out being just a handful of examples of widespread damages to infrastructure. As previously outlined in Section 3, direct impacts to infrastructure can lead to indirect impacts to business and this forensic analysis aimed to identify and quantify these impacts and their root causes. Figure 1 The most significant vulnerability that resulted in damage was systemic vulnerability due to the fact that many people in the area were reliant on a single source of drinking water. In addition to this, the treatment plant was unable to draw on other source of water and reservoirs. Systemic vulnerability was followed by organisational vulnerability in terms of significance. The range of organisational factors was not confined to just those of the plant management but also to decisions made about emergency management and flood defences. One of the most significant factors was the inadequacy of the emergency plan for the plant that was in place prior to the flood in dealing with the scale and extent of the flooding. Physical and social vulnerabilities are the least significant of the identified vulnerabilities. The physical vulnerability in this case was minimal mainly due to some decisions such as the one to shut down the water treatment plant helped to reduce the overall impact of the flooding. Social vulnerabilities were less significant due to the isolated and technical nature of infrastructure. In this case social aspects became involved mostly in the emergency operation and provision of water to customers. Although significant in their own right, they contribute less to damage than the others In a regional context as is the case in Gloucestershire, the most important elements are electricity, water supply, wastewater drainage systems, transportation, and communication systems. Welfare and social systems such as food distribution centres and financial centres as well as emergency services are also of importance for the functioning of normal services during a flood event. , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) Figure 1 be rescued and 13 people died. Nearly 500,000 people were left without water or electricity and the bill was expected to be more than £3 billion. 4.1 Infrastructure Due to the complex interdependencies of infrastructure networks, with many elements relying on the operation of one or many others, it is difficult to equate the impact the failure of one element has on a region or the businesses in that region. For this reason it was important to carry out forensic investigations for each sector of infrastructure in order to establish any indirect impacts that occurred between different infrastructures. 4 The Severn Trent Floods This particular case study focuses on the Gloucestershire area of the U.K. which has experienced severe flooding in recent years, most notably in 2007 which resulted in the largest emergency operation in the U.K since World War II. In June and July 2007 more than 55,000 properties were flooded, 7,000 people had to In a regional context, the most important elements are electricity, water supply, wastewater drainage systems, 7 FLOODrisk 2016 - 3rd European Conference on Flood Risk Management %& ' , #** -* ( # &#* .- * ! / ! + ! ,0 * ! ( &%&/.# ! ! #* ! ** ! 1* ! 2 ( '2 Table 7 List of most significant contributing factors affecting Severn Trent Water Chipping Campden, Gloucester, Cheltenham, and Tewkesbury to help people that could not get home. These rest centres collectively accommodated around 2000 people at the height of the emergency.9 The forensic analysis of the network revealed the following results. Figure 3 Figure 3 As can be seen, the most significant vulnerability is the physical characteristic of the system. This relates mostly to the deterioration of assets on the network, physical damage to roads, a lack of maintenance, restricted access caused by water on the roads, congestion and the resulting difficulties for emergency operations that resulted from congestion. Table 7 List of most significant contributing factors affecting Severn Trent Water Table 7 List of most significant contributing factors affecting Severn Trent Water As was the case in the water network, the hazard is again significant but since it is out of the control of the infrastructure sector and therefore must be considered separately to the other vulnerabilities. The sectors that were most affected indirectly by the damages to the water infrastructure were residences, businesses, other infrastructure, emergency services and government agencies such as the EA and district councils. The graph below illustrates the weight of the impacts on each sector with residents and businesses being the most impacted. Systemic vulnerabilities were the next most significant aspect for the transport network with restrictions in the network that lead to congestion. 4.1.1 Water Following these vulnerabilities was the hazard itself. Without the flood there would be no flood damage. In this sense, the hazard, although amplified by certain factors, is unavoidable from the perspective of the water company. Water treatment plants represent a critical infrastructure in their provisions of healthy drinking water to a large number of people across the country. In the case of the flooding in Gloucestershire, this is the service that was most disrupted and which consequently had the biggest impact on the businesses and public as a whole. As a particularly clear and poignant example, the failure of the Mythe water treatment plant owned by Severn Trent Water (STW), resulted in loss of drinkable water to over 350,000 people for up to 16 days. This extreme incident was coupled with widespread minor incidents and damage to other assets such as the sewerage system and other smaller pumps. Severn Trent Water estimate the total cost to the company due to direct damage to be in the region of £25 million and £35 million. In terms of the most significant individual factors, the list below illustrates the contributing weight of each. It is worth noting that the weighted factor considers both contributing and reducing factors. For this reason the shutting down of the plant, although it affected many people in the short term, reduced the long term damage that would have resulted if essential equipment was damaged. infrastructure was carried out as outlined in Section 2.2 and the following results emerged. 8 8 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences 7 16004 ( 2016) FLOODrisk 2016 - 3rd European Conference on Flood Risk Management FLOODrisk 2016 - 3rd European Conference on Flood Risk Management It was noted that improved use of secondary roads and dual-lane motorways would have led to fewer disruptions. The rail network by its nature is quite susceptible to systemic vulnerabilities and was affected greatly. Figure 2 4.1.2 Roads and Transport Organisational vulnerability is the next most significant vulnerability and relates to decisions taken within the sector that lead to disruptions such as the inefficiency in the communication of information to motorists, cooperation with local authorities and emergency services which were hindered by a lack of understanding due to communication issues and inefficiencies in emergency plans in dealing with an event of such a scale. However, actions to provide support for those stranded on roads and railways were overall quite effective. 4.1.2 Roads and Transport As a result of the flooding the roads and transport network was seriously disrupted. Motorists and public transport users found themselves stranded as the transport network struggled to cope with the conditions ! an estimated 10,000 motorists were stranded overnight on the M5 between junctions 10 and 12 and on some other roads, and approximately 500 people were stranded at Gloucester Railway Station as the railway network failed. Rest centres were established in Moreton-in-Marsh, " of knowledge about how to react in such events were less significant but nonetheless did exist. In terms of the most significant individual factors, the list below illustrates the contributing weight of each. It is worth noting that the weighted factor considers both contributing and reducing factors. For this reason, while access to emergency supplies for example, was restricted, 9 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences 7 16004 ( 2016) FLOODrisk 2016 - 3rd European Conference on Flood Risk Management Figure 4 the network in general operated well and resulted in very few threats to life. The biggest impact was to the normal operation of businesses and daily life. -' /! 3 4 5 $ 6!5 * 1* 7- * ! FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 4 . * 8 ! (5 ! - ! 293 ! , ! , * ! * ! ! 2 5 : ;<;; ; ; ! % ! & -3 ! 2 * ( $ #-))( # (93 # % * -* - - = -* - + + % & Table 8 List of contributing factors affecting roads and transport network Figure 4 , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) Figure 6 The physical vulnerability of assets on the network to flooding is a major area of concern for the electricity supply companies who spend a large amount of time and money maintaining these during normal operational periods. In times of crisis this maintenance is much more resource intensive and some assets were either damaged or, shut down in order to avoid damage. The nature of electrical equipment makes it very susceptible to damage from flooding Organisational decisions made during the emergency lead to large impacts. The decision to take the Castlemeads power station off line may have saved potential long-term damage but it affected several thousand customers and resulted in large costs to the company and disruption to operations. Figure 6 Somewhat surprisingly, this analysis reveals that systemic vulnerabilities are not as significant as others. This may be down to a lack of evidence or information but it is clear that it is significant. Perhaps systemic resilience resulted in comparatively small impacts compared to the closure of the water treatment plant for example. 4.2 Business Having carried out forensic investigations for each of the previously outlined infrastructure sectors, the factors that affected businesses indirectly were translated to a forensic investigation of the business sector. These factors were categorised as indirect factors while direct factors were added which were not related to infrastructure. From this point a similar process was followed as for the infrastructure sectors. The aim of the forensic investigation for businesses was to compare and contrast the effect of direct and indirect vulnerabilities. In terms of the most significant individual factors, the list below illustrates the contributing weight of each. It is worth noting that the weighted factor considers both contributing and reducing factors. 4 1* ! .- ! ! & -3 ! ( (* * #* (93 Table 9 List of contributing factors affecting electricity network 4.1.3 Electricity Impacts to the electricity supply network were also widespread. However, less information was available regarding the factors and impacts as were available for the water and transport networks. However a forensic analysis was still able to be carried out. During the flood event in the summer of 2007 there was a high risk that the Walham electricity sub-station, which serves Gloucestershire and parts of Wales and Herefordshire, could be flooded leading to electricity failure. Gloucestershire Fire and Rescue Service led the efforts to save Walham from flooding, calling in flood defence barriers and additional pumping equipment. The massive operation continued on 23rd July to prevent serious flooding at Walham sub-station up until the predicted high tide at 3am. The flood defences withstood the high tide and the immediate threat of loss of electricity passed. However, at the Castlemeads electricity sub-station, operated by Central Networks, the situation had deteriorated and surge water had overwhelmed the pumps. This led to a controlled switch off of power, initially leaving about 42,000 people in Gloucester without power. By the end of the day the number of homes without power was reduced to 1,800. The most significant vulnerabilities aside from the hazard were organisational and physical vulnerability followed by systemic vulnerabilities. Figure 5 Table 8 List of contributing factors affecting roads and transport network As shown in Figure 4 the sectors most affected by direct damage to the transport network were residents/motorists and emergency services, followed by businesses and public agencies. This is in contrast with the water network which affected businesses most. Figure 5 10 4.2.1 Indirect damages Having accumulated and analysed the impact of indirect damages to businesses caused by direct damage to infrastructure it can be seen that organisational vulnerabilities contributed more to damage than any other. Figure 7 Table 9 List of contributing factors affecting electricity network As shown in Figure 6 the sectors most affected by direct damage to the electricity supply network were both the residents and businesses equally with other infrastructures, emergency costs and public agencies. Figure 7 Organisational vulnerabilities of infrastructure refer to the decisions taken by management in the infrastructure companies that lead to increased impact on businesses. These decisions include the decision to shut the water treatment plant and the electricity supply plant for example. In relation to roads and transport they relate to the lack of communication to motorists about what to do in emergency situations. Organisational vulnerabilities of infrastructure refer to the decisions taken by management in the infrastructure companies that lead to increased impact on businesses. These decisions include the decision to shut the water treatment plant and the electricity supply plant for example. In relation to roads and transport they relate to the lack of communication to motorists about what to do in emergency situations. Physical vulnerabilities were the second most significant for businesses. This includes the location of the water treatment plants and the power supply plants and roads which were all in physically vulnerable 11 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) , E3S Web of Conferences 7 16004 ( 2016) locations. Also the nature of the emergency was particularly hazardous for both electricity and water treatment as water can be disastrous for both. cost of repairs and recovery and to difficulties in accessing insurance and public funding. Again in contrast to direct damage, systemic vulnerability is less significant. This is due to the nature of business in comparison with the nature of infrastructure networks which depend on many individual elements of the system to operate functionally I order to maintain constant supply of services. Following physical vulnerability is systemic vulnerability which in infrastructure relates to reliance on one single element of a system which exposes end users to the risk of losing service in the case of that one element being damaged or isolated. In this case each of the infrastructure networks experienced this. 4.3 Conclusions of forensic analysis Several conclusions can be drawn from this in-depth analysis. Firstly, the clearest difference between indirect and direct vulnerabilities is the increased significance of economic factors. Businesses are reliant on cash flow and continuity of business in order to minimise damages. Not only does damage to stock and premises have an economic impact, so too does the loss of custom. However, the difficulty in accessing insurance and public funding and the problems related to identifying direct and indirect costs due to flooding mean that the economic vulnerabilities can leave businesses struggling for a long time after a flood. Secondly, systemic vulnerabilities, while extremely significant for infrastructure and indirect impacts, are less so for businesses themselves. This means that improvements in the systemic resilience of networks would benefit businesses more than increasing the systemic resilience of businesses. Economic vulnerabilities refer to the impact that reduced custom had on business as people were occupied with dealing with the emergency. 4.2.1 Indirect damages Social vulnerabilities include such factors as the misuse of water tanks that were provided by the water company once the water treatment plant had been shut down. This resulted in loss of water and damage to equipment. Additionally, the increased use of water once the closure was announced as imminent affected emergency supplies. It also refers to the ability of businesses to respond to the indirect damages as many had no previous experience of flooding of this scale and emergency services were overwhelmed. The fact that many customers were lost due to being occupied with managing their own situations affected business as did the fact that many thought that particular towns were flooded and avoided shopping there for some time after they were cleared. 4.2.2 Direct damages Direct damages are those that directly affected businesses such as the flooding of premises, loss of stock, closure of business due to flooding, and relocation costs. 5 Findings ! " #$$% Figure 8 5.1 Assessment of forensic investigation The forensic investigation methodology developed is very useful in identifying the root causes of damage, direct and indirect. Comparisons can be made between different sectors and how they are impacted in different ways. Also, this methodology allows us to assign contributing weights to the different sectors of the infrastructure network. Figure 8 When it comes to direct vulnerabilities physical vulnerability is again the most significant. This relates mostly to flooding of properties and development of floodplains which led to properties being located in high flood risk areas. The methodology gives a comprehensive overview of the reality of the situation by drawing together factors from a wide range of perspectives thus illuminating areas that are often overlooked when it comes to damage impact analysis. The multi-disciplinary approach covers areas that relate to social, physical, organisational, economic and systemic aspects of vulnerabilities. This is very beneficial in identifying the best area for investment of funds and mitigation measures. Organisational vulnerabilities are the second most significant vulnerability and include the lack of knowledge with regard to recovery methods. Additionally, many businesses were not prepared for flooding and did not have business continuity plans or emergency action plans in place which led to increased damage and longer recovery times. However, it is clear from the case study approach that any forensic analysis is only as good as the data that is used. In this case it was difficult to access primary data that would allow for more in-depth analysis, in particular In contrast to indirect damages, economic vulnerabilities are more significant. This relates to the 12 , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) , E3S Web of Conferences 7 16004 ( 2016) , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conferenc 7 16004 ( 2016) DOI: 10.1051/ 6 e3sconf/201 0716004 ) ' with regard to the actual impact on businesses. While some figures can be found that refer to national estimates and some local statistics, without comprehensive knowledge of a further dataset, actual impacts must be assumed. 5.1 Assessment of forensic investigation This reduces the validity of the findings. 0 . ) Secondly, the weights assigned to factors are assigned based on informed judgement and are subjective. An attempt is made to address this by using controlled parameters but often it is left open to the judgement of the investigator. # 1 / 2 &3 5.2.2 Categories of data * 8 . 4 / / . ( #$$% ' ) Timeliness of collection can significantly affect the quality and usability of data. With many organisations reporting on the difficulties of getting staff to cut-off areas during flooding, much data is not collected at the most appropriate time, when the flood waters are at their highest. Additionally, continuous monitoring after the event to establish recovery and drying out times etc. would greatly improve the ability to estimate actual damage and impact. 5.2.2 Categories of data The importance of a common platform for data sharing as well as of shared procedures for data collection (that envisages also the integration of present missing information) has then been identified as a key objective for the IDEA project. Ultimately, judgements must be made regarding the reliability of any data used and this is in the hands of investigating team and ultimate responsibility lies with them. 7 References 1. Integrated Research on Disaster Risk. (2011). Forensic Investigations of Disasters: The FORIN Project (IRDR FORIN Publication No. 1). Beijing: Integrated Research on Disaster Risk. Acknowledgement 9 4 , ) 4 The research described in the paper has been developed within the project "IDEA" (Improving Damage assessments to Enhance cost-benefit Analyses, EU prevention and preparedness project in civil protection and marine pollution) funded by DG-ECHO, G.A.N. ECHO/SUB/2014/694469). Authors acknowledge with gratitude all the people involved in the project. 5.2.1 Sources of data comprehensive view over political, social, economic, structural, physical and systemic vulnerabilities that is innovative and extremely useful in improving the targeting of mitigating measures that can build overall resilience. 5 + &0$ ( 67#$$% This approach is very beneficial in an area where perspectives are often limited and isolated within particular fields of expertise and where investment can be fragmented and inefficient. The aim of such a forensic approach is to improve the cost-benefit of limited resources in tackling the underlying issues that often result in widespread damages during hazard events. However, this approach is limited by the quantity and quality of data available and suitable data can only be relied upon if it is being collected at the right time and with the correct parameters. Quality control of data starts at collection and continues through storage, analysis and sharing. Effective collaboration across all agencies and bodies involved in data collection is a key factor in assuring reliable and useable data for forensic analysis. 5.2 Data requirements for forensic investigation Figure 9 Range of data needed for forensic analysis in relation to businesses & & ' " () * * + , , " - * - Figure 9 Range of data needed for forensic analysis in relation to businesses * 4 . / / Figure 10 Categories of data and agency responsible Figure 10 Categories of data and agency responsible Figure 10 Categories of data and agency responsible 13 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 7 16004 ( 2016) 6 Conclusions !$ " 4+$>!?$/ * @ $ 5!(A$ Forensic disaster investigations offer a valuable tool that offers unique insight into the root causes of disaster damage. This methodology brings together expert knowledge from a wide range of disciplines and casts a 14 DOI: 10.1051/ 6 e3sconf/201 0716004 , E3S Web of Conferences 7 16004 ( 2016) FLOODrisk 2016 - 3rd European Conference on Flood Risk Management FLOODrisk 2016 - 3rd European Conference on Flood Risk Management 3. Smock, R. (2002) Reducing subjectivity in qualitative risk assessments, GSEC Practical Requirements (v.1.3) (December 2001) Requirements (v.1.3) (December 2001) q 4. Heilemann, K., Balmand, E., Lhomme, S., De Brujin, K., Linmei, N. & Serre, D. (2013). Identification and analysis of most vulnerable infrastructure in respect to floods. Floodprobe D2, 1. 5. Tierney, K. (1997a). Impacts of recent disasters on businesses: The 1993 Midwest floods and the 1994 Northridge Earthquake (No. NCEER-SP- 0001). Buffalo: Multidisciplinary Center for Earthquake Engineering and Research, State University of New York at Buffalo. 6. McBain, W., Wilkes, D. & Retter, M. (2010). Flood resilience and resistance for critical infrastructure, CIRIA. 7. Allenby, B. & Fink, J. (2005). Toward inherently secure and resilient societies. Science, 309, 1034-1036. 8. Allen, C. R., Gunderson, L. & Johnson, A. (2005). The use of discontinuities and functional groups to assess relative resilience in complex systems. Ecosystems, 8, 958-966 8. Allen, C. R., Gunderson, L. & Johnson, A. (2005). The use of discontinuities and functional groups to assess relative resilience in complex systems. Ecosystems, 8, 958-966 9. Gloucestershire County Council, (2007). Scrutiny final report. Overview and Scrutiny Management Committee. 15
https://openalex.org/W3098837797	https://link.springer.com/content/pdf/10.1007/s43039-020-00015-4.pdf	English	null	The great Millennials’ trouble: leading or confused green generation? An Italian insight	Italian journal of marketing	2,020	cc-by	9,490	Italian Journal of Marketing (2020) 2020:289–308 https://doi.org/10.1007/s43039-020-00015-4 Italian Journal of Marketing (2020) 2020:289–308 https://doi.org/10.1007/s43039-020-00015-4 ORIGINAL ARTICLE * Anna Paola Codini anna.codini@unibs.it The great Millennials’ trouble: leading or confused green generation? An Italian insight Michelle Bonera1 · Anna Paola Codini1 · Giulia Miniero2 Received: 23 March 2020 / Accepted: 2 November 2020 / Published online: 17 November 2020 © The Author(s) 2020 Michelle Bonera michelle.bonera@unibs.it 1 Dipartimento di Economia e Management, Università degli Studi di Brescia, C.da S. Chiara 50, 25122 Brescia, Italy Keywords Green consumption · Millennials · Green generation · Cluster analysis · Green values 2 Franklin University Switzerland, Via Ponte Tresa 29, 6904 Lugano, Switzerland * Anna Paola Codini anna.codini@unibs.it Michelle Bonera michelle.bonera@unibs.it Giulia Miniero Gminiero@fus.edu 1 Dipartimento di Economia e Management, Università degli Studi di Brescia, C.da S. Chiara 50, 25122 Brescia, Italy 2 Franklin University Switzerland Via Ponte Tresa 29 6904 Lugano Switzerland 1 Introduction Over the past decades, increasing attention has been paid to the future of the planet (Kim and Choi 2005; Synodinos and Bevan-Dye 2014). Environmental sustainability has been raised at the top of the international political agenda (United Nations 2017) and has been recognized as a critical driver of innovation for companies (Nidumolu et al. 2009; Hopkins 2010). Consumers say that manufacturers are responsible for the future of the planet (Young 2018), and this leads all companies to face the challenge of integrating environ- mental issues into business strategy and activities (Nidumolu et al. 2009). However, to fully realize the true potential of the green market, businesses must help consumers to change their behaviours. Green consumer behaviour is vital to the sustainability of the environment (Tobler et al. 2012). Consequently, the identification of consumers’ driv- ers for pro-environment behaviour and the explanation of the relationship between such drivers and the pro-environment behaviour has become relevant in the literature (Wan- del and Bugge 1997; Tariq 2014; Wong et al. 2014; Miniero et al. 2014). Among consumers, Millennials are regularly considered the ones driving the sus- tainable movement with their lifestyle and behavioural changes (Sheahan 2005; Smith 2011; Hanson-Rasmussen and Lauver 2018). Therefore, they are also referred to as the “Green Generation”. Furthermore, Millennials are more likely than any other genera- tion to say that they would pay extra for eco-friendly and sustainable products (Young 2018). According to this trend affecting Millennials and other generations, many brands tried to catch the appeal and quickly capitalize on these changes. For instance, UK sandwich chain Pret A Manager has now three entirely vegetarian outlets, L’Oreal has recently launched its first vegan hair colour range, and Unilever rolled out its new brand “Love Beauty and Planet”, an eco and vegan-friendly brand using bottles made from recycled plastics. This experience has been fruitful, as, in 2018, Unilever declared that its “sustainable living” brands grew faster than the other businesses, leading to 70% of its turnover growth. Despite its green image, the Millennial’s consumption of green products has not been deeply researched (Smith and Brower 2012). In particular, no many studies focused on the differences within Millennials generation in terms of green consump- tion (Coşkun and Özbük 2019) and motivations to buy or not to buy green products. Abstract The paper purpose is to investigate the green consumption behaviour of the Millen- nials generation. The paper aims to understand if all Italian Millennials are similar in terms of green consumption and if there are differences in adopting and consuming green products. As Millennials are considered the driving generation of the sustain- able movement, thanks to their lifestyle and behaviours, our study tries to compre- hend whether these consumers can be considered the leading “Green Generation”. We performed a cluster analysis, using the non-hierarchical method by applying the k-medium algorithm, segmenting Millennials. The segmentation was performed according to the reasons underlying and against green consumption. Then, green values and green consumer behaviour of the segments were analyzed. The results of our research reveal the existence of different clusters of Millennial consumers in terms of green attitude. Some confusion about green issues in the Millennials gen- eration emerges, contrasting with the literature. Our results do not necessarily imply a negative attitude towards green values by Millennials, but different green attitudes among the clusters. Keywords Green consumption · Millennials · Green generation · Cluster analysis · Green values 1 Dipartimento di Economia e Management, Università degli Studi di Brescia, C.da S. Chiara 50, 25122 Brescia, Italy 2 Franklin University Switzerland, Via Ponte Tresa 29, 6904 Lugano, Switzerland (0123456789) 1 3 456789) 3 Italian Journal of Marketing (2020) 2020:289–308 290 2.1 Millennials generation Millennials have been indicated by the demographers Strauss and Howe (1991) as the generation born between 1982 and 2003. Despite the Millennials’ years of birth cutoff points, that can differ according to other authors and oversimplify the differences between groups, the cohort is defined by the differentiating cultural traits as they appear to be special, sheltered, confident, conventional, team-ori- ented, pressured, and achieving (Strauss and Howe 1991). They are also known as Generation Y, Next Generation, Net Generation (Shap- ira 2008), Generation me (Twenge and Me 2006), Echo Boomer, or New Boomer (Carlson 2009). Currently, they represent around 27% of the world population (Peterson et al. 2017) and, according to the demographic projections of the U.S. Census Bureau (Vespa and Medina 2018), in the US are on the verge of surpass- ing the largest generation of the last 100 years, the Baby Boomers, becoming, in turn, the largest generation of living adults in the nation. For these reasons, soon, it will find itself having a greater weight in terms of workforce, consumption, and spending capacity (Debevec et al. 2013). Concerning Millennials, it emerged that most of the research was conducted in the United States, starting from the early 1990s (Corvi et al. 2007). Equivalent studies in Europe appear to be much rarer also in 2019. Considering the characteristics that can identify Millennials, these can differ according to the birthplace, social and economic conditions, culture, and life experiences, but due to globalization, social media, the export of Western culture, they present many common traits. y p y It is possible to describe the Millennials as a generation with higher levels of education than previous age groups; Millennials are struggling to achieve eco- nomic independence from their family by carrying out jobs of a lower level than their qualification. They grew up during the Internet development and globali- zation process. Therefore, they are the most informed, demanding, and aware generation ever, they are accustomed to thinking and acting quickly (Junco and Mastrodicasa 2007; Kilian et al. 2012; Nielsen 2017). They are multitasking, and they have widespread social networks (Parment 2009; Hewlett et al. 2009). They are self-confident and goal-oriented (Howe and Strauss 2000; Twenge 2014), and they aspire to a healthy and balanced lifestyle (Valentine and Powers 2013).f Millennials show greater acceptance of cultural differences and changes with a different way of perceiving equal rights and equality (Howe 2010). 1 Introduction In order to fill this gap, this paper aims at describing green consumer behaviour of the Millennials generation, in order to understand if Millennials can be considered as the “Green Generation.” Thanks to a cluster analysis, this paper will try to answer to this research question: are all Millennials the same in terms of green consumption? Are there differences in terms of the adoption and consumption of green products? 1 3 1 3 Italian Journal of Marketing (2020) 2020:289–308 291 2.1 Millennials generation Millenni- als value freedom of speech, self-expression, but also society by being extremely group-oriented (Muralidharan et al. 2016; Nielsen 2017). The causes behind the greater solidarity of the Millennials towards their neighbour are not attributable to a feeling of empathy. However, they lie in a less rigorous education that they have received, characterized by fewer formalities in dealing with teachers and parents. This led to a de-legitimization of the authority of institutions: the rules and way of thinking of previous generations are being considered less influencing, with 1 3 Italian Journal of Marketing (2020) 2020:289–308 292 the consequent abandonment of prejudices, on gender, race and sexual orientation (Twenge and Me 2006).i They have confidence in the possibility of creating a better future and of cor- recting the faults of previous generations (Eisner 2005). They accomplish tasks in order to get an endorsement for their efforts. Moreover, as they are self-absorbed and reliant and demonstrate a high sense of independence (Eisner 2005), they are less involved with behaviours that do not make them feel important or do not appeal to them (Alexander and Sysko 2012), also showing negative traits such as narcissism, cynicism and skepticism (Twenge and Me 2006). 2.2 Millennials consumption and green consumption features Relatively to the Millennials’ consumer cohort, they are capable of maintaining remote relationships at any time (Table 1), with friends, customers, suppliers or col- laborators, breaking down all the space–time barriers existing in the past (Dimock 2018). To a lesser attachment to political and religious institutions, Millennials Gen- eration compensates with a network of friends, colleagues and affinity groups of interest (Fry 2018; Censis 2016). They want to know where the products they buy come from, which are the com- panies’ policies, and they do not hesitate to compare companies, using social media (Nielsen 2017). Technology and social networks allow them to dispose of a large amount of information (Wolburg and Pokrywczynski 2001). Millennials can eas- ily do extended online research before buying a product. It is, therefore, a dynamic target, difficult to conquer, and characterized by impatience (Alexander and Sysko 2012), being very inclined to change, to research, to constant innovation (Parment 2009; Reisenwitz and Iyer 2009; Eurispes 2017).i Moreover, marketing is a well-known field for them, and marketing activities are viewed with suspicion (Tsui and Hughes 2001). Millennials have grown up in a Table 1 Millennials main consumption features. Source Authors elaboration Ability to maintain remote relationships Dimock (2018), Fry (2018) and Censis (2016) Disposability of a large amount of information Wolburg and Pokrywczynski (2001) Willingness to get information about products they buy and to compare companies, using social media Nielsen (2017) A dynamic target, difficult to conquer, and character- ized by impatience Alexander and Sysko (2012) Very inclined to change, to research, to constant innovation Parment (2009), Reisenwitz and Iyer (2009) and Eurispes (2017) Marketing is a well-known field for them, and market- ing activities are viewed with suspicion Tsui and Hughes (2001) Respond to company announcements with low levels of tolerance, more brand awareness and the ability to distinguish illusion from reality Engebretson (2004), Ciminillo (2005), Pesquera (2005) and Valentine and Powers (2013) More aware of targeted advertisements Aschemann Witzel and Niebuhr Aagaard (2014) Brands tend to be less important in the purchasing decision Caplan (2005) and Phillips (2007) Table 1 Millennials main consumption features. Source Authors elaboration 1 3 Italian Journal of Marketing (2020) 2020:289–308 293 media-saturated world, bombarded since their early teens with thousands of adver- tisements a year (Valentine and Powers 2013). 2.2 Millennials consumption and green consumption features For these reasons, they respond to company announcements in a different way, with low levels of tolerance, more brand awareness and the ability to distinguish illusion from reality (Engebretson 2004; Ciminillo 2005; Pesquera 2005; Valentine and Powers 2013). Generation Y members are becoming more aware of targeted advertisements, and this makes them immune to certain communications, albeit digital (Aschemann Witzel and Niebuhr Aagaard 2014). Even brands tend to be less important in the purchasing decision (Caplan 2005; Phillips 2007). Furthermore, Millennials represent an important tar- get for marketers concerning their high spending power (Howe and Strauss 2007). The historical period of maximum expansion of sustainable environmental prob- lems was precisely in the period of the birth of the Millennial generation, which grew from infancy in a climate dedicated to pro-sustainability propaganda, which was able to make them more likely to possess a green conscience (Hyllegard et al. 2011; Nielsen 2017) (Table 2). This generation has better knowledge of the environ- mental issue and is more concerned about saving the planet (Schmeltz 2012; Kan- chanapibul et al. 2014; Casaló and Escario 2016). As Millennials are aware of the problems concerning the ecosystem, even if few young people have real in-depth knowledge on the subject, it is logical to expect that they adopt new behaviours that support sustainable development (Ivanova et al. 2019). Many studies iden- tify a greater propensity to sustainable buying for Millennials than previous gen- erations (Fischer et al. 2017; Hanson-Rasmussen and Lauver 2018). The results of the research by Kanchanapibul et al. (2014) show that there is a good relationship between the Millennials and the world of ecology. They want companies to have a positive impact on society more generally (Deloitte 2018). Conversely, Millenni- als seem to have a higher green consciousness, not necessarily leading to a green behaviour (Uyeki and Holland 2000; Diamantopoulos et al. 2003; Johnson et al. 2004; Grønhøj and Thøgersen 2012; Hume 2010). This phenomenon is known as the “green gap” (Kennedy et al. 2009; Nielsen 2017). Even if they are more likely to purchase sustainably, other personal factors can take over. Among these, par- ent–child relationship (Wilcock et al. 2004; Jang et al. 2011; Lappänen et al. 2012; Anvar and Venter 2014; Meeusen 2014; Muralidharan et al. 2016; Casaló and Escario 2016; Naderi and Van Steenburg 2018) modifying interest, awareness and therefore the intention to purchase green products (Kanchanapibul et al. ble 2 Millennials main positive and negative green consumption features. Source Authors elaboration Table 2 Millennials main positive and negative green consumption features. Source Authors elaboration Have a better knowledge of the environmental issue and are more concerned about saving the planet Schmeltz (2012), Kanchanapibul et al. (2014), Casaló and Escario (2016), Hyllegard et al. (2011), Nielsen (2017) and Kanchanapibul et al. (2014) Greater propensity to sustainable buying Ivanova et al. (2019), Fischer et al. (2017) and Hanson-Rasmussen and Lauver (2018) Want companies to have a positive impact on soci- ety more generally Deloitte (2018) Environmental concern not necessarily leading to green behaviour Uyeki and Holland (2000), Diamantopoulos et al. (2003), Johnson et al. (2004), Grønhøj and Thøgersen (2012), Hume (2010), Kennedy et al. (2009) and Nielsen (2017) The influence of ecological knowledge is weaker than personal motivations when it comes to mak- ing the purchase decision Wilcock et al. (2004), Jang et al. (2011), Lappänen et al. (2012), Anvar and Venter (2014), Meeusen (2014), Muralidharan et al. (2016), Casaló and Escario (2016), Naderi and Van Steenburg (2018), Kanchanapibul et al. (2014) and Heo and Muralidharan (2017) Age does not seem to be a sufficient structural vari- able to guarantee eco-compatible behaviour Muralidharan et al. (2016) The price of green products could hinder the impact of green attitudes on sustainable purchase behaviour Aschemann Witzel and Niebuhr Aagaard (2014) and Covino (2017) The quality and the availability of green products could hinder the impact of green attitudes on sustainable purchase behaviour Bucic et al. (2012) Green products are often perceived as more expan- sive and even less performant Lu et al. (2013) Traditional factors must be satisfied first, and only later ecological ones Bollani et al. (2017) Green characteristics are not part of the main consideration criteria during the purchase of a product Bucic et al. (2012) Have a better knowledge of the environmental issue and are more concerned about saving the planet Schmeltz (2012), Kanchanapibul et al. (2014), Casaló and Escario (2016), Hyllegard et al. (2011), Nielsen (2017) and Kanchanapibul et al. (2014) characteristics are not part of the main consideration criteria during the purchase of a product (Bucic et al. 2012). characteristics are not part of the main consideration criteria during the purchase of a product (Bucic et al. 2012). 2.2 Millennials consumption and green consumption features 2014; Heo and Muralidharan 2017) can be listed.fi Age, therefore, does not seem to be a sufficient structural variable to guarantee eco-compatible behaviour (Muralidharan et al. 2016). The statistical analysis shows how the influence of ecological knowledge of the younger generations is weaker than personal motivations when it comes to making the purchase decision. In par- ticular, the price (Aschemann Witzel and Niebuhr Aagaard 2014; Covino 2017), the quality, and the availability (Bucic et al. 2012) of green products are reasons that could hinder the impact of green attitudes on sustainable purchase behaviour of the young generations. Green products are often perceived as more expansive and even less performant (Lu et al. 2013). This does not mean that other eco-sustaina- ble characteristics are not so important for Millennials. However, traditional factors must be satisfied first, and only later ecological ones (Bollani et al. 2017) and green 1 3 Italian Journal of Marketing (2020) 2020:289–308 294 3.1 Research context The empirical analysis carried out describes the behaviour of Millennials generation and investigates how these consumers could be clustered according to the reasons underlying and against green consumption. Literature dealt with green consumption through market research evidenced how some consumers show, unlike others, a clear propensity to environmentally 1 3 Italian Journal of Marketing (2020) 2020:289–308 295 friendly products (Haws et al. 2014). This suggests the existence of differences in attitudes, values, psychological characteristics affecting the factors underly- ing green purchasing decisions, leading to disparities consistent with purchas- ing behaviour. According to Burke and Davis (2014) and other authors (Mainieri et al. 1997; Roberts 1996; De Pelsmacker et al. 2005; Bezencon and Blili 2010; Leiserowitz et al. 2012; Bucic et al. 2012; Wright et al. 2014), segmentation is beneficial for companies that aim to cater their products to green consumers, as to increase the chances of doing that successfully.i friendly products (Haws et al. 2014). This suggests the existence of differences in attitudes, values, psychological characteristics affecting the factors underly- ing green purchasing decisions, leading to disparities consistent with purchas- ing behaviour. According to Burke and Davis (2014) and other authors (Mainieri et al. 1997; Roberts 1996; De Pelsmacker et al. 2005; Bezencon and Blili 2010; Leiserowitz et al. 2012; Bucic et al. 2012; Wright et al. 2014), segmentation is beneficial for companies that aim to cater their products to green consumers, as to increase the chances of doing that successfully.i Our analysis was based on a survey. The questionnaire was divided into five sections matching scales validated in other studies (Bailey et al. 2016; Burke and Davis 2014; Haws et al. 2014).i The first part consisted of seven questions collecting socio-demographic infor- mation on the respondents (gender, age, education, marital status, profession, nationality). The second part was aimed at understanding the general consumers’ attitude and sensitivity towards green issues based on the Green consumption values scale (Haws et al. 2014). The main aim of these authors was to develop a scale of values that could measure proneness to environmental sustainability and consequently to green behaviour, defined as “the tendency to express the value assigned to envi- ronmental protection through its purchasing and consumption behaviour” (Haws et al. 2014, p. 337).ii Specifically, Haws et al. 3.1 Research context (2014) identified a scale of 6 green values, articulated in six statements using a Likert scale ranging from 1 to 7, according to the level of agreement/disagreement. After examining the relationship between green and other existing environmental measures, Haws’ et al. (2014), demonstrate the abil- ity of 6 green values scale to predict relevant purchase behaviours. So, the second section of the questionnaire reported all the six items from the Haws et al. (2014) scale (a) It is important to me that the products I use do not harm the environ- ment; (b) I consider the potential environmental impact of my actions when mak- ing many of my decisions; (c) my purchasing habits are affected by my concern for our environment; (d) I would describe myself as environmentally responsi- ble; (e) I am concerned about wasting the resources of our planet; (f) I am will- ing to be inconvenienced in order to take actions that are more environmentally friendly). The third and the fourth section of the questionnaire included 25 questions about the reasons for and against green consumption, based on the research of Burke and Davis (2014). The preliminary analysis conducted by Burke and Davis (2014) con- firmed that consumers employ those reasons used for and against ethical consump- tion, as documented in the literature. Besides, the innovativeness of ethical prod- ucts—as a motivating reason for ethical purchases –, and other additional (negative) reasons (trialability, packaging, indifference, availability, confusion, and stigma) were included, as mentioned by a participant to the preliminary analysis.f The different reasons for and against ethical consumption were measured in terms of agreement/disagreement using a scale from 1 to 7. Regarding the reasons for green consumption, the question was articulated as fol- lows. “I purchase green products because”: 1 3 3 Italian Journal of Marketing (2020) 2020:289–308 296 • it helps me fit in with my peers; • it helps make a difference; • they are easy to find; • it can make me an opinion leader; • I can save money; • they are of a higher quality; • they are healthier for me; • it can help me with the creation of my positive identity; • I genuinely care about the issues they deal with; • they may grant me a higher status; • they utilize innovative technology; • I am very informed and am able to distinguish between ethical and non-ethical products. 3.2 Data collection The questionnaire, including the five previous sections, was submitted directly and online via the Google Moduli involving Italian consumers from November 2018 to June 2020. Specifically, two different surveys were conducted using the same ques- tionnaire. The first started in November 2018 and finished in January 2019, while the second started at the end of May 2020 and finished at the end of June 2020. Overall, the sample consisted of 261 respondents of Italian individuals in the 18–38 age group, so Millennials (more precisely, 86% in 18–27 class, and 14% in 28–38 class (Table 3). 3.1 Research context Regarding the reasons against green consumption, the question was articulated as follows. “I reject green products, because”: • there is a monetary risk in trialing them; • their packaging is unattractive or unappealing; • they offer no other benefit (or product features) othe • I am skeptical as to how ethical these products really • I don’t really care about the consequences of not buy • I don’t give them much thought; • they are harder to find in stores; • you have to go to specialty stores to buy them; • I am confused as to what makes a product ethical; • they are of a lower quality; • they are too expensive for the value received; • people who buy them are given a negative stigma; • I am suffering from ethical overload; • I am sick of hearing about it. I am suffering from ethical overload; f • I am sick of hearing about it. These reasons pro and against green consumption are the main variables employed for the cluster analysis. The fifth part of the questionnaire aimed to understand what kind of green products respondents buy and how often, using a scale ranging from 1 (never) to 5 (often). The 17 product categories selected to understand ethical purchasing behaviour were the same included in previous studies, which identified, for example, products purchased daily such as cof- fee (De Pelsmacker et al. 2005), chocolate (Didier and Lucie 2008) and chicken (Teisl et al. 2008). The selection also includes examples of ethical products that respondents mentioned explicitly in Burke’s qualitative research (2014), includ- ing recycled paper for printers and safely caught dolphin tuna. The list was then supplemented with categories frequently appearing in online guides and mobile phone applications (e.g. GoodGuide, Inc.) informing consumers about ethical purchases (Burke and Davis 2014). 1 3 Italian Journal of Marketing (2020) 2020:289–308 297 Table 3 Socio-demographic features of the sample 3.3 Data analysis After data collection, data were processed, and the cluster analysis was performed with the Systat programme, using the non-hierarchical method by applying the k-medium algorithm, and considering the reasons underlying green consumption and the reasons against green consumption as variables to segment the sample. Table 3 Socio-demographic features of the sample Variables Number of respondents Percentage of respondents (%) Gender Males 86 33 Females 174 67 Age 18–38 261 100 Education Lower than high school 1 0 High school 153 59 Graduate degree 70 27 Postgraduate degree 34 13 PhD 1 0 Marital status Unmarried 233 89 Married/engaged 25 6 Widowed/divorced/separated 3 1 Kids Yes 16 6 No 245 94 Work status Student (including student worker) 200 77 Unemployed 17 7 Employee 44 17 298 Italian Journal of Marketing (2020) 2020:289–308 The variables taken into consideration for the segmentation were the driv- ers underlying green behaviour and the reasons against green consumption (see Table 4). The variables taken into consideration for the segmentation were the driv- ers underlying green behaviour and the reasons against green consumption (see Table 4). The cluster analysis allowed to identify three clusters. After clustering the sample, the single clusters were investigated, matching the results of the cluster analysis with the results of the answers provided by respondents to the other sec- tions of the questionnaire (especially Sect. 2 on green values and Sect. 5 on green consumption). Data analysis enabled to describe the feature of the identified clus- ter in terms of green values and green consumption. This led us to a better under- standing of the green consumption behaviour of Millennials. Table 4 List of variables used performing cluster analysis. Source Burke and Davis (2014) p g y ( ) I purchase green products because 1. It helps me fit in with my peers 2. It helps make a difference 3. They are easy to find 4. It can make me an opinion leader 5. I can save money 6. They are of a higher quality 7. They are healthier for me 8. It can help me with the creation of my positive identity 9. I genuinely care about the issues they deal with 10. They may grant me a higher status 11. They utilize innovative technology 12. I am very informed and I am able to distinguish between ethical and non-ethical products I reject green products because 1. 3.3 Data analysis There is a monetary risk in trialing them 2. Their packaging is unattractive or unappealing 3. They offer no other benefit (or product features) other than being ethical 4. I am skeptical as to how ethical these products really are 5. I don’t really care about the consequences of not buying ethical products 6. I don’t give them much thought 7. They are harder to find in stores 8. You have to go to specialty stores to buy them 9. I am confused as to what makes a product ethical 10. They are of lower quality 11. They are too expensive for the value received 12. People who buy them are given a negative stigma 13. I am suffering from ethical overload—I am sick of hearing about it Italian Journal of Marketing (2020) 2020:289–308 299 4.1.3 Cluster 3: “Really Green” (27% of the total sample) For this group of consumers, purchasing green products helps make a difference. They also declare to care genuinely about the issues these products deal with. For these reasons, the label assigned to these consumers is “Really Green”. Thanks to the data collected during the survey regarding the socio-demographic features, green attitudes, and green consumption, clusters were further described allowing us to address the research question better, as to say: can Millennials be con- sidered the leading and real “Green Generation”?. 4.1.2 Cluster 2: “Surely not Green” (37% of the total sample) In this case, respondents clearly identify specific reasons for rejecting green prod- ucts. First, they state that they do not really care about the consequences of not buy- ing green products, they have to go to specialty stores to buy them, and these prod- ucts are too expensive for the value received. Otherwise, no specific reasons to buy green products emerge. For these reasons, the label assigned to these consumers is “Surely not Green”. 4.1.1 Cluster 1: “Social Green” (29% of the total sample) These consumers state that purchasing green products helps them fit in with their peers, makes them opinion leaders, helps them to save money, to create a positive identity, and grants a higher status. Besides, they declare to buy green products also because of their innovative technology. According to these answers, these consum- ers seem to be green not for the planet but just for themselves. They strongly care about showing off their green consumption, as this empowers their social status. This general attitude is also supported by the reasons not to buy green products. These consumers identify the unattractive and unappealing packaging and the nega- tive stigma as the main reasons for rejecting green products. For these reasons, the label assigned to these consumers is “Social Green”. 4.1 Cluster analysis The analysis revealed 3 clusters: cluster 1 with 76 Millennials (29% of the sam- ple); cluster 3 with 70 Millennials (27%), and cluster 2 with 97—the majority of the sample (37% of the sample), as shown in Table 5. According to the rea- sons behind green consumption, clusters were labelled and described as follows. Specifically, the table reports the reasons that trigger consumers to purchase and to reject green products that mostly characterize each of the three clusters. For instance, Millennials in cluster 1 purchase green products because it helps fitting Table 5 Final cluster centers. Source Authors elaboration Table 5 Final cluster centers. Source Authors elaboration Cluster 1 (29% of the total sample) Cluster 2 (37% of the total sample) Cluster 3 (27% of the total sample) Assigned cluster Reasons to purchase green products Peers 4 2 3 1 Difference 5 4 6 3 Easy to find 4 3 4 Opinion leader 4 2 3 1 Save money 4 2 3 1 Higher quality 5 4 5 Healthier 6 5 6 Positive identity 5 2 4 1 Genuinely care 5 3 6 3 Higher status 4 2 3 1 Innovative technology 5 4 4 1 Informed 4 3 4 Reasons to reject green products Monetary risk 4 4 3 Packaging 4 3 2 1 No benefit 4 4 2 Sceptical 4 4 2 No care 3 4 2 2 No thought 4 4 2 Find in store 4 4 3 Specialty store 4 5 4 2 Confused 4 4 2 Lower quality 3 3 2 2 Too expensive 4 5 3 1 Negative stigma 3 2 1 Overload 3 3 2 Table 5 Final cluster centers. Source Authors elaboration 1 3 300 Italian Journal of Marketing (2020) 2020:289–308 in with peers. The right column in the table, indeed, reports for each reason for buying/not buying green products, the “assigned cluster”. 1 3 4.2 Millennials’ green values Regarding the green values scale, the average scores (Table 6) assigned to each proposition of the second section of the questionnaire were analyzed to understand what are the attitude and opinion, within each cluster. The scores refer to a scale going from a minimum of 1 (“totally disagree”) to a maximum of 7 (“totally agree). The high scores assigned by all the clusters to the green values support a gen- eral sensitivity and a positive attitude towards green issues by all the Millennials included in the analysis. 4.2 Millennials’ green values At the same, the highest average scores are in cluster 1, defined as the cluster of “Social Green” Millen- nials, confirming this cluster as a “green” cluster. This is the same for cluster 3, reporting the higher average scores after cluster 1, also confirming this as a “green” cluster. Green”), assuming low values compared to the average score of the sample and the average scores assigned by the other two clusters. At the same, the highest average scores are in cluster 1, defined as the cluster of “Social Green” Millen- nials, confirming this cluster as a “green” cluster. This is the same for cluster 3, reporting the higher average scores after cluster 1, also confirming this as a “green” cluster. Having a look at the single items included in the table, something interesting regarding the green consumption in Millennials generation emerges. Regardless the cluster, for all the groups, scores are higher (> 5) for the items affecting the green attitude (“It is important to me that the products I use do not harm the environ- ment”, “I would describe myself as environmentally responsible”, “I am concerned about wasting the resources of our planet”). On the other side, the scores affecting green behaviour (“I consider the potential environmental impact of my actions when making many of my decisions”, “My purchasing habits are affected by my concern for our environment”, “I am willing to be inconvenienced to take actions that are more environmentally friendly”), even if high, generally decrease (< 5). This might be consistent with an attitude-behaviour gap in Millennials, as, despite a positive attitude towards green issues, this attitude does not result in concrete behaviour. 4.2 Millennials’ green values Regardless, looking at the average scores in the different clusters, the lowest scores assigned to green values were in cluster 2 (“Surely not 1 3 Italian Journal of Marketing (2020) 2020:289–308 301 Table 6 Average scores assigned to green values Source Authors elaboration Green values (scale 1–7) Cluster 1 Cluster 2 Cluster 3 Total sample It is important to me that the products I use do not harm the environment 5.71 5.51 5.64 5.53 I consider the potential environmental impact of my actions when making many of my decisions 4.86 4.67 4.67 4.67 My purchasing habits are affected by my concern for our environment 4.56 4.23 4.50 4.23 I would describe myself as environmentally responsible 5.10 5.00 5.06 5.01 I am concerned about wasting the resources of our planet 5.59 5.39 5.52 5.38 I am willing to be inconvenienced in order to take actions that are more environmentally friendly 4.94 4.73 4.89 4.72 Table 6 Average scores assigned to green values Source Authors elaboration Green values (scale 1–7) Cluster 1 Cluster 2 Cluster 3 Total sample It is important to me that the products I use do not harm the environment 5.71 5.51 5.64 5.53 I consider the potential environmental impact of my actions when making many of my decisions 4.86 4.67 4.67 4.67 My purchasing habits are affected by my concern for our environment 4.56 4.23 4.50 4.23 I would describe myself as environmentally responsible 5.10 5.00 5.06 5.01 I am concerned about wasting the resources of our planet 5.59 5.39 5.52 5.38 I am willing to be inconvenienced in order to take actions that are more environmentally friendly 4.94 4.73 4.89 4.72 1 Table 6 Average scores assigned to green values Source Authors elaboration Green values (scale 1–7) Cluster 1 Cluster 2 Cluster 3 Total sample It is important to me that the products I use do not harm the environment 5.71 5.51 5.64 5.53 I consider the potential environmental impact of my actions when making many of my decisions 4.86 4.67 4.67 4.67 My purchasing habits are affected by my concern for our environment 4.56 4.23 4.50 4.23 I would describe myself as environmentally responsible 5.10 5.00 5.06 5.01 I am concerned about wasting the resources of our planet 5.59 5.39 5.52 5.38 I am willing to be inconvenienced in order to take actions that are more environmentally friendly 4.94 4.73 4.89 4.72 Table 6 Average scores assigned to green values Source Authors elaboration Green values (scale 1–7) Cluster 1 Cluster 2 Cluster 3 Total sample It is important to me that the products I use do not harm the environment 5.71 5.51 5.64 5.53 I consider the potential environmental impact of my actions when making many of my decisions 4.86 4.67 4.67 4.67 My purchasing habits are affected by my concern for our environment 4.56 4.23 4.50 4.23 I would describe myself as environmentally responsible 5.10 5.00 5.06 5.01 I am concerned about wasting the resources of our planet 5.59 5.39 5.52 5.38 I am willing to be inconvenienced in order to take actions that are more environmentally friendly 4.94 4.73 4.89 4.72 1 3 Italian Journal of Marketing (2020) 2020:289–308 302 Green”), assuming low values compared to the average score of the sample and the average scores assigned by the other two clusters. 1 3 4.3 Millennials’ green consumer behaviour As previously described, the value assigned to environmental protection does not necessarily lead to green consumer behaviour. It is, therefore, necessary to ana- lyze the green consumption in each cluster. The data of the fifth part of the ques- tionnaire were elaborated to show the frequency of green purchasing in each of the identified clusters. i The percentage of those who sometimes, often, or always buy green products in “Social Green” (48,99%) and “Really Green” (56,35%) clusters is higher com- pared to those who buy green products with the same frequency belonging to the “Surely not Green” cluster (31,62%). This supports the fit of cluster analysis con- ducted in the first part, as those who assigned high scores to green values in the second part of the questionnaire, and who belong to “Social Green” and “Really Green” clusters, actually show a greener consumer behaviour than the respond- ents belonging to the second cluster. Secondly, it is interesting to observe how, comparing the buying behaviours of consumers belonging to the same cluster, the green consumer behaviour mostly differs. f The trends of the frequencies reported in Fig. 1 refer to the purchasing of envi- ronmentally safe dishwashing liquid. Even though results slightly change according to the kind of product selected for the analysis, it is surprising that, for instance in the “Social Green” segment, Millennials declaring to never or rarely buy environ- mentally safe dishwashing liquid are 32.89% of the cluster. In “Really Green” clus- ter, this percentage is 35.71% of the cluster, while in “Surely not Green” cluster this goes up to 68.04%. This is consistent with the general features of the cluster. 1 3 1 3 Italian Journal of Marketing (2020) 2020:289–308 303 32,89% 6 65,79% 8,04% 35,71% 31,96% 64,29% Cluster 1 Cluster 2 Cluster 3 Never or rarely Sometimes, often, always Fig. 1 Consumption of environmentally safe dishwashing liquid. Source Authors elaboration Fig. 1 Consumption of environmentally safe dishwashing liquid. Source Authors elaboration 6 Limitation of the research First, investigating the drivers of green consumption and the reasons against green consumption, the differences emerging according to the different product categories were not analyzed. For example, the health concern might prevail in the consump- tion of product categories such as food or detergents, while the status might be more relevant for other categories such as clothing. Another limitation of our study is that the interviewees may have overestimated or underestimated the reasons behind the positive or negative attitudes, as well as the frequency with which certain products are purchased. Finally, the behaviour and attitudes of Millennials in the cluster analysis were not compared with other categories of respondents. In order to overcome this limitation, future research will compare Millennials with all the other generations, including Z generation, completely neglected in this analysis.i Moreover, since the participants in this first survey are all Italian, it would be interesting to include a comparison with consumers of different nationalities. Funding Open access funding provided by Università degli Studi di Brescia within the CRUI-CARE Agreement. 5 Conclusions The purpose of the empirical study was to investigate the attitude and the reasons behind the green consumption behaviour of the Millennials generation. Data analysis performed hitherto gives an interesting contribution to the literature devoted to green consumption and suggests some managerial implications.i The first result of the empirical analysis contrasts with the literature and empiri- cal evidence, usually describing Millennials as pro-environment oriented (Smith and Brower 2012; Eumetra Monterosa 2017; GWI 2018). On the other hand, our find- ings do not support the theories evidencing the existence of a negative correlation between young generations and environmental sensitivity (Van Liere 1981; Zimmer et al. 1994; Finisterra do Paço and Raposo 2010).f In particular, different green attitudes and behaviours were evidenced by the high number of respondents included in the second cluster of “Surely not Green” (37%). Similar evidence emerged even analyzing the scores assigned by the respondents to green values, and those regarding the green consumer behaviour. Besides, the high percentage of consumers belonging to cluster 1 defined as “Social Green” reveals something interesting about the green consumption in Millennials generation. This cluster, indeed, seems to be “green” not for feeling a devotion towards the planet, but mostly for showing off this attitude. This might be consistent with the general attitude of Millennials to be “self-oriented” (Naderi and Van Steenburg 2018) that, in this case, lead to exhibit green consumption as synonymous of social status. Our results do not necessarily imply a negative attitude towards green values by Millennials, but of course different green attitudes in the three clusters. This is also supported by the analysis of green consumption in each cluster. Focusing on managerial implications, although the segment of “Surely not green” seems to be the least attractive for green marketers, these are the ones requiring more attention. Information and knowledge are necessary to ensure that these con- sumers develop an adequate sensitivity to the surrounding environment so that they 3 3 304 Italian Journal of Marketing (2020) 2020:289–308 can no longer judge based on prejudices, sometimes depending on previous experi- ences of “greenwashing”. Additionally, “Social Green” might be a strategic target, influencing skeptical consumers going green. Funding Open access funding provided by Università degli Studi di Brescia within the CRUI-CARE Agreement. References Alexander, C. S., & Sysko, J. M. (2012). A study of the cognitive determinants of generation Y’s entitl ment mentality. Academy of Educational Leadership Journal, 16(2), 63–68. Anvar, M., & Venter, M. (2014). Attitudes and purchase behaviour of green products among generation consumers in South Africa. Mediterranean Journal of Social Sciences, 5(21), 183–194. Aschemann Witzel, J., & Niebuhr Aagaard, E. M. (2014). Elaborating on the attitude–behaviour gap regarding organic products: Young Danish consumers and in-store food choice. International Jour- nal of Consumer Studies, 38(5), 550–558. Bailey, A., Mishra, A., & Tiamiyu, M. (2016). GREEN consumption values and Indian consumer response to marketing communications. Journal of Consumer Marketing, 33(7), 562–573. response to marketing communications. Journal of Consumer Marketing, 33(7), 562–573. Bezencon, V., & Blili, S. (2010). Ethical products and consumer involvement: What’s new? European Bezencon, V., & Blili, S. (2010). Ethical products and consumer involvement: What’s new? Europea Journal of Marketing, 44(9/10), 1305–1321. Bollani, L., Peira, G., Varese, E., Nesi, E., Pairotti, M. B., & Bonadonna, A. (2017). Labelling and sus- tainability in the green food economy: Perception among Millennials with a good cultural back- ground. Rivista di studi sulla sostenibilità. ground. Rivista di studi sulla sostenibilità. Bucic, T., Harris, J., & Arli, D. (2012). Ethical consumers among the Millennials: A cross-national stud Journal of Business Ethics, 110(1), 113–131. Journal of Business Ethics, 110(1), 113–131. f ( ), Burke, P. F., & Davis, C. E. S. (2014). Segmenting consumers’ reasons for and against ethical consump- tion. European Journal of Marketing, 48(11/12), 2237–2261. Caplan, E. (2005). Brand loyalty. Dealerscope, 60, May 20, 60. E. (2005). Brand loyalty. Dealerscope, 60, May 20, 6 Caplan, E. (2005). Brand loyalty. Dealerscope, 60, May 20, 60. Carlson E (2009) 20th century US generations Washington DC: Population Reference Bure Caplan, E. (2005). Brand loyalty. Dealerscope, 60, May 20, 60. C l (2009) 20 h S hi Caplan, E. (2005). Brand loyalty. Dealerscope, 60, May 20, 60. Carlson, E. (2009). 20th-century US generations. Washington, DC: Population Reference Bureau. Casaló, L. V., & Escario, J. J. (2016). Intergenerational association of environmental concern: Evidence of parents’ and children’s concern. Journal of Environmental Psychology, 48, 65–74. Censis. (2016). Vita da Millennial: web, new media, startup e molto altro. Nuovi soggetti della ripresa italiana alla prova. Ciminillo, J. A. (2005). Elusive Generation Y demands edgier marketing. Automotive News, 79(6144), 28B.f Corvi, E., Bonera, M., & Bigi, A. (2007). Conflict of interest All the authors declare that they have no conflict of interest. Conflict of interest All the authors declare that they have no conflict of interest. Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Hel- sinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com- mons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licen ses/by/4.0/. 1 3 Italian Journal of Marketing (2020) 2020:289–308 305 References Are the Italian Millennials Similar or Different from the US Millennials?. Conference proceedings Seventh International Business Conference, Sidney, Aus- tralia, 3–6 December.i Coşkun, A., & Özbük, R. M. Y. (2019). Environmental segmentation: Young Millennials’ profile in a emerging economy. Young Consumers. g g y g Covino, R. M. (2017). Are you appealing to fresh and healthy Millennials? Convenience Store News. De Pelsmacker, P., Driesen, L., & Rayp, G. (2005). Do consumers care about ethics? Willingness to pay for fair-trade coffee. The Journal of Consumer Affairs, 39(2), 363–385. f f ff Debevec, K., Schewe, C., Madden, T., & Diamond, W. (2013). Are today’s Millennials splintering into a new generational cohort? Maybe! Journal of Consumer Behaviour, 12, 20–31. Deloitte. (2018). Deloitte Millennial survey. Retrieved from https://www2.deloitte.com/content/dam/ Deloitte/global/Documents/About-Deloitte/gx-2018-millennial-survey-report.pdf. downloaded 2019, December 15. Diamantopoulos, A., Schlegelmilch, B. B., Sinkovics, R. R., & Bohlen, G. M. (2003). Can socio-demo- graphics still play a role in profiling green consumers? A review of the evidence and an empirical investigation. Journal of Business Research, 56(6), 465–480. Didier, T., & Lucie, S. (2008). Measuring consumer’s willingness to pay for organic and fair trade pro ucts. International Journal of Consumer Studies, 32(5), 479–490.i Dimock, M. (2018). Defining generations: Where Millennials end and post-Millennials begin. Pew Research Center. http://www.pewresearch.org/fact-tank/2018/03/01/defining-generations-where -millennials-end-and-post-millennials-begin/. p g Eisner, S. (2005). Managing generation Y. S.A.M. Advanced Management Journal, 70(4), 4–15. Engebretson, J. (2004). Odd gen out. American Demographics, 24(4), 14–18. Eumetra Monterosa. (2017). https://www.lifegate.it/persone/news/osservatorionazionale-sostenib ita-2017. Eurispes. (2017). Ri2017 Documento di sintesi: 29° rapporto Italia. Finisterra do Paço, A. M., & Raposo, M. L. B. (2010). Green consumer market segmentation: Empirical findings from Portugal. International Journal of Consumer Studies, 34, 429–436. 1 3 Italian Journal of Marketing (2020) 2020:289–308 306 Fischer, D., Böhme, T., & Geiger, S. M. (2017). Measuring young consumers’ sustainable consumption behaviour: Development and validation of the YCSCB scale. Young Consumers, 18, 312–326. Fry, R. (2018). Millennials are the largest generation in the U.S. Labor force, Pew Research Center. http www.pewresearch.org/fact-tank/2018/04/11/millennials-largest-generation-us-labor-force/. Global Web Index. (2018). The rise of green consumerism: What do brands need to know?f Grønhøj, A., & Thøgersen, J. (2012). Action speaks louder than words: The effect of personal attitudes and family norms on adolescents’ pro-environmental behaviour. Journal of Economic Psychology, 33, 292–302. Hanson-Rasmussen, N. J., & Lauver, K. J. (2018). Environmental responsibility: Millennial values and cultural dimensions. Journal of Global Responsibility, 9(1), 6–20. Haws, K. L., Winterich, K. P., & Naylor, R. References W. (2014). Seeing the world through GREEN-tinted glasses: Green consumption values and responses to environmentally friendly products. Journal of Con- sumer Psychology, 24(3), 336–354. Heo, J., & Muralidharan, S. (2017). What triggers young Millennials to purchase eco-friendly products? The interrelationships among knowledge, perceived consumer effectiveness, and environmental concern. Journal of Marketing Communications, 1–17. Hewlett, S. A., Sherbin, L., & Sumberg, K. (2009). How generation and baby boomers will reshape your agenda. Harvard Business Review, 71–77. Hopkins, M. S. (2010). How sustainability fuels design innovation. MIT Sloan Management Review, 52(1), 75–81. Howe, N. (2010). Meet Mr. and Mrs. Gen X: A new parent generation. AASA—The School Superintendents Association. Howe, N., & Strauss, W. (2000). Millennials rising: The next greatest generation. New York, NY: Vintage Books. Howe, N., & Strauss, W. (2007). The next 20 years: How customer and workforce attitudes will evolve. Har- vard Business Review, 85(7–8), 41–52. Hume, M. (2010). Compassion without action: Examining the young consumers consumption and attitude to sustainable consumption. Journal of World Business, 45(4), 385–394.l Hyllegard, K., Yan, R., Ogle, J., & Attmann, J. (2011). The influence of gender, social cause, charitable sup- port, and message appeal on Gen Y’s responses to cause-related marketing. Journal of Marketing Man- agement, 27(1/2), 100–123.ii Istat. (2016). Classificazione delle generazioni. Statistiche sperimentali. https://www.istat.it/it/files//2011/01/ Generazioni-nota.pdf.f Ivanova, O., Flores-Zamora, J., Khelladi, I., & Ivanaj, S. (2019). The generational cohort effect in the conte of responsible consumption. Management Decision, 57(5), 1162–1183. Jang, Y. J., Kim, W. G., & Bonn, M. A. (2011). Generation Y consumers’ selection attributes and behavio- ral intentions concerning green restaurants. International Journal of Hospitality Management, 30(4), 803–811. Johnson, C. Y., Bowker, J. M., & Cordell, H. K. (2004). Ethnic variation in environmental belief and behav- ior: An examination of the new ecological paradigm in a social psychological context. Environment and Behavior, 36, 157–186. Junco, R., & Mastrodicasa, J. M. (2007). Connecting to the net generation: What highereducation professio als need to know about today’s students. Washington, DC: NASPA. Kanchanapibul, M., Lacka, E., Wang, X., & Chan, H. K. (2014). An empirical investigation of green pur- chase behaviour among the young generation. Journal of Cleaner Production, 66, 528–536.f Kennedy, E. H., Beckley, T. M., McFarlane, B. L., & Nadeau, S. (2009) mental concern in Canada. Rural Sociology, 74(3), 309–329. Kennedy, E. H., Beckley, T. M., McFarlane, B. L., & Nadeau, S. (2009). References Reaching Generation Y. San Antonio Express-Newsz, 29(1).f Peterson, E. R., Sillman, A., & McCaffrey, C. R. (2017). Where are the global Millennials?. AT Kearney- Global Business Policy Council (GBPC). Available at www.atkearney.com/web/global-businesspolicy- council/article. Phillips, C. (2007). Millennials: Clued in or clueless. Advertising age, 78(46), 12–13.f Reisenwitz, T. H., & Iyer, R. (2009). Differences in generation x and generation y: Implications for the orga ization and marketers. Marketing Management Journal, 19(2), 91–103. Roberts, J. A. (1996). Will the real socially responsible consumer please step forward? Business Horizons, 39(1), 79–84. Schmeltz, L. (2012). Consumer-oriented CSR communication: Focusing on ability or morality? Corpora Communications: An International Journal, 17(1), 29–49. Shapira, I. (2008). What comes next after generation X? The Washington Post, 6. Sheahan, P. (2005). Generation Y: Thriving and surviving with generation Y at work. Prahan: Hardie Grant Books. Smith, B. (2011). Who shall lead us? How cultural values and ethical ideologies guide young marketers’ evaluations of the transformational manager–leader. Journal of Business Ethics, 100(4), 633–645.l Smith, K. T., & Brower, T. R. (2012). Longitudinal study of green marketing strategies that influence Mille nials. Journal of Strategic Marketing, 20(6), 535–551. Smith, K. T., & Brower, T. R. (2012). Longitudinal study of green marketing strategies that influence Millen- nials. Journal of Strategic Marketing, 20(6), 535–551. Strauss W & Howe N (1991) Generations: The history of America’s Future 1584 to 2069 New York: Strauss, W., & Howe, N. (1991). Generations: The history of America’s Future, 1584 to 2069. New Yor William Morrow and Company. Synodinos, C., & Bevan-Dye, A. (2014). Determining African Generation Y students’ likelihood of engaging in pro-environmental purchasing behaviour. Mediterranean Journal of Social Sciences, 5(21), 101–109. p p g f Tariq, M. Z. (2014). Impact of green advertisement and green brand awareness on green satisfaction with mediating effect of buying behavior. Journal of Managerial Sciences, 8(2), 274–289.f f Teisl, M. F., Radas, S., & Roe, B. (2008). Struggles in optimal labelling: How different consumers react to various labels for genetically modified foods. International Journal of Consumer Studies, 32(5), 447–456. Tobler, C., Visschers, V. H., & Siegrist, M. (2012). Addressing climate change: Determinants of consum- ers’ willingness to act and to support policy measures. Journal of Environmental Psychology, 32(3), 197–207. Tsui, B., & Hughes, L. Q. (2001). Generation next. Advertising Age, 72(3), 14–16. Twenge, J. M. (2014). References Rural urban differences in environ- mental concern in Canada. Rural Sociology, 74(3), 309–329. Kilian, T., Hennigs, N., & Langner, S. (2012). Do Millennials read books or blogs? Introducing a med usage typology of the Internet generation. Journal of Consumer Marketing, 29(2), 114–124. Kilian, T., Hennigs, N., & Langner, S. (2012). Do Millennials read books or blogs? Introducing a media usage typology of the Internet generation. Journal of Consumer Marketing, 29(2), 114–124. Kim, Y., & Choi, S. M. (2005). Antecedents of green purchase behavior: An examination of collectivism, environmental concern, and PCE. ACR North American Advances. Kim, Y., & Choi, S. M. (2005). Antecedents of green purchase behavior: An examination of collectivism environmental concern, and PCE. ACR North American Advances. Lappänen, J. M., Haahla, A. E., Lensu, A. M., & Kuitunen, M. T. (2012). Parent–child similarity in environ- mental attitudes: A pairwise comparison. The Journal of Environmental Education, 43(3), 162–176. Leiserowitz, A., Maibach, E., Roser-Renouf, C., & Hmielowski, J. (2012). Global warming’s six Americas, Yale University and George Mason University, New Haven, CT, Yale Project on Climate Change Com- munication. http://environment.yale.edu/climatecommunication/files/Six-Americas-March-2012.pdf. Accessed 25 September 2013. 1 3 Italian Journal of Marketing (2020) 2020:289–308 307 Lu, L., Bock, D., & Joseph, M. (2013). Green marketing: What the Millennials buy. Journal of Business Strategy, 34(6), 3–10. Mainieri, T., Barnett, E. G., Valdero, T. R., Unipan, J. B., & Oskamp, S. (1997). Green buying: The influence of environmental concern on consumer behaviour. The Journal of Social Psychology, 137(2), 189–204.l Meeusen, C. (2014). The intergenerational transmission of environmental concern: The influence of parents and communication patterns within the family. Journal of Environmental Education, 45(4), 77–90. Miniero, G., Codini, A., Bonera, M., Corvi, E., & Bertoli, G. (2014). Being green: from attitude to actual consumption. International Journal of Consumer Studies, 38(5), 521–528. Muralidharan, S., Rejón-Guardia, F., & Xue, F. (2016). Understanding the green buying behaviour of younger Millennials from India and the United States: A structural equation modeling approach. Jour- nal of International Consumer Marketing, 28(1), 54–72.i Naderi, I., & Van Steenburg, E. (2018). Me first, then the environment: Young Millennials as green consum- ers. Young Consumers, 19(3), 280–295. Nidumolu, R., Prahalad, C. K., & Rangaswami, M. R. (2009). Why sustainability is now the key driver innovation. Harvard Business Review, 43, 57–64. Nielsen. (2017). Millennial: Dai!, The Nielsen Company (Italy) S.r.l.. 3, 337–347. Parment, A. (2009). Die Generation Y-Mitarbeiter der Zukunft. Wiesbaden: Gabler. Pesquera, A. (2005). References Generation me-revised and updated: Why today’s young Americans are more con dent, assertive, entitled—and more miserable than ever before. New York: Simon and Schuster.i Twenge, J., & Me, G. (2006). Why today’s young Americans are more confident. Assertive, entitled—and more miserable than ever before. Atria Paperback.i United Nation. (2017). World population prospects. Key findings and advance tables. https://esa.un.org/unpd/ wpp/publications/files/wpp2017_keyfindings.pdf.f ii Uyeki, E. S., & Holland, L. J. (2000). Diffusion of pro-environment attitudes? American Behavioral Scientis 43(4), 646–662. Valentine, D. B., & Powers, T. L. (2013). Generation Y values and lifestyle segments. Journal of Consum Marketing, 30(7), 597–606. 1 3 Italian Journal of Marketing (2020) 2020:289–308 308 Van Liere, (1981). Environmental concern: Does it make a difference how it’s measured? Environment and Behavior, 13(6), 651–676. Vespa, J., Armstrong D. M., & Medina, L. (2018). Demographic turning points for the United States: Popu- lation projections for 2020 to 2060. Current Population Reports, P25-1144, U.S. Census Bureau, Wash- ington, DC. Wandel, M., & Bugge, A. (1997). Environmental concern in consumer evaluation of food quality. Foo Quality and Preference, 8(1), 19–26. Wilcock, A., Pun, M., Khanona, J., & Aung, M. (2004). Consumer attitudes, knowledge and behaviour: review of food safety issues. Trends in Food Science and Technology, 15(2), 56–66. Wolburg, J. M., & Pokrywczynski, J. (2001). A psychographic analysis of Generation Y college students. Journal of Advertising Research, 41(5), 33–52. f g Wong, C. W. Y., Lai, K. H., Shang, K. C., & Lu, C. S. (2014). Uncovering the value of green advertising for environmental management practices. Business Strategy and the Environment, 23(2), 117–130. Wright, M. J., Teagle, D. A. H., & Feetham, P. M. (2014). A quantitative evaluation of the public response to climate engineering. Nature Climate Change, 4, 106–110. Young, K. (2018). The rise of green consumerism: What do brands need to know? GlobalWebIndex Q2 2018. https://www.globalwebindex.net.ff Zimmer, M. R., Stafford, T. F., & Stafford, M. R. (1994). Green issues: Dimensions of environmental concern. Journal of Business Research, 30(1), 63–74. 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https://openalex.org/W3086075392	https://estudogeral.sib.uc.pt/bitstream/10316/103845/1/Recent-advances-on-properties-and-utility-of-nanomaterials-generated-from-industrial-and-biological-activities2021CrystalsOpen-Access.pdf	English	null	Recent Advances on Properties and Utility of Nanomaterials Generated from Industrial and Biological Activities	null	2,020	cc-by	19,176	Citation: Yadav, V.K.; Malik, P.; Khan, A.H.; Pandit, P.R.; Hasan, M.A.; Cabral-Pinto, M.M.S.; Islam, S.; Suriyaprabha, R.; Yadav, K.K.; Dinis, P.A.; et al. Recent Advances on Properties and Utility of Nanomaterials Generated from Industrial and Biological Activities. Crystals 2021, 11, 634. https:// doi.org/10.3390/cryst11060634 Academic Editors: Ewa Wierzbicka and Karolina Syrek Received: 24 March 2021 Accepted: 2 May 2021 Published: 1 June 2021 g * Correspondence: virendra.yadav@jnujaipur.ac.in (V.K.Y.); marinacp@ua.pt (M.M.S.C.-P.); pdinis@dct.uc.pt (P.A.D.); samreen.heena.khan@gmail.com (S.H.K.); luisa2diniz@gmail.com (L.D.) g Correspondence: virendra.yadav@jnujaipur.ac.in (V.K.Y.); marinacp@ua.pt (M.M.S.C.-P.); di i @d t t (PA D ) h kh @ il (S H K ) l i 2di i @ il (L D ) Citation: Yadav, V.K.; Malik, P.; Khan, A.H.; Pandit, P.R.; Hasan, M.A.; Cabral-Pinto, M.M.S.; Islam, S.; Suriyaprabha, R.; Yadav, K.K.; Dinis, P.A.; et al. Recent Advances on Properties and Utility of Nanomaterials Generated from Industrial and Biological Activities. Crystals 2021, 11, 634. https:// doi.org/10.3390/cryst11060634 Abstract: Today is the era of nanoscience and nanotechnology, which ﬁnd applications in the ﬁeld of medicine, electronics, and environmental remediation. Even though nanotechnology is in its emerging phase, it continues to provide solutions to numerous challenges. Nanotechnology and nanoparticles are found to be very effective because of their unique chemical and physical properties and high surface area, but their high cost is one of the major hurdles to its wider application. So, the synthesis of nanomaterials, especially 2D nanomaterials from industrial, agricultural, and other biological activities, could provide a cost-effective technique. The nanomaterials synthesized from such waste not only minimize pollution, but also provide an eco-friendly approach towards the utilization of the waste. In the present review work, emphasis has been given to the types of nanomaterials, different methods for the synthesis of 2D nanomaterials from the waste generated from industries, agriculture, and their application in electronics, medicine, and catalysis. Received: 24 March 2021 Accepted: 2 May 2021 Published: 1 June 2021 Keywords: nanomaterials; carbon nanotubes; rice husk; agriculture waste; carbon nanoﬁbres Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Virendra Kumar Yadav 1,2,, Parth Malik 2, Afzal Husain Khan 3 , Priti Raj Pandit 4, Mohd Abul Hasan 5, Marina M. S. Cabral-Pinto 6, , Saiful Islam 5 , R. Suriyaprabha 2, Krishna Kumar Yadav 7 , Pedro A. Dinis 8,* , Samreen Heena Khan 2,* and Luisa Diniz 6,* Virendra Kumar Yadav 1,2,, Parth Malik 2, Afzal Husain Khan 3 , Priti Raj Pandit 4, Mohd Abul Hasan 5, Marina M. S. Cabral-Pinto 6, , Saiful Islam 5 , R. Suriyaprabha 2, Krishna Kumar Yadav 7 , Pedro A. Dinis 8,* Samreen Heena Khan 2,* and Luisa Diniz 6,* 1 School of Lifesciences, Jaipur National University, Jaipur 302017, Rajasthan, India 2 School of Nanosciences, Central University of Gujarat, Gandhinagar 382030, Gujarat, Ind parthmalik1986@gmail.com (P.M.); sooriyarajendran@gmail.com (R.S.) 2 School of Nanosciences, Central University of Gujarat, Gandhinagar 382030, Gujarat, India; parthmalik1986@gmail.com (P.M.); sooriyarajendran@gmail.com (R.S.) p g ( ) y j g ( ) 3 Civil Engineering Department, College of Engineering, Jazan University, Jazan 114, Saudi Arabia; ahkhan@jazanu.edu.sa j 4 Bioxcentre, IIT-Mandi, Mandi 175005, Himachal Pradesh, India; panditashanu@gmail.com 5 Civil Engineering Department, College of Engineering, King Khalid University, P.O. Box 394, Abha 61421, Saudi Arabia; mohad@kku.edu.sa (M.A.H.); sfakrul@kku.edu.sa (S.I.) 6 Geobiotec Research Centre, Department of Geoscience, University of Aveiro, 3810-193 Aveiro, Portugal p y 7 Faculty of Science and Technology, Madhyanchal Professional University, Ratibad, Bhopal 462044, Madhya Pradesh, India; envirokrishna@gmail.com p y 7 Faculty of Science and Technology, Madhyanchal Professional University, Ratibad, Bh l 462044 M dh P d h I di i k i h @ il 8 Marine and Environmental Sciences Centre (MARE), Department of Earth Sciences, University of Coimbra, Rua, Silvio Lima, 3030-790 Coimbra, Portugal crystals crystals 1. Introduction Nanotechnology is providing new solutions and opportunities to ensure sustain- able energy and environments for the future. Nanotechnology deals with the design and development of the materials at the nanoscale (1–100 nm) or one dimension in the nanoscale [1,2]. The word nano was derived from the Greek word meaning “dwarf” [3] and denoted as nm. By using such measurement, the size of viruses are about 100 nm (30–100) nm [4] and that of a human hair is 1000 nm in diameter. Nanotechnology and nanoscience allow researchers to manipulate the properties of materials at the atomic level [5]. Nanomaterials are typically those materials having at least anyone dimension at the nanoscale (<100 nm). Nanomaterials can be produced in a variety of methods like chemical, physical and biological with different classes such as carbon-based nanomaterials: carbon-based nanomaterials [6,7], nanocomposites [8], metals, alloys, nanopolymers [9,10], nanoglassses [11], and nanoceramics [9,12]. Nanomaterials can be either synthesized in Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/crystals Crystals 2021, 11, 634. https://doi.org/10.3390/cryst11060634 Crystals 2021, 11, 634 2 of 26 the laboratory or could be derived from the natural resources [13]. The nanomaterial synthesized from the commercial precursor materials makes the product as well as process expensive. Moreover, the source of nanomaterial is also depleting, so there is a need to rely on the natural and alternative sources of nanomaterial [14]. The natural nanomaterial [15] act as a potential candidate for the development of nanomaterials. The nanomaterial derived from such processes are cost-effective [16], biocompatible [17] and environmentally friendly [13]. The waste materials that are commonly used for nanomaterial synthesis include industrial waste like ﬂy ash [7,18], red mud, agricultural waste [19,20] (rice husk and straw, wheat husk and straw, coconut shell), and plastic waste [21,22]. Most of these waste materials mainly act as a pollutant to the environment, which are produced in tonnes annually around the globe. The utilization of such products for the synthesis of carbon nanomaterials reduces the pollution from the environment and simultaneously provides an environment-friendly and economical approach. Moreover, nanomaterials derived from such waste products will have a greater impact in the industries when these are surface functionalized or transformed into some isomeric forms. 1. Introduction The surface functional- ization is mainly done for a speciﬁc function, by modifying functional groups, etc. These nanostructured materials based on their purities can ﬁnd applications in electronics [23], wastewater treatment [24], medicine [25], and catalysis [25]. The present review focused on the classiﬁcation of nanomaterials, synthesis of carbon-based nanomaterials from industrial and agricultural wastes, and their utilization for environmental applications. r could be derived from the natural resources [13]. The nanomaterial synthesized from he commercial precursor materials makes the product as well as process expensive. Moreover, the source of nanomaterial is also depleting, so there is a need to rely on the atural and alternative sources of nanomaterial [14]. The natural nanomaterial [15] act as potential candidate for the development of nanomaterials. The nanomaterial derived om such processes are cost-effective [16], biocompatible [17] and environmentally iendly [13]. The waste materials that are commonly used for nanomaterial synthesis in- ude industrial waste like fly ash [7,18], red mud, agricultural waste [19,20] (rice husk nd straw, wheat husk and straw, coconut shell), and plastic waste [21,22]. Most of these aste materials mainly act as a pollutant to the environment, which are produced in nnes annually around the globe. The utilization of such products for the synthesis of arbon nanomaterials reduces the pollution from the environment and simultaneously rovides an environment-friendly and economical approach. Moreover, nanomaterials erived from such waste products will have a greater impact in the industries when these e surface functionalized or transformed into some isomeric forms. The surface function- ization is mainly done for a specific function, by modifying functional groups, etc. These anostructured materials based on their purities can find applications in electronics [23], astewater treatment [24], medicine [25], and catalysis [25]. The present review focused n the classification of nanomaterials, synthesis of carbon-based nanomaterials from in- ustrial and agricultural wastes, and their utilization for environmental applications. 2.1. Zero-Dimensional Nanomaterial (0D Nanomaterial) 2.1. Zero-Dimensional Nanomaterial (0D Nanomaterial) In 0D material (quantum dot) [QD], there is conﬁnement of electrons in all three directions [28]. Zero dimensional nanomaterial has gained huge attention in the ﬁeld of research and in material-based industries [29]. Such material ﬁnds applications in the light- emitting diodes (LEDs) [30], solar cells [31], single-electron transistors [32], and lasers. The common example of zero-dimensional nanomaterial are spheres (including hollow spheres) and nanoclusters [33], quantum dots that includes core-shell QDs also [34], heterogeneous particles arrays, onions [35], and nanolenses. Carbon-based materials such as Fullerene like (FL) structures are having extraordinary mechanical properties and they are being used in multiple applications like biomedicine and microelectronics [36]. 2.2. One Dimensional Nanomaterial (1D Nanomaterial) One dimensional nanomaterial is those materials which are conﬁned in two dimen- sion but free in one dimension [37]. Some of the common examples of 1D nanomaterial are wires, nanowires [38], nanotubes, nanoﬁbres [39], nanobelts [40], nanoribbons [40], nanorods [41], and hierarchical nanostructures. For the last decade, such nanomaterials have garnered huge attention because of their remarkable properties and such wider ap- plicability in terms of research and development. Such materials have a wider impact in nanoelectronics [42], nanodevices, and nanosystems [43], nanocomposite materials [44], and alternative resources of energy. The 1D nanomaterials are the most preferred material for exploring the properties at the nanoscale. It is also used for the investigation of size and dimensionality dependence of functional properties [45]. 2. Classiﬁcation of Nanostructured Materials Classification of Nanostructured Materials Nanostructured materials (NSMs) have gained a huge consideration in fundamental science and technological applications due to their multifunctionality and unique chemical, physical, electronic and magnetic properties at the nanoscale [26]. Every day, novel nano- materials are synthesized, so classiﬁcation is of the utmost necessity. Figure 1 presents the broad classiﬁcation of nanomaterials. Nanostructured materials (NSMs) have gained a huge consideration in fundamental ience and technological applications due to their multifunctionality and unique chemi- al, physical, electronic and magnetic properties at the nanoscale [26]. Every day, novel anomaterials are synthesized, so classification is of the utmost necessity. Figure 1 pre- ents the broad classification of nanomaterials. Figure 1. Classification of nanomaterials. The density of the state varies considerably for different nanomaterials which are based on the degree of freedom/confinement [27]. Based on the nanostructural elements Figure 1. Classiﬁcation of nanomaterials. The density of the state varies considerably for different nanomaterials which are based on the degree of freedom/conﬁnement [27]. Based on the nanostructural elements and their physical and chemical properties, the nanomaterials have been classiﬁed into four classes, i.e., 0D, 1D, 2D, and 3D, by Pokropivny. gure 1. Classification of nanomaterials. Figure 1. Classiﬁcation of nanomaterials. The density of the state varies considerably for different nanomaterials which are ased on the degree of freedom/confinement [27]. Based on the nanostructural elements The density of the state varies considerably for different nanomaterials which are based on the degree of freedom/conﬁnement [27]. Based on the nanostructural elements and their physical and chemical properties, the nanomaterials have been classiﬁed into four classes, i.e., 0D, 1D, 2D, and 3D, by Pokropivny. The density of the state varies considerably for different nanomaterials which are ased on the degree of freedom/confinement [27]. Based on the nanostructural elements The density of the state varies considerably for different nanomaterials which are based on the degree of freedom/conﬁnement [27]. Based on the nanostructural elements and their physical and chemical properties, the nanomaterials have been classiﬁed into four classes, i.e., 0D, 1D, 2D, and 3D, by Pokropivny. Crystals 2021, 11, 634 3 of 26 2.3. Two-Dimensional Nanomaterials (2D Nanomaterials) 2D nanomaterials have only one dimension in the nano range while the other two dimensions are out of the nano range [46]. They are said to be the thinnest materials, which possess the highest surface area, and are increasingly gaining global interest from fundamentals of physical sciences, chemistry, to materials engineering. Graphene was the ﬁrst 2D nanomaterial to trigger the research on 2D nanomaterial. Other than graphene, researches also focus on other 2D nanomaterials, such as boron nitride, transition metal dichalcogenide, mono-elemental 2D semiconductors, i.e., silicene, germanene, stanene, and phosphorene, and 2D oxide/hydroxide materials. In recent years, not only the syn- thesis, but also the applications of 2D NSMs have drastically drawn attention in materials research because of their several interesting properties at the nanoscale. In comparison with bulk materials, two-dimensional (2D) nanomaterials own rare physiochemical assets develops due to their high aspect ratio (SVR) [47], distinctive surface chemical properties, and quantum conﬁnement effect [48]. The 2D NSMs ﬁnds applications in sensing mate- rials, photocatalysis, nanocontainers and nanoreactors [34]. Most preferably, the metallic based 2D NSMs have been exploited widely in sensing, catalysis, photothermal therapy, surface-enhanced Raman scattering (SERS), bioimaging, and solar cells [49], due to their phenomenal properties. The common examples of 2D nanomaterials are nanoprisms [50], nanoplates [51], nanosheets [52,53], nanowalls [53], and nanodisks [54], which are shown in Figure 2. 4 of 26 4 of 27 Crystals 2021, 11, 634 Crystals 2021, 11, x FOR Figure 2. Examples of 2D materials. 2 4 Three Dimensional Nanomaterials (3D Nanomaterials) Figure 2. Examples of 2D materials. 2.4. Three Dimensional Nanomaterials (3D Nanomaterials) Figure 2. Examples of 2D materials. Figure 2. Examples of 2D materials. Figure 2. Examples of 2D materials. Figure 2. Examples of 2D materials. 2 4 Three Dimensional Nanomaterials (3D Nanomaterials) 2.4. Three Dimensional Nanomaterials (3D Nanomaterials) 2.4. Three Dimensional Nanomaterials (3D Nanomaterials) 2.4. Three Dimensional Nanomaterials (3D Nanomaterials) The 3D NSMs three dimensional nanomaterials are those materials that have their free dimensions in all three directions and there is no confinement and limitations [34]. The common examples of three 3D nanomaterials are powders [55], multilayer [56], fi- brous and polycrystalline material [57]. The 3D nanomaterials exhibit a large specific sur- face area [58], because of which such nanostructures provide adequate surface absorption sites for the molecules in a small area. 2.3. Two-Dimensional Nanomaterials (2D Nanomaterials) The 3D NSMs are extensively used for catalysis in nanomaterials finds applications in the field of catalysis [59], magnetism and for the de- velopment of electrode material for batteries [60]. Additionally, the porosity in the three dimensions supports the easy transport of the molecules. Examples of 3D NMs include nanoballs (dendritic structures) [61], nanocoils [62], nanocones [34], nanopillers [63], and nanoflowers [63] The 3D NSMs three dimensional nanomaterials are those materials that have their free dimensions in all three directions and there is no conﬁnement and limitations [34]. The common examples of three 3D nanomaterials are powders [55], multilayer [56], ﬁbrous and polycrystalline material [57]. The 3D nanomaterials exhibit a large speciﬁc surface area [58], because of which such nanostructures provide adequate surface absorption sites for the molecules in a small area. The 3D NSMs are extensively used for catalysis in nanomaterials ﬁnds applications in the ﬁeld of catalysis [59], magnetism and for the development of electrode material for batteries [60]. Additionally, the porosity in the three dimensions supports the easy transport of the molecules. Examples of 3D NMs include nanoballs (dendritic structures) [61], nanocoils [62], nanocones [34], nanopillers [63], and nanoﬂowers [63]. 3.1. Physical Methods for Synthesis of 2D NSMs 3.1. Physical Methods for Synthesis of 2D NSMs y f y f The 2D NSMs could be synthesized by various physical methods [84] such as evapo- ration [85], lithography [86], sputtering, phase condensation, hot and cold plasma spray pyrolysis [87], inert gas phase condensation [88], pulsed laser ablation method [89], and sonochemical reduction [90]. These methods (physical) are generally used for the synthe- sis of nanowalls [53], nanoprisms [91], nanosheets [92], nanoplates [93], and nanodisks [34]. The nanomaterials synthesized by the physical method are homogenous in nature and ordered. Dai et al., 2002 developed the SnO nanodisks [64] alumina plates using the thermal evaporation method under optimized environmental conditions [94]. Here, firstly, SnO or SnO2 powders were kept in an alumina boat, which was in turn placed in a quartz tube reactor (evaporation source), where alumina acted as a substrate which was placed one by one downstream. The physical techniques provide an environment-friendly approach for the development of 0D, 1D, 2D, and 3D nanomaterials, which are shown The 2D NSMs could be synthesized by various physical methods [84] such as evapo- ration [85], lithography [86], sputtering, phase condensation, hot and cold plasma spray pyrolysis [87], inert gas phase condensation [88], pulsed laser ablation method [89], and sonochemical reduction [90]. These methods (physical) are generally used for the synthesis of nanowalls [53], nanoprisms [91], nanosheets [92], nanoplates [93], and nanodisks [34]. The nanomaterials synthesized by the physical method are homogenous in nature and ordered. Dai et al., 2002 developed the SnO nanodisks [64] alumina plates using the thermal evaporation method under optimized environmental conditions [94]. Here, ﬁrstly, SnO or SnO2 powders were kept in an alumina boat, which was in turn placed in a quartz tube reactor (evaporation source), where alumina acted as a substrate which was placed one by one downstream. The physical techniques provide an environment-friendly approach for the development of 0D, 1D, 2D, and 3D nanomaterials, which are shown below in Figure 4. Figure 3. Different methods of synthesis of nanomaterials. Figure 3. Different methods of synthesis of nanomaterials. Figure 3. Different methods of synthesis of nanomaterials. Figure 3. Different methods of synthesis of nanomaterials. below in Figure 4. 3.2. Chemical Methods for Synthesis of Nanomaterials Chemical methods have contributed to the fabrication of materials at the nanoscale [95]. The Chemical methods have several advantageous properties over physical methods as the previous one involves mixing of chemical at the molecular level which ensures good chemical homogeneity [74,96]. Chemical reduction methods are reported to have numerous drawbacks for instance utilization toxic reagents and solvents, generation of unwanted by-products due to which there are several extra steps is needed for removal of impurities, time-consuming [97]. The most common chemical methods are electroless deposition [98], lyotropic liquid crystal templates [34], hydrothermal and solvothermal method, sol-gel technique, electrochemical deposition, chemical vapor deposition (CVD), laser pyrolysis, and laser chemical vapor deposition techniques (LCVD), which are utilized frequently for the production of different NSMs. The above-mentioned techniques are shown in Figure 5. Chen et al., in 2018, reported the synthesis of two-dimensional metallic nanomaterials from various routes [99]. Yang et al., in 2019, reported the synthesis, engineering, and applications of ﬂy ash from various routes like physical and chemical but the emphasis was given mainly on the precursor mediated synthesis, not on the waste-based materials [100]. In 2015, Paul et al. reported the thin ﬁlm deposition of Feo on the Pt(111) by the ferrocene adsorption and oxidation method [101]. Zhang et al. reported the synthesis of multifunc- Chemical methods have contributed to the fabrication of materials at the nanoscale [95]. The Chemical methods have several advantageous properties over physical methods as the previous one involves mixing of chemical at the molecular level which ensures good chemical homogeneity [74,96]. Chemical reduction methods are reported to have numerous drawbacks for instance utilization toxic reagents and solvents, generation of unwanted by-products due to which there are several extra steps is needed for removal of impurities, time-consuming [97]. The most common chemical methods are electroless deposition [98], lyotropic liquid crystal templates [34], hydrothermal and solvothermal method, sol-gel technique, electrochemical deposition, chemical vapor deposition (CVD), laser pyrolysis, and laser chemical vapor deposition techniques (LCVD), which are utilized frequently for the production of different NSMs. The above-mentioned techniques are shown in Figure 5. p q g Chen et al., in 2018, reported the synthesis of two-dimensional metallic nanomaterials from various routes [99]. 3 Diff t M th d f N t i l S th i 3. Different Methods of Nanomaterials Synthesis 3. Different Methods of Nanomaterials Synthesis The nanomaterials could be developed by all three means i.e., chemical, physical and biological methods which is shown in Figure 3. Among them, the physical approaches include sputtering [64], laser ablation [65], pyrolysis [66], lithography [67], and hot and cold plasma [67]. Meanwhile, the chemical methods that are used most frequently are lyo- tropic liquid crystal templates [68], electrochemical deposition [69], electroless deposition [70], hydrothermal [71] and solvothermal techniques [72], sol gel technique [73,74], laser chemical vapor deposition technique [75], laser pyrolysis [76], and chemical vapor depo- sition [77]. The nanomaterials could also be synthesized by biological approaches like mi- crobial [78] and plant derived materials [79]. The microbial synthesis of nanomaterials [80] employs the utilization of microorganisms like algae [81], fungi [53], and bacteria [82]. The main drawback is that when there is a utilization of commercial precursor for the synthe- sis of nanomaterials by any of the above-mentioned approaches, the process, as well as The nanomaterials could be developed by all three means i.e., chemical, physical and biological methods which is shown in Figure 3. Among them, the physical approaches include sputtering [64], laser ablation [65], pyrolysis [66], lithography [67], and hot and cold plasma [67]. Meanwhile, the chemical methods that are used most frequently are lyotropic liquid crystal templates [68], electrochemical deposition [69], electroless deposition [70], hy- drothermal [71] and solvothermal techniques [72], sol gel technique [73,74], laser chemical vapor deposition technique [75], laser pyrolysis [76], and chemical vapor deposition [77]. The nanomaterials could also be synthesized by biological approaches like microbial [78] and plant derived materials [79]. The microbial synthesis of nanomaterials [80] employs the utilization of microorganisms like algae [81], fungi [53], and bacteria [82]. The main drawback is that when there is a utilization of commercial precursor for the synthesis of nanomaterials by any of the above-mentioned approaches, the process, as well as the product, become expensive. So, in order to obtain a cost-effective material, the precursor should be lower in terms of cost. One such material is industrial waste [83], biological waste, or agricultural waste [19]. 5 of 26 recur- logical Crystals 2021, 11, 634 , g [ ] Figure 3. Different methods of synthesis of nanomaterials. Figure 3. Different methods of synthesis of nanomaterials. below in Figure 4. 3.2. Chemical Methods for Synthesis of Nanomaterials Yang et al., in 2019, reported the synthesis, engineering, and applications of ﬂy ash from various routes like physical and chemical but the emphasis was given mainly on the precursor mediated synthesis, not on the waste-based materials [100]. In 2015, Paul et al. reported the thin ﬁlm deposition of Feo on the Pt(111) by the ferrocene adsorption and oxidation method [101]. Zhang et al. reported the synthesis of multifunc- Crystals 2021, 11, 634 6 of 26 tional ﬂexible 2-dimensional carbon nanostructured N-nets reported their importance in electronics, energy, and the environment [102]. EW 6 of 27 tional ﬂexible 2-dimensional carbon nanostructured N-nets reported their importance in electronics, energy, and the environment [102]. W 6 of 27 Figure 4. Physical methods for the synthesis of 2D nanomaterial. Figure 4. Physical methods for the synthesis of 2D nanomaterial. Figure 4 Physical methods for the synthesis of 2D nanomaterial Figure 4. Physical methods for the synthesis of 2D nanomaterial. 3.2. Chemical Methods for Synthesis of Nanomaterials Chemical methods have contributed to the fabrication of materials at the nanoscale [95]. The Chemical methods have several advantageous properties over physical methods as the previous one involves mixing of chemical at the molecular level which ensures good chemical homogeneity [74,96]. Chemical reduction methods are reported to have numer- ous drawbacks for instance utilization toxic reagents and solvents, generation of un- wanted by-products due to which there are several extra steps is needed for removal of impurities, time-consuming [97]. The most common chemical methods are electroless deposition [98], lyotropic liquid crystal templates [34], hydrothermal and solvothermal method, sol-gel technique, electrochemical deposition, chemical vapor deposition (CVD), laser pyrolysis, and laser chemical vapor deposition techniques (LCVD), which are uti- lized frequently for the production of different NSMs. The above-mentioned techniques are shown in Figure 5. Among all the metallic nanoparticles silver nanoparticles has gained used considera- tion due to their exceptional properties and applications. Silver nanoparticles of different shapes and sizes have an important role in medicine, the biomedical ﬁeld and drug deliv- ery [103]. Till now silver NPs of various shapes and sizes has been reported by numerous investigators. Nanoprisms are one of the examples of 2D nanomaterial, which had gained huge attention in the biomedical ﬁeld [103]. below in Figure 4. 3.2. Chemical Methods for Synthesis of Nanomaterials Silver nanoprisms were synthesized silver salts by chemical reduction and photochemical method where the earlier method is more preferred than the later one due to their more controlled growth of nanoprisms which ﬁnds application in the industries [104]. Monodispersed hematite (a-Fe2O3) nanodiscs of size (50 ± 10 nm in diameter and thickness of 6.5 nm) synthesized under mild conditions through a facile hydrothermal method, i.e., hydrolysis of ferric chloride [105]. The reported method was quite unique as there was no use of surfactants, no toxic or hazardous chemi- cal precursors, and no high temperature decomposition of iron precursors in non-polar solvents. The synthesized hematite nanodiscs were further characterized by atomic force microscopy (AFM), X-ray diffraction (XRD), scanning electron microscopy (SEM), trans- mission electron microscopy (TEM), Brunauer–Emmett–Teller (BET), and superconducting quantum interface device (SQUID). The synthesis of Ta3N5 nanoplates was reported by Jie Fu and Sara E. Skrabalak, 2016, for the photocatalytic application [106]. A simple technique developed for the production of hexagonal-shaped Ag nanoplates whose diameter was in the range of 15–20 nm with a smooth nanobulk of 120 nm [107]. The silver nanoplates were prepared by a kinetically controlled solution growth method under the following conditions: polyvinyl pyrrolidone (PVP) as a capping agent, dextrose as a reducing agent, Crystals 2021, 11, 634 7 of 26 and urea as a habit modiﬁer at 50 ◦C and the crystalline structure of silver nanoplates analyzed by the XRD and TEM. EW 7 of 27 and urea as a habit modiﬁer at 50 ◦C and the crystalline structure of silver nanoplates analyzed by the XRD and TEM. IEW 7 of 27 analyzed by the XRD and TEM. Figure 5 Chemical methods of synthesis of nanomaterials Figure 5. Chemical methods of synthesis of nanomaterials. Figure 5 Chemical methods of synthesis of nanomaterials Figure 5. Chemical methods of synthesis of nanomaterials. g y Chen et al., in 2018, reported the synthesis of two-dimensional metallic nanomaterials from various routes [99]. Yang et al., in 2019, reported the synthesis, engineering, and applications of fly ash from various routes like physical and chemical but the emphasis was given mainly on the precursor mediated synthesis, not on the waste-based materials [100]. In 2015, Paul et al. reported the thin film deposition of Feo on the Pt(111) by the ferrocene adsorption and oxidation method [101]. Zhang et al. below in Figure 4. 3.2. Chemical Methods for Synthesis of Nanomaterials reported the synthesis of multifunctional flexible 2-dimensional carbon nanostructured N-nets reported their im- portance in electronics, energy, and the environment [102]. Among all the metallic nanoparticles silver nanoparticles has gained used consider- ation due to their exceptional properties and applications. Silver nanoparticles of different shapes and sizes have an important role in medicine, the biomedical field and drug deliv- ery [103]. Till now silver NPs of various shapes and sizes has been reported by numerous investigators. Nanoprisms are one of the examples of 2D nanomaterial, which had gained huge attention in the biomedical field [103]. Silver nanoprisms were synthesized silver Xin He et al., 2009 synthesized triangular/hexagonal silver nanoplates, nanobelts and chain-like nanoplate assemblies by utilizing N,N-dimethylformamide (DMF) along with PVP [108]. The results revealed that due to the strong interaction between Ag+ and PVP, there was the formation of individual nanoplates and external features of nanoplates were controlled by the ratio of AgNO3 and PVP. Sial et al., in 2018, synthesized multimetallic nanosheets which were utilized for the manufacturing of fuel cells [109]. Zheng et al., 2011 synthesized Palladium NSs by using CO as a reducing agent [110]. Yan-song Zhou et al., in 2016, reported an ultra-facile and generalized approach for the synthesis of metal oxide nanosheets (TiO2, Co3O4, Fe2O3, ZnO) with a larger surface and applied them for energy applications [111]. Jianxing Liu et al. reported the synthesis of hematite nanosheets by using a large-sized particles of iron red and found that the shape of hematite have important effect on the magnetic and optical properties [112]. All the above-mentioned chemical processes revealed a simple, reliable, and useful approach towards the synthesis of 2D NSMs. The shape, size, and composition of the 2D NSMs can be varied by precursor solutions, conditions of deposition and substrate materials [84]. g [ ] p y salts by chemical reduction and photochemical method where the earlier method is more preferred than the later one due to their more controlled growth of nanoprisms which finds application in the industries [104]. Monodispersed hematite (a-Fe2O3) nanodiscs of Besides all the above-mentioned techniques for the synthesis of nanomaterials, there are a few less applied chemical mediated approaches. One such technique is electrochemical synthesis mainly by anodization and cathodization. Though both the techniques are Crystals 2021, 11, 634 8 of 26 commonly used in an electrochemical based industry but rarely known for the synthesis of nanomaterials. 4. Carbon Nanomaterials Carbon is not only the most abundant element on Earth’s crust, but it also acquires exceptional properties because of its hybrid orbitals. The allotropes of carbon are mainly due to the hybridization of bonds formed after the combination of atomic orbitals (s and p) into new hybrid orbitals as sp, sp2, and sp3. The different allotropes of carbon are bucky- balls (0D), CNTs (1D), graphene sheets (2D), and diamond (3D) [119]. Due to the allotropy, carbon forms a separate class of 2D nanomaterials that includes graphene, GO, CNTs, buckyballs and its derivatives which are shown in Figure 6 and the properties of graphene oxide (GO) is shown in Figure 7. All these nanocarbons ﬁnd applications in electronics, environmental cleanup, drug delivery, agriculture, research and catalysis [120]. The wider applications of nanocarbons are also due to the presence of their wide range of structural and textural properties. Above all, nanocarbons, CNTs, and graphene are the most widely used nanomaterials in the ﬁeld of nanotechnology [120]. below in Figure 4. 3.2. Chemical Methods for Synthesis of Nanomaterials Several investigators have reported the synthesis of 1D, 2D, and 3D nanomaterials by using electrochemical methods. Dai et al., in 2019, reported the synthesis of a 1D nanomaterial by anodization method, and also highlighted their importance for manufacturing energy storage devices. Anodization is an electrochemical oxidation technique for depositing metal, metal oxides or semiconductors on a surface in order to increase the thickness of the metal. Nowadays, porous materials are also synthesized for enhanced applications in the ﬁeld of energy and wastewater treatment. By using this technique, mainly nanotubes are synthesized. The anodization mechanism depends on the various physical parameters like pH, time, potential, electrolyte temperature and water content. All these factors govern the morphology, porosity, wall thickness, and length of the synthesized nanomaterials. Till now, by applying such a technique, the following metal and non-metal oxides have been synthesized: ZnO, ZrO2, α-Fe2O3, WO3, Ta2O5, Nb2O5, HfO2, CuO/Cu2O, and NiO [113]. Kawde et al. reported the synthesis of AuNPs modiﬁed- graphite pencil electrode by cathodization. Further, it was used for the non-enzymatic sensitive voltametric detection of glucose [114]. Numerous investigators also reported the electrochemical based synthesis of either 1D, 2D, or 3D nanomaterials [115–117]. 3.3. Biological Methods for the Synthesis of 2D NSMs The biological synthesis of nanomaterials involves the synthesis from plants and their parts, microbes, e.g., algae, fungi, and bacteria. In comparison to the chemical and physical methods, biological methods are eco-friendly and there is a minimum utilization of hazardous chemicals. Besides this, the nanomaterials synthesized by biological methods are biocompatible. There are several reports wherein the nanomaterials have been synthesized by biological methods [116,118]. 4.1. Synthesis of GO from Agro Waste Sugarcane bagasse (SB) [123] is an agricultural waste that is rich in carbon content. Every year it is produced in MTs around the whole world and challenges a potential threat to the environment. The recovery of nanocarbons and GO from such waste will reduce pollution from the environment. The recovery of GO from sugarcane bagasse includes the following steps, collection of the ﬁber, crushing followed by grinding to obtain a powder, repetition of these two steps in order to increase the ﬁneness of the powder [124]. Grounded SB and ferrocene were mixed in a 5:1 ratio by weight, in a crucible, and calcination was performed in a mufﬂe furnace at 300 ◦C for 10 min under atmospheric conditions. The as-produced black solid was collected and the ﬁnal product was analyzed. Crystals 2021, 11, 634 9 of 26 4.2. Synthesis of Fullerenes (OD NMs) from Fly Ash an Industrial Waste Fly ash is an industrial waste, which is produced during the production of electricity from the pulverized coal in the thermal power plants (TPPs) (TPPs) [18]. Every year, a million tonnes (MTs) of coal ﬂy ash (CFA) are produced throughout the whole world, out of which about 50–60% are utilized while the rest are dumped in the ﬂy ash ponds in the near vicinity of TPPs. These ﬂy ashes act as a rich source of carbon materials like fullerene, CNTs, graphene etc. Such carbon nanomaterials are formed from the organic content of the coal and incomplete combustion of coal, soots, and charcoal combustion end products [12,121]. The unburned carbon could be either directly reused in the TPPs furnace as a fuel or it could be processed further for the synthesis of fullerenes. Other than coal combustion, industrial activities such as mining and metallurgical operations also contribute to ﬂy ash generation. Compositionally, ﬂy ash comprises diverse minerals and carbon materials either in single or combined form. The toxicity risk of ﬂy ash has recently been reported in relation to a deteriorating environmental quality in many developed and developing nations across the globe. In these circumstances, a potential utilization of these materials towards preparation of nanomaterials like CNTs [122], fullerenes [64] and several others could be a signiﬁcant breakthrough remedy to improve the pollution and toxicity extents and contents of environment. There are numerous researches where ﬂy ash have been used either for the recovery of fullerenes or synthesis of fullerenes. 4 1 Sy the i of Fulle e e (OD NM ) f o Fly A h a 4.3. Synthesis of Carbon Nanotubes/Carbon Nanoﬁbers 4.1. Synthesis of Fullerenes (OD NMs) from Fly Ash an Industrial Waste Fly ash is an industrial waste, which is produced during the production of electricity from the pulverized coal in the thermal power plants (TPPs) (TPPs) [18]. Every year, a million tonnes (MTs) of coal fly ash (CFA) are produced throughout the whole world, out of which about 50–60% are utilized while the rest are dumped in the fly ash ponds in the near vicinity of TPPs. These fly ashes act as a rich source of carbon materials like fullerene, CNTs, graphene etc. Such carbon nanomaterials are formed from the organic content of the coal and incomplete combustion of coal, soots, and charcoal combustion end products [12,121]. The unburned carbon could be either directly reused in the TPPs furnace as a fuel or it could be processed further for the synthesis of fullerenes. Other than coal combustion, industrial activities such as mining and metallurgical operations also contribute to fly ash generation. Compositionally, fly ash comprises diverse minerals and carbon materials ei- ther in single or combined form. The toxicity risk of fly ash has recently been reported in relation to a deteriorating environmental quality in many developed and developing na- tions across the globe. In these circumstances, a potential utilization of these materials towards preparation of nanomaterials like CNTs [122], fullerenes [64] and several others could be a significant breakthrough remedy to improve the pollution and toxicity extents and contents of environment. There are numerous researches where fly ash have been used either for the recovery of fullerenes or synthesis of fullerenes. Alam et al. reported the recovery and synthesis of fullerenes from CFA [7]. The nature of such fullerenes are impure which could be processed further to obtain highly pure fullerene. Yadav and Fule- kar also reported the presence and recovery of fullerenes from the fly ash [12]. 4.2. Synthesis of GO from Agro Waste Sugarcane bagasse (SB) [123] is an agricultural waste that is rich in carbon content. Every year it is produced in MTs around the whole world and challenges a potential threat One of the most systematically studied nanostructures, carbon nanotubes (CNTs) are cylindrically shaped materials with lengths in the order of few microns while the cross- sectional diameters are in the nanometer range [125,126]. The elongated surface of these materials makes them robustly versatile for their functionalization has driven need-based applications. 4.1. Synthesis of GO from Agro Waste Alam et al. reported the recovery and synthesis of fullerenes from CFA [7]. The nature of such fullerenes are impure which could be processed further to obtain highly pure fullerene. Yadav and Fulekar also reported the presence and recovery of fullerenes from the ﬂy ash [12]. EW 9 of 27 of hazardous chemicals. Besides this, the nanomaterials synthesized by biological meth- ods are biocompatible. There are several reports wherein the nanomaterials have been synthesized by biological methods [116,118]. 4. Carbon Nanomaterials Carbon is not only the most abundant element on Earth’s crust, but it also acquires exceptional properties because of its hybrid orbitals. The allotropes of carbon are mainly due to the hybridization of bonds formed after the combination of atomic orbitals (s and p) into new hybrid orbitals as sp, sp2, and sp3. The different allotropes of carbon are buck- yballs (0D), CNTs (1D), graphene sheets (2D), and diamond (3D) [119]. Due to the allo- tropy, carbon forms a separate class of 2D nanomaterials that includes graphene, GO, CNTs, buckyballs and its derivatives which are shown in Figure 6 and the properties of graphene oxide (GO) is shown in Figure 7. All these nanocarbons find applications in electronics, environmental cleanup, drug delivery, agriculture, research and catalysis [120]. The wider applications of nanocarbons are also due to the presence of their wide range of structural and textural properties. Above all, nanocarbons, CNTs, and graphene are the most widely used nanomaterials in the field of nanotechnology [120]. Figure 6. Different types of nanocarbons. Figure 6. Different types of nanocarbons. Figure 6. Different types of nanocarbons. Figure 6. Different types of nanocarbons. 10 of 26 10 of 27 Crystals 2021, 11, 634 Crystals 2021, 11, x FOR Figure 7. Properties of graphene. Figure 7. Properties of graphene. Figure 7. Properties of graphene. Figure 7. Properties of graphene. Figure 7. Properties of graphene. Figure 7. Properties of graphene. 4 1 S th i f F ll (OD NM ) f Fl A h 4.3. Synthesis of Carbon Nanotubes/Carbon Nanoﬁbers 4 1 Sy the i of Fulle e e (OD NM ) f o Fly A h a 4.3. Synthesis of Carbon Nanotubes/Carbon Nanoﬁbers Although the hybridization of constitutive carbon atoms is sp2 (similar to graphene), the arrangement of atoms is relatively distinct (that does not form layers). The two known variations are single-walled and multiwalled, with a high purity and cost of the former. The most extraordinary feature of these materials is their structural toughness, imparted by inherently high rigidity, Young’s Modulus, coefﬁcient of elasticity which together are the reasons for their robust suitability in civil, defense, aeronautic and many other strategic applications [126,127]. It is because of such remarkable properties that these materials are widely preferred for developing immobilization-based assays, with high detection sensitivities. An interesting aspect of these nanomaterials is that based on their geometry and chiral carbon vicinity, these can be electrically conductive, semi-conductive, or insulated [126]. These adjustable electronic properties form the basis of their usage in single electronic transistors, ﬂexible automated diodes where electron ﬂow needs to be manipulated [128]. Comprised of only carbon, a variety of substrates have been used to obtain nanotubes via differently explored mechanisms. The most widely used methods of preparation are laser ablation, CVD [129], and electric discharge, which necessitate the provision of a speciﬁc stoichiometric mix of precursors. Though there are some concerns regarding the drug delivery application of these materials (with a potential risk of toxicity initiation), still the ability of functionalization has minimized such concerns and enabled a dose and location-speciﬁc drug delivery with them. Readers are suggested to consider more speciﬁc literature sources regarding the biological applications of these nanomaterials. Substrates as common as biscuits, chocolates, waste tyres, rubber, and manifold carbon- comprising substances have been used to prepare carbon nanotubes [130–132]. Crystals 2021, 11, 634 11 of 26 11 of 26 4.4. Synthesis of CNTs from Fly Ash 4.4. Synthesis of CNTs from Fly Ash Several studies report the preparation of CNTs from ﬂy ash, with a 2016 study claimed the utility and aptness of Saudi Arabian ﬂy ash to provide CNTs using chemical vapor deposition method, provided all reaction conditions are maintained [133]. The preparation of CNTs from ﬂy ash could be considered as an alternative to the famous electric arc- discharge method, with signiﬁcant reports of transition metals (Mn, Mg, Ca, Na, Pb, Cd, Cr, Co, Ni, Zn, and Mo), present as traces in the ﬂy ash. Depending on the regional geography and parent source of generation, the transition metal composition and diversity extents may vary amongst different sources. A generalized idea of typical ﬂy ash composition is mentioned in Table 1. This synthesis of CNTs serves a dual purpose, one being the minimization of hazardous waste in the environment, the other being the cost-effectiveness and minimized use of energy. So, this approach is ﬁttingly a green solution to minimize the undesired environmental risks of ﬂy-ash by means of a sustainable approach. Research on particulate matter pollution does pose a concern of signiﬁcant respiratory complications from inhaling ﬂy-ash. Table 1. Elemental composition of ﬂy ashes. Elements Composition (wt. %) SiO2 40–60% Al2O3 20–40% Fe2O3-Fe3O4 5–15% TiO2 2–5% Carbon 5–20% CaO, BaO, MgO, MnO, P2O5 Traces Table 1. Elemental composition of ﬂy ashes. 4.5. Synthesis of CNTs from Plastic Waste and Tyres Carbon based everyday gadgets, such as plastic materials, tyres [75], rubber end products, and several other forms, can be readily used for making CNTs, using several modiﬁcations in their subsequent chemical treatment approaches. The generation of plastic wastes to the tune of billion tones on an everyday basis is one of the most pulsating concerns since plastic wastes often encounter a disposal problem due to their biodegradability concerns. Plastics are viciously produced as waste products from industries, household routines, laboratories, hospitals and eateries. Although the non-biodegradable nature of these materials has resulted in their substantial recycling, recycled plastics often lose their plasticity. Many studies have nevertheless used the plasticity intact waste materials to make CNTs via processing under varying oxygen environments. In one such study, plastic waste was readily decomposed to propylene which subsequently catalyzed the MWCNT formation over the surface of metal catalysts [134]. The growth mechanism is well known, reportedly following a tip-growth or base-growth pattern in the course of a vapor–liquid–solid reaction [135,136]. Nevertheless, there is still no clarity regarding the utilization of carbon atoms whether in the bulk catalyst or react within the top surface of the catalyst. The reaction was mediated by the utilization of reactor material (SS 316 tube of a CVD reactor), with the conﬁrmatory studies revealing that removal of Cr from the reactor vessel resulted in MWCNT growth. Similar studies on SS 316 mesh surface found the involvement of Fe and Ni in the CNT formation. The results were in agreement with the works by Levendis and co-workers with a further ensuring of metal catalyst prevalence along the tip of MWCNTs inside the tubes [137]. g p Similarly, a 2016 study by Zhang and Williams reports the synthesis of MWCNTs along with hydrogen generation by the catalytic pyrolysis of waste tyres. The study employed a catalyst system comprising of a Ni/Al2O3 prepared via impregnation of Ni on the Al2O3 surface. The experimental procedure was optimized by varying the temperature from (700 to 900) ◦C, alongside varying the tyre to catalyst ratios from 1:0.5 to 1:1 and 1:2 and Crystals 2021, 11, 634 12 of 26 12 of 26 using steam input via injection of hot water at 0.2 and 5 mL per hour injection rates. 4.5. Synthesis of CNTs from Plastic Waste and Tyres Estimation of the carbon fractions (formed as product) revealed 253.7 mg per gram tyre to be comprising of ﬁlamentous carbons at 1:1 tyre to catalyst ratios at a catalyst temperature of 900 ◦C. Microscopic screening of the product showed a signiﬁcant proportion of deposited ﬁlamentous carbons as MWCNTs. The procedure also released hydrogen at compatible rates that met the fuel and energy scarcity, making this overall approach a reliable and efﬁcient methodology to utilize the tyre waste. An important aspect of this approach was that it ﬁrstly processed the nickel nitrate as nickel precursor by its dissolution in ethanol on alumina support that gradually converted into slurry via continuous stirring. The ﬁnal catalyst was prepared on overnight drying of this slurry at 90 ◦C in an oven, at 2 ◦C per minute till the temperature reached 750 ◦C. This process took nearly 3 h of holding time, following which the solid material collected was crushed into (0.05–0.18) mm sized granules. It is interesting to note that the smaller size of catalyst particles conferred a higher surface area to the reacting species, so whether a different physical form of the particles would be able to provide the product in the same morphology with a similar yield remains a signiﬁcant concern [132]. g Quite recently, the synthesis of CNTs was reported from waste rubber-based substrates, with the experimental procedure utilizing the blended form of acrylonitrile butadiene and styrene-butadiene rubbers (NBR and SBR). The blend could not be conventionally decomposed due to its stronger mechanical strength and thermal resistance, however, the pyrolysis of the disposable form of the blend was optimized at 450 ◦C in a horizontal CVD pyrolyzer with a cautiously maintained nitrogen supply to yield hydrocarbon fractions. Upon allowing the CVD of these hydrocarbon fractions on different catalytic systems at 850 ◦C for half an hour, the screening of formed product using HRTEM, thermo gravimetric analysis (TGA), and Raman spectroscopy inferred a signiﬁcant formation of SWCNTs to an efﬁcient extent. Subsequently, in the course of physical analysis, it was noted that adjusting the crystallinity of Fe-Ni catalyst on different zeolites was a critical factor affecting the structure and diameter of as formed CNTs [75]. So, approaches like these are all potential solutions to synthesize nanotubes in desired yields from robust, cheap, and biocompatible materials ensuring, minimal pollution risk and higher output yields compared to costly and energy intensive conventional methods. 4.6. Synthesis of CNTs from Agro Waste: Rice Husk In the different parts of the world, rice husk (RH) shows as one of the most dom- inant crop residues and the disposal of which often results in crucial environmental risks [138–140]. The major constituent of RH as well as its burnt ash is silica (up to 90%) (widely used as fillers and area enhancement specific applications). So, efforts to utilize RH in its native as well burnt forms as a reliable material providing energy are on a rapid high. Furthermore, the global RH production registered a nearly 6% increase from 2010 to 2014, which represent an alarming threat as an environmental hazard [141]. The utilization of rice husk (substantially comprising carbon, nitrogen, and hydrogen) commences with gasification (or pyrolysis), which generates fragments suitable for power generation and biologically compatible charcoal. The one deemed fit for power generation could be utilized as such via landfilling and fertilizer application. However, the fraction acting as bio-reduced char contributes significantly to industrial activities. This fraction provides three potential materials, active carbon, porous carbon and amorphous silica, all of which have highly good absorption characteristics conferred by their significant surface area contributions. While amorphous silica finds peculiar suitability in soil improvement and the cement in- dustry the active and porous carbon fractions are highly efficient adsorbents and used for wastewater treatment applications. So, with a carbon texture, the normally waste RH could be potentiated into manifold useful industrial products. Readers can have a detailed look at the RH utilization and processing methodology in a highly informative contribution made by Nguyen et al. in 2019. This is a review article that comprehensively discusses the engi- neering and industrial potential of RH and its derivative fractions (such as silicon nitride, Crystals 2021, 11, 634 13 of 26 13 of 26 magnesium silicide and others) as refractory materials, filler agents in thermoplastics, as reinforcement agent, the adsorbent in polymer composites and many others. Osman et al. reported the synthesis of activated carbon and CNTs by using Miscanthus × giganteus which is a high silicate containing common perennial grass. Both the particles were characterized by the sophisticated instrument, i.e., mainly for their pore size prior to their application in wastewater treatment [142]. 4.7. Synthesis of CNTs and Graphene from Oil Oils represent some of the most used commodities which are basically natural hy- drocarbon precursors having varying carbon chain fatty acids. The carbon skeleton of oils, accompanied by a range of physical and chemical modifying technologies such as fractional crystallization, fractional distillation, chromatographic separation, aqueous two- phase attraction are the incentives for their reduction procedures that could enable a range of products. Several kinds of oils, such as turpentine, eucalyptus, palm, turpentine, neem (Azadirachta indica), and sunﬂower, have been reported to enable efﬁcient scale synthesis yields of CNTs and graphene [143]. The use of turpentine oil in the making of CNTs has been proposed by Chatterjee et al. through its decomposition on the surface of ﬁnely dispersed Co catalyst at 675 ◦C optimized the CVD method to synthesize CNTs. The study also proposed the application of synthesized CNTs in making efﬁcient electrochemical double-layer capacitor [144]. In several interesting modiﬁcations, scientists have optimized the use of neem, sun- ﬂower, sesame, camphor and castor oils as the parent carbon sources for CNT synthesis. The utilization of sesame oil has attracted signiﬁcant scientiﬁc attention, owing to its ed- ible nature, clean methodology and formation of hollow CNTs with diverse shapes and morphology [145]. The formed nanotubes had no Fe nanoparticles in the interior, had diameters within 50–60 nm, and a sheet-like structure showing an intricate long-range array of folds. Thus, synthesis of nanotubes from oil represents the renewable, energy-efﬁcient, cost-effective and most importantly, much more compatible to environment and laboratory personnel [146]. So, since the CNTs inception, making CNTs in big yields is now no more a herculean task than in the beginning years. 4.8. Synthesis of CNTs and Graphene from Poultry Waste Poultry products or waste are also rigorous sources of carbon materials and their derivatives and are mostly comprised of carbohydrates and proteins, along with a dense supplement in the form of calcium [147]. Regarding the utilization of these materials to meet the energy concerns, eggshell material promises to be a very rich source of providing carbon skeleton, it has been used with signiﬁcant interest to optimize the microbial growth for designated yields of biofuels. Though CNTs are concretely not reported as being synthesized from these materials, yet a modiﬁed version, namely, C-dots (inherently carbon comprising quantum dots) have successfully been synthesized using this natural resource. The primary advantage of these nanomaterials compared to conventional quantum dots is their low toxicity. A 2012 study reported from China has optimized the microwave assisted approaches (providing intensive and efﬁcient energy) to process egg shell material for a reduced reaction time to obtain C-dots [148]. The study aimed at the microwave treatment of eggshell material to form C-dots, having a maximum ﬂuorescence peak (at 450 nm) alongside a quantum yield of ~14%. The modiﬁcation of operational parameters like reaction time (microwave duration), temperature, and the relative contents of eggshell material could be the signiﬁcant leads to obtaining many other variations in the products, and for obtaining the biologically and biophysically more robust product designs. 4.9. Carbon-Based Fullerene-Like (FL) Solid Compounds These compounds are the new class of materials, which have remarkable mechanical properties which can be tuned very easily in order to dope with numerous dopants. Cecilia et al. reported the synthesis of Fullerene like sulfocarbide studied with difunctional Crystals 2021, 11, 634 14 of 26 14 of 26 theory (DFT). They studied their formation, energetic and structural effects of sulphur atoms at carbon sites in a graphene-like network and many other properties [36]. FL like CPx was also reported by Furlan et al., in 2006 and studied the relative stability of precursors and defect energetics during synthetic growth [149]. 4.10. Surface Functionalization and Modiﬁcation of Graphene The synthesis of 2D nanoparticles from agricultural and industrial waste not only makes the product economical, but it also minimizes the solid waste pollution arising every year. Government organizations expands a large amount of money on the handling of such waste. So, synthesis of nanoparticles by using all the above industrial and agricultural waste makes an ecofriendly and economical approach. However, their acceptability among industries can be drastically enhanced by either surface modiﬁcation or by isomerization. Both steps will bring a change to the external surface of 2D nanoparticles. One of the most common 2D nanoparticles which can be easily surface functionalize is graphene. The surface modiﬁcation of GO can be carried out by several methods for instance; modiﬁcation by means of chemical, covalent and non-covalent modiﬁcation, electrochemical modiﬁca- tion, and decoration of graphene by metal and metal oxide nanoparticles [13]. All these surface functionalization techniques makes the graphene speciﬁc for instance in biomedi- cal, wastewater treatment and agricultural applications [150]. For the elimination of any speciﬁc pollutant from the wastewater, graphene can be modiﬁed, which will enhance the speciﬁcity of the graphene [151]. Similarly, in the medical ﬁeld, such graphene can also be decorated with the antibacterial nanoparticles, e.g., Ag, ZnO, etc., which will have effective and efﬁcient antimicrobial effects [152]. By surface modiﬁcation and isomerization, the applicability of the 2D nanoparticles like graphene could be increased for industrial and daily purposes. 4.11. D Carbon Nanomaterials Carbon-based 3D nanomaterials are very promising for various applications, i.e., electrochemical energy conversion and storage etc. Various carbon allotropes doped heteroatoms can be utilized for low-cost mass production of electrode materials. Porous 3D carbon provides multiple advantages, such as large surface area, maximized exposure to active sites, 3D conductive pathways for efﬁcient electron transport, and porous channels to facilitate electrolyte diffusion. It is difﬁcult to synthesize and functionalize isotropic 3D carbon structures still very useful material for various applications. There are several wastes generated from biological activities like agriculture, or from the industry, e.g., red mud, ﬂy ash, etc., which could be used as a precursor material for the synthesis of 3D carbon nanomaterials. There is also incense sticks ash, produced after burning incense sticks at houses and temples in South Asian countries. Since such incense sticks are ignited at low temperature, about 20–30% ash has unburned carbon, which could be processed further for the synthesis of an economical and sustainable source of carbon nanomaterials [153]. Paul et al. reported the synthesis of highly porous 3D CNT foam as an anode for the Li-ion based batteries for energy-based applications [154]. Paul et al. reported the synthesis of 3D heteroatom doped CNMs, as multifunctional ferrous free catalyst for the applications of enery storage devices [155]. Paul et al. synthesized BN co-doped CNT based nanoporous brushes for energy-based applications especially in the form of supercapacitors at elevated temperatures [156]. Maria et al. emphasized the importance of polymers in the design of 3D CNTs-based scaffolds for biomedical purposes [7,157]. 5.1. Catalytic Applications of 2D Nanomaterials in Fuel Cells In the present-day energy-savvy scenario, everyone is anxious to obtain quicker and greater product formation, minimizing not only the operational steps, but also the energy requirements. 2D NMs serve as ideal solutions to all these concerns in having a high aspect ratio, high electron mobility, unsaturated surface coordination, and unique material properties (especially physical, chemical and electronic) [161]. The ultraﬁne thickness of these materials confers upon them ultrahigh speciﬁc surface areas and high surface energy, making them appropriate towards numerous surface-active applications such as those in fuel cells. For the efﬁcient function of these cells, oxygen generation and transport have to take place at reasonably good rates. The catalytic approaches in most general cases employ platinum (Pt) nanoparticles (NPs) immobilized on the surface of the carbon substrate. However, due to their high costs and slow reaction kinetics, the use of Pt NPS is not economically as well as commercially viable. To tackle these issues, the development of new methods like alloying and nanostructured engineering which could ensure maximum activity, stability along with cost minimization has emerged to be a priority [109]. Amongst the several different shapes attainable by noble metal alloys, ultrathin 2D sheet-like struc- tures having a single or few atoms thickness are acquiring signiﬁcant interest because of their large size, high electron mobility and surface energy. These features confer a high surface area to volume ratio to the ultrathin 2D sheet-like materials thereby giving rise to a high density of unsaturated atoms. For instance, Hong et al. have reported faster ethanol oxidation using ultrathin free-standing Pd-Pt-Ag (ternary) noble metal alloy [162]. Similarly, Din et al. proposed the suitability of quaternary noble metal alloy Pt-Cu-Bi- Mn (porous nanosheets) having 3–4 nm thickness as novel catalysts having high oxygen (reduction and oxidation) capabilities apart from a signiﬁcant methanol tolerance [163]. Paul et al., 2019, highlighted the importance of carbon NMs based metal-free electrocatalyts for various applications [164]. Further, Paul et al. reported the applications of nanoporous graphitic carbon for supercapacitors and similar purposes [165]. Cheng et al. reported the 3D printing functional NMs for the electrochemical and energy storage purposes [166]. p g gy g p p Bismuth-based nanomaterials are a unique category of materials that holds interesting properties like chemical, electrical and catalytic activities. Bismuth-based nanoparticles, including bismuth chalcogenides, bismuth vanadate, and bismuth oxyhalides, continue to show excellent photocatalytic activity in wastewater treatment. 5. Applications of 2D Nanomaterials The speciality of nanomaterials lies in their tunable nanoscale dimensionality, on the basis of which these are considered as one, two or three dimensional [158]. Thus, two-dimensional nanomaterials are typically those materials that have two of their three di- mensions restricted to <100 nm [159]. There is no clear consensus regarding the upper limit of this restriction. This implies that, in these materials, it is feasible to retrieve the quantum Crystals 2021, 11, 634 15 of 26 15 of 26 scale effects on two dimensions, i.e., the restriction of the electronic motions of excited state electrons (more conventionally known by the terminology “quantum conﬁnement”). Examples of these materials include nanosheets, ﬁbrous networks having nanometric widths and heights with lengths in the order of micrometres. Popular applications of these materials include their inclusion as catalysts, electronic/battery devices, hydrogen sensing, laser protection, magnetic memory devices, and other domains, based on surface plasmon resonance (SPR) attributes [160]. 5.1. Catalytic Applications of 2D Nanomaterials in Fuel Cells Bi-based materials like halogen combine Bismuth materials are showing very good topological applications. Freitas et al., 2015 synthesized 2D-Bi containing single layers which were preserved by hydrogenation and concluded that the hydrogenation step, provides a ﬂexible chemical ten- ability that has the potential to preserve the band topology of the pristine XBi phases [167]. Sial et al., have rigorously compiled the several methods of making nanosheets (NSs) and their limitations in the present scenario (pertaining to energy considerations and economic constraints). Different methods of synthesizing 2D NSs are carbon monoxide (CO) conﬁned growth, hydrothermal/solvothermal synthesis, wet chemical synthesis, self-assembly of NPs, topochemical reduction method, template-based synthesis, seeded growth, and microwave-assisted growth. Even though each of the methods provides speciﬁc characteristics of products in terms of morphology, the unanimous factors affecting their implementation are the need for robust catalysis (which offers lesser reaction time and is less costly) and the requirement of energy from an external agency. For example, CO assisted growth method allows the preferential growth on the substrate due to good Crystals 2021, 11, 634 16 of 26 16 of 26 surface adsorption of CO. These methods are workable through the feasibility of interac- tional distinctions of water and non-aqueous solvents, such as viscosity and dissociation constant. The process is characterized by selective oxidative etching enabling attainment of speciﬁc anisotropic growth. Two critical requirements of these methods are optimum reaction temperature maintenance alongside the steady action of a reducing agent. Like- wise, wet chemical synthesis offers layered patterns of ultrathin NSs, with industrially scalable products allowing no CO requirement (unlike the CO assisted growth method and hydrothermal/solvothermal method). Another mechanism of interest is self-assembly which provides NSs regulated by weak binding interactions and comparatively larger sizes. However, the advantage in this method is that requirement of energy from the external end is very low and the constituent species themselves acquire a minimum en- ergy conﬁguration. Like-wise, the topochemical reduction approach is speciﬁcally suited for making single crystalline metal alloy NS utilizing Ni and Co as a combined catalyst in an aqueous medium while the template synthesis method is an efﬁcient strategy to obtain layered nanostructures and extensively utilizes graphene and its derivatives as templates. 5.1. Catalytic Applications of 2D Nanomaterials in Fuel Cells Comparing the basic requirements of these two methods, it is quite evident that the template synthetic approach offers much higher control with every successive step being regulated by the chemical composition of the preceding deposited material layer. Another benign approach for making 2D NSs is the use of microwave technology, which is especially preferred for making inorganic nanomaterials having high quantum yield and high precision. Although this is a green approach, it is yet again dependent on energy input from outside. Often template-based synthesis mechanisms utilizing hydrothermally fabricated catalysts have relied on for commercial purposes [168]. y p p The working of fuel cell involves rigorous electrochemical processes, characterized by an electrocatalytic oxygen reduction reaction (ORR) and hydrogen evolution reaction (HER), involving formic acid oxidation and alcoholic oxidation at cathode and anode. The major problems encountered in the commercialized application of fuel cells are improvements in the electrode preparation with minimized use of precious metals, controlling the kinetics of the electrochemical process which collectively reduces the output efﬁciency of a fuel cell. So, in general, faster, more efﬁcient, and rigorous catalysis with minimized expenditure and care requirements are the key. With continuous better understanding, several alternative mechanisms have emerged as steady sources of energy provision, like microbial driven fuel cells which utilize the energy generated from microbial metabolism (the functioning of enzymes and key pathways). However, this recourse is also not free of constraints as there is a constant need to ensure optimum microbial activities through providing speciﬁc pH, temperature, humidity and minimizing the ion concentration [169]. Recently, a new methodology making use of CNT-based composite materials has emerged. The concurrent hindrances related to dependence on water for conductivity, high methanol permeability, frequent disintegration (of conventionally used materials) in the presence of –OH radicals and low to moderate chemical stability have been the reasons to screen a safer, more reliable and efﬁcient alternative. A novel attempt in this direction has been the use of naﬁon based membranes and its composite with the inclusion of CNTs as polymer electrolyte material (PEM) has provided a solution to recurrent limitations, through its greater mechanical stability, greater tensile strength and stronger physical texture [170]. Thus, nanomaterials provide numerous structural beneﬁts to improve the fuel cell working through improvement in catalysis and energy-savvy functioning. 5.3. Nanotechnology and Solar Energy Probably, the cleanest, unanimously accessible and even most used form of energy, solar energy is a rigorous input agency for most of daily life activities. From microbes to plants, animals and even human beings, all require solar energy directly or indirectly for the sustenance of life. Commercial usage of solar energy presents exciting prospects, which are often limited by its low efﬁciency (substantially attributed to the uncertainty of availability) and inabilities to be scaled up. Lots of progress has been made via the use of nanomaterials in native and engineered form, to increase the absorption efﬁcacy of the sun’s energy radiations. The most popular area has been the use of solar cell panels to provide electricity in which the functional circuit comprises an assembly of solar cells in a rectangular pattern. The efﬁciency of original assembly is quite low owing to which Si wafers (with amicable impurities) are added to it, which collectively not only improve the absorption but also manifold the utilization extents. Similarly, nanoscale attenuators and converters have been drafted into calculators to improve their charging efﬁciencies and performance. Lots of bioassays and drug carrier systems are in the market working through photothermal attributes of metallic NPs and their constitutive assemblies. Thin layers or assemblies of nanomaterials have emerged as carriers of more uniform and regulated solar energy absorption that remain localized to the surface and do not cause any serious effect in the bulk. Piezoelectric materials (such as MgO and ZnS based nanostructures) have come to the forefront, making use of pressure inﬂuences from solar energy (as input) to conduct the electricity or perform mechanical works. Many of these conceptualizations are in the research phase, with delays in optimization studies, meeting the scale-up regulations and constraints. Owing to this, the commercialization of such innovations is being delayed. Considering the energy crisis scenario (in particular for the developing world), these solutions could be potential remedies to eradicate the inadequate energy availability. Recently, the use of nanoﬂuids (typically having either solid NPs or (1–100) nm-sized nanoﬁbers suspended in a liquid) has been on the peculiar rise to enhance the utilization potential of solar energy [172]. These ﬂuids, having dissolved nanomaterial(s), are able to enhance the outlet temperature by 30–100 K, enabling an enhanced potential to absorb the sunlight without any damage to native structures of the base material. 5.2. Applications Related to Surface Plasmon Resonance SPR is the characteristic phenomenon driven by the dominant surface effect of nano- materials, and more speciﬁcally the metal or metal oxide NPs. These entities absorb light at maximum at a peculiar wavelength after which the constituent ions are excited and progressively move to a high energy state. As the temperature increases (due to the input heat or light energy or via intermolecular frictional activities), these excited particles rapidly move with a net charge and remain in the semi-solid state, termed as plasma. The Crystals 2021, 11, 634 17 of 26 17 of 26 terminology plasmon is originated from the essence of ions existing in the plasmonic state. The resonance implies an instant where the light energy absorption is maximum, owing to which the manifested surface effects are also greater. Each nanoparticle has a characteristic SPR corresponding to a particular kind of incident light, so the SPR wavelengths are often used as identiﬁers for the formation of speciﬁc NPS. Since there is maximum energy absorbance in the SPR event, the nanoscale effects are also highest at this particular instant, giving rise to maximum bioactivities or quantum conﬁnement dependent properties. The applications of nanomaterials have been signiﬁcantly improved after a clear understanding of this phenomenon, with bulk species or sensing moieties being swiftly re-placed either by individual NPs (bound in membranes) or by the combination of nanomaterials (such as assembled nanostructures or hybrid NPs and thin layers of nanomaterials. For detailed insights into SPR and its consequent applications, readers are suggested to refer to more speciﬁc literature [171]. 5.3. Nanotechnology and Solar Energy One study claimed a more than 100% enhancement in photo thermal efﬁciency of 0.01% graphite-based nanoﬂuid than without using it (normal functioning mode involving the coating of an absorbing collector). The use of these ﬂuidic materials has enabled improved photovoltaic application via long-lasting existence in non-agglomerated form, having high stability without undergoing signiﬁcant chemical changes in the base ﬂuid [173]. The use of nanoﬂuids has signiﬁcantly improved the efﬁciency of electrolysis manifolds by the replacement of conventional electrolytes, allowing the faster and smoother conduct of chemical reactions [174] Crystals 2021, 11, 634 18 of 26 18 of 26 6. Conclusions & Future Prospects The progress and better understanding of nanotechnology and its functional principles have slowly entered into the multiple inter and cross-domain disciplines, to improve the product life, design, performance and overall quality by a considerable reduction in the raw materials. The use of nanostructures like CNTs, 2D nanosheets, several different kinds of NPs have provided stronger and more efﬁcient materials, enabling multifunctional performance and increased outputs. The incorporation of CNTs along with graphene and their derivatives have bettered the present performance of materials by a substantial improvement in their structural responses, mechanical strength, stress-bearing capacity and physical load-bearing capacities. The availability of these materials in multiple nanoscale dimensions has enabled the synthesis of desired materials with robust self-adjusting responses and ﬂexibilities. Though much remains unknown and even unpredictable, which sometimes poses a risk in terms of their nanoscale manifested enhanced chemical reactivity, regular research and the continuous merging of scientiﬁc cross disciplines have signiﬁcantly improved the understanding with respect to the use of nanomaterials. The market scenario predicts hopeful aspects from a consumer point of view, while at the same time appearing a little gloomy for the reduced manpower requirement. So, a better understanding of nano-material usage and applications deﬁnitely presents a brighter future and better living standard for mankind. • Every year, tonnes of industrial wastes are produced with no use. Scientists are now starting to utilize the wastes for the synthesis of nanoparticles such as metals and metal oxide nanoparticles, nano-cellulose, carbon-based nanoparticles, and nano-ﬁbres. p p • These nanoparticles are further used to solve various environmental problems, espe- cially nanoremediation. The futures prospects of waste-generated nanomaterials are in a wide range of po- tential applications as sustainable, ecofriendly, and low-cost alternatives to conventional nanomaterials. Author Contributions: Conceptualization, V.K.Y., P.M. and P.R.P.; methodology, V.K.Y., K.K.Y., S.I., L.D. and R.S.; validation, S.H.K., P.R.P., L.D., M.M.S.C.-P. and A.H.K.; formal analysis, P.A.D., K.K.Y., P.A.D., S.H.K. and R.S.; resources, M.A.H., S.I., M.M.S.C.-P., L.D. and A.H.K.; writing—original draft preparation, V.K.Y.; S.H.K., R.S., P.M. and P.R.P., writing—review and editing, V.K.Y., M.A.H., P.A.D. and K.K.Y.; supervision, V.K.Y., A.H.K., L.D., M.M.S.C.-P. and S.I.; project administration V.K.Y., L.D., P.A.D., P.R.P. and P.M.; funding acquisition, A.H.K., S.I., M.A.H., L.D., P.A.D. and M.M.S.C.-P.; software, S.H.K., M.A.H., M.M.S.C.-P., K.K.Y., S.I., R.S. and P.M. All authors have read and agreed to the published version of the manuscript. Author Contributions: Conceptualization, V.K.Y., P.M. and P.R.P.; methodology, V.K.Y., K.K.Y., S.I., L.D. References Synthesis of γ-alumina (Al2O3) nanoparticles and their potential for us th l f th l bl d f i d t i l t t N l Ad 2019 1 213 218 [C R f] , ; , ; , ; , y γ ( 2 3) p p the removal of methylene blue dye from industrial wastewater. Nanoscale Adv. 2019, 1, 213–218. [CrossRef] 7. Alam, J.; Yadav, V.K.; Yadav, K.K.; Cabral-Pinto, M.M.; Tavker, N.; Choudhary, N.; Shukla, A.K.; Ali, F.A.; Alhoshan, M.; Hamid, A.A. Recent Advances in Methods for the Recovery of Carbon Nanominerals and Polyaromatic Hydrocarbons from Coal Fly Ash and Their Emerging Applications. Crystals 2021, 11, 88. [CrossRef] 8. Pyun, J.; Matyjaszewski, K. Synthesis of Nanocomposite Organic/Inorganic Hybrid Materials Using Polymerization. Chem. Mater. 2001, 13, 3436–3448. [CrossRef] y , , [ ] 9. Hanemann, T.; Szabó, D.V. Polymer-Nanoparticle Composites: From Synthesis to Modern Applications. Materials 2010, 3, 3468–3517. [CrossRef] 9. Hanemann, T.; Szabó, D.V. Polymer-Nanoparticle Composites: From Synthesis to Modern Applic 3468–3517. [CrossRef] 10. Tavker, N.; Yadav, V.K.; Yadav, K.K.; Cabral-Pinto, M.M.; Alam, J.; Shukla, A.K.; Ali, F.A.; Alhoshan, M. Removal of Cadmium and Chromium by Mixture of Silver Nanoparticles and Nano-Fibrillated Cellulose Isolated from Waste Peels of Citrus Sinensis. Polymers 2021, 13, 234. [CrossRef] 11. Marghussian, V. Nano-Glass Ceramics: Processing, Properties and Applications; Elsevier: Alpharetta, GA, USA, 2015; pp. 1–282. 12. Yadav, V.K.; Fulekar, M.H. Advances in Methods for Recovery of Ferrous, Alumina, and Silica Nanoparticles from Fly Ash Waste. Ceramics 2020, 3, 34. [CrossRef] 12. Yadav, V.K.; Fulekar, M.H. Advances in Methods for Recovery of Ferrous, Alumina, and Silica Nanoparticles from Fly Ash Waste. Ceramics 2020, 3, 34. [CrossRef] 13. Yoriya, S.; Intana, T.; Tepsri, P. Separation of Cenospheres from Lignite Fly Ash Using Acetone–Water Mixture. Appl. Sci. 2019, 9, 3792. [CrossRef] 14. Yadav, V.K.; Yadav, K.K.; Gnanamoorthy, G.; Choudhary, N.; Khan, S.H.; Gupta, N.; Kamyab, H.; Bach, Q.-V. A novel synthesis and characterization of polyhedral shaped amorphous iron oxide nanoparticles from incense sticks ash waste. Environ. Technol. Innov. 2020, 20, 101089. [CrossRef] 15. Rana, S.; Fangueiro, R.; Thakur, V.K.; Joshi, M.; Thomas, S.; Fiedler, B. Nanomaterials from Natural Products for Industrial Applications. Available online: https://downloads.hindawi.com/journals/specialissues/743287.pdf (accessed on 30 May 2021). 16. Dutta, T.; Kim, K.-H.; Deep, A.; Szulejko, J.; Vellingiri, K.; Kumar, S.; Kwon, E.; Yun, S.-T. Recovery of Nanomaterials from Battery and Electronic Wastes: A New Paradigm of Environmental Waste Management. Renew. Sustain. 6. Conclusions & Future Prospects and R.S.; validation, S.H.K., P.R.P., L.D., M.M.S.C.-P. and A.H.K.; formal analysis, P.A.D., K.K.Y., P.A.D., S.H.K. and R.S.; resources, M.A.H., S.I., M.M.S.C.-P., L.D. and A.H.K.; writing—original draft preparation, V.K.Y.; S.H.K., R.S., P.M. and P.R.P., writing—review and editing, V.K.Y., M.A.H., P.A.D. and K.K.Y.; supervision, V.K.Y., A.H.K., L.D., M.M.S.C.-P. and S.I.; project administration V.K.Y., L.D., P.A.D., P.R.P. and P.M.; funding acquisition, A.H.K., S.I., M.A.H., L.D., P.A.D. and M.M.S.C.-P.; software, S.H.K., M.A.H., M.M.S.C.-P., K.K.Y., S.I., R.S. and P.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Deanship of Scientiﬁc Research, King Khalid University, Abha, Kingdom of Saudi Arabia, grant number RGP.1/174/42. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available within the article. Acknowledgments: The authors thankfully acknowledge the Deanship of Scientific Research, King Khalid University, Abha, Kingdom of Saudi Arabia, for funding the research through RGP.1/174/42 grant. Conﬂicts of Interest: The authors declare no conﬂict of interest. 19 of 26 Crystals 2021, 11, 634 Al2O3 aluminum oxide BaO barium oxide BET Brunauer–Emmett–Teller ◦C degree Celsius Ca calcium CaO calcium Oxide Cd cadmium CO carbon monoxide Co3O4 cobalt(II,III) oxide CNTs carbon nanotubes Cr chromium CVD chemical vapor deposition DMF N,N-dimethylformamide Fe ferrous Fe2O3 ferric oxide Fe3O4 ferrous ferric oxide Ga gallium GO grapheme oxide HER hydrogen evolution reaction HRTEM high resolution transmission electron microscopy InSe indium selenide K kelvin LCVD laser chemical vapor deposition techniques LEDs light emitting diodes Mg magnesium MgO magnesium oxide µm micrometer Mn manganese MnO manganese oxide Mo molybdenum MoO3 molybdenum trioxide MoS2 molybdenum disulﬁde MWCNT multi walled carbon nanotubes Na sodium Ni nickel Nm nanometer NMs nanomaterials NSMs nano structured materials NSs nanosheets NPs nanoparticles NSs nanostructures ORR oxygen reduction reaction Pb lead Pd palladium PEM polymer electrolyte material P2O5 phosphorus pentoxide Pt platinum PVP polyvinyl pyrrolidone QD quantum dots Rice Husk rice husk SB sugarcane bagasse Se selenium SEM scanning electron microscopy SERS surface enhanced Raman scattering SiO2 silicon dioxide (silica) SnO stannous oxide SnO2 stannic oxide 20 of 26 Crystals 2021, 11, 634 TEM transmission electron microscopy TGA thermogravimetric analysis TiO2 titanium dioxide TPPs thermal power plants WO3 tungsten trioxide WS3 tungsten (VI) sulﬁde XRD X-ray diffraction Zn zinc ZnO zinc oxide ZnS zinc sulﬁde References 1. Mansoori, G.A.; Soelaiman, T.A.F. Nanotechnology—An Introduction for the Standards. J. ASTM Int. 2005, 2, 1–21. Mansoori, G.A.; Soelaiman, T.A.F. Nanotechnology—An Introduction for the Standards. J. ASTM Int. 2005 2. Yadav, V.K.; Ali, D.; Khan, S.H.; Gnanamoorthy, G.; Choudhary, N.; Yadav, K.K.; Thai, V.N.; Hussain, S.A.; Manhrdas, S. Synthesis and Characterization of Amorphous Iron Oxide Nanoparticles by the Sonochemical Method and Their Application for the 2. Yadav, V.K.; Ali, D.; Khan, S.H.; Gnanamoorthy, G.; Choudhary, N.; Yadav, K.K.; Thai, V.N.; Hussain, S.A.; Manhrdas, S. Synthesis and Characterization of Amorphous Iron Oxide Nanoparticles by the Sonochemical Method and Their Application for the Remediation of Heavy Metals from Wastewater. Nanomaterials 2020, 10, 1551. [CrossRef] [PubMed] 2. Yadav, V.K.; Ali, D.; Khan, S.H.; Gnanamoorthy, G.; Choudhary, N.; Yadav, K.K.; Thai, V.N.; Hussain, S.A and Characterization of Amorphous Iron Oxide Nanoparticles by the Sonochemical Method and T y 3. Malshe, A. Nanotechnology. In CIRP Encyclopedia of Production Engineering; The International Academy for Produ, Laperrière, L., Reinhart, G., Eds.; Springer: Berlin, Heidelberg, 2016. [CrossRef] y 3. Malshe, A. Nanotechnology. In CIRP Encyclopedia of Production Engineering; The International Academy for Produ, Laperrière, L., Reinhart, G., Eds.; Springer: Berlin, Heidelberg, 2016. [CrossRef] p g g 4. Lošdorfer Božiˇc, A.; Šiber, A.; Podgornik, R. Statistical analysis of sizes and shapes of virus capsids and their resulting elastic properties. J. Biol. Phys. 2013, 39, 215–228. [CrossRef] [PubMed] 4. Lošdorfer Božiˇc, A.; Šiber, A.; Podgornik, R. Statistical analysis of sizes and shapes of virus capsids and their resulting elastic properties. J. Biol. Phys. 2013, 39, 215–228. [CrossRef] [PubMed] p p y 5. Mansoori, G.A. Advances in Atomic and Molecular Nanotechnology. Available online: http://trl.lab.uic.edu/1.OnlineMaterials/ nano.publications/AdvancesInAtomicNMolecularnanotech.pdf (accessed on 30 May 2021). 5. Mansoori, G.A. Advances in Atomic and Molecular Nanotechnology. Available online: http://trl.lab.uic.edu/1.OnlineMaterials/ nano.publications/AdvancesInAtomicNMolecularnanotech.pdf (accessed on 30 May 2021). , gy p // / / nano.publications/AdvancesInAtomicNMolecularnanotech.pdf (accessed on 30 May 2021). p p y 6. Ali, S.; Abbas, Y.; Zuhra, Z.; Butler, I.S. Synthesis of γ-alumina (Al2O3) nanoparticles and their potentia in the removal of methylene blue dye from industrial wastewater. Nanoscale Adv. 2019, 1, 213–218. [Cr p p y i, S.; Abbas, Y.; Zuhra, Z.; Butler, I.S. References Energy Rev. 2017, 82, 3694–3704. [CrossRef] 17. Ali, H.; Hassaan, M. Applications of Bio-waste Materials as Green Synthesis of Nanoparticles and Water Puriﬁcation. Adv. Mater. 2017, 6, 85–101. [CrossRef] 18. Yadav, V.K.; Fulekar, M.H. The current scenario of thermal power plants and ﬂy ash production an India. Int. J. Adv. Eng. Res. Dev. 2018, 5, 768–777. 19. Sangeetha, J.; Thangadurai, D.; Hospet, R.; Purushotham, P.; Manowade, K.R.; Mujeeb, M.A.; Mundaragi, A.C.; Jogaiah, S.; David, M.; Thimmappa, S.C.; et al. Production of Bionanomaterials from Agricultural Wastes. In Nanotechnology: An Agricultural Paradigm; Springer: Singapore, 2017; pp. 33–58. [CrossRef] g p g g p pp 20. Cabral-Pinto, M.M.S.; Inácio, M.; Neves, O.; Almeida, A.A.; Pinto, E.; Oliveiros, B.; Ferreira da Silva, E.A. Human Health Risk Assessment Due to Agricultural Activities and Crop Consumption in the Surroundings of an Industrial Area. Expo. Health 2020, 12, 629–640. [CrossRef] 21. Zheng, Y.; Zhang, H.; Ge, S.; Song, J.; Wang, J.; Zhang, S. Synthesis of Carbon Nanotube Arrays with High Aspect Ratio via Ni-Catalyzed Pyrolysis of Waste Polyethylene. Nanomaterials 2018, 8, 556. [CrossRef] 21 of 26 Crystals 2021, 11, 634 22. Cabral-Pinto, M.M.S.; Marinho-Reis, P.; Almeida, A.; Pinto, E.; Neves, O.; Inácio, M.; Gerardo, B.; Freitas, S.; Simões, M.R.; Dinis, P.A.; et al. Links between Cognitive Status and Trace Element Levels in Hair for an Environmentally Exposed Population: A Case Study in the Surroundings of the Estarreja Industrial Area. Int. J. Environ. Res. Public Health 2019, 16, 4560. [CrossRef] y g j J 23. Khan, I.; Saeed, K.; Khan, I. Nanoparticles: Properties, applications and toxicities. Arab. J. Chem. 2019, y g j 23. Khan, I.; Saeed, K.; Khan, I. Nanoparticles: Properties, applications and toxicities. Arab. J. Chem. 2019, 12, 908–931. [CrossRef] 24. Yadav, V.K.; Fulekar, M.H. Biogenic synthesis of maghemite nanoparticles (γ-Fe2O3) using Tridax leaf extract and its application for removal of ﬂy ash heavy metals (Pb Cd) Mater Today Proc 2018 5 20704–20710 [CrossRef] p p pp 24. Yadav, V.K.; Fulekar, M.H. Biogenic synthesis of maghemite nanoparticles (γ-Fe2O3) using Tridax leaf extract and its application for removal of ﬂy ash heavy metals (Pb, Cd). Mater. Today Proc. 2018, 5, 20704–20710. [CrossRef] y y ( ) y 25. Nikalje, A. Nanotechnology and Its Applications in Medicine. Med. Chem. 2015, 5, 081–089. [CrossRef] j gy pp 26. Jeevanandam, J.; Barhoum, A.; Chan, Y.S.S.; Dufresne, A.; Danquah, M. Review on Nanoparticles and Nanostructured Materials: History, Sources, Toxicity, and Regulations. References Goyenola, C.; Stafström, S.; Hultman, L.; Gueorguiev, A.G. Structural Patterns Arising during Synthetic Sulfocarbide. J. Phys. Chem. C 2012, 116, 21124–21131. [CrossRef] oyenola, C.; Stafström, S.; Hultman, L.; Gueorguiev, A.G. Structural Patterns Arising during Synthetic Growth ulfocarbide. J. Phys. Chem. C 2012, 116, 21124–21131. [CrossRef] 36. Goyenola, C.; Stafström, S.; Hultman, L.; Gueorguiev, A.G. Structural Patterns Arising during Synthetic Growth of Fullerene Like Sulfocarbide. J. Phys. Chem. C 2012, 116, 21124–21131. [CrossRef] Edelstein, A.S. Nanomaterials. In Encyclopedia of Materials: Science and Technology; Buschow, K.H.J., Cahn, 37. Edelstein, A.S. Nanomaterials. In Encyclopedia of Materials: Science and Technology; Buschow, K.H.J., Ca Ilschner B Kramer E J Mahajan S Veyssière P Eds ; Elsevier: Oxford UK 2001; pp 5916–5927 [C Ilschner, B., Kramer, E.J., Mahajan, S., Veyssière, P., Eds.; Elsevier: Oxford, UK, 2001; pp. 5916–5927. [CrossRef] 38. Whitby, R.L.D.; Acquah, S.F.A.; Ma, R.; Zhu, Y. 1D Nanomaterials. J. Nanomater. 2010, 2010, 597851. [CrossRef] 38. Whitby, R.L.D.; Acquah, S.F.A.; Ma, R.; Zhu, Y. 1D Nanomaterials. J. Nanomater. 2010, 2010, 597851. [CrossRef] 39. Zhao, Y.; Hong, H.; Gong, Q.; Ji, L. 1D Nanomaterials: Synthesis, Properties, and Applications. Available online: https: //downloads.hindawi.com/journals/jnm/2013/101836.pdf (accessed on 30 May 2021). 40. Fang, X.; Zhang, L. One-Dimensional (1D) ZnS Nanomaterials and Nanostructures. J. Mater. Sci. Technol. 2006, 22, 721–736. 41. Zhang, C.; Yan, Y.; Zhao, Y.; Yao, J. Synthesis and Applications of Organic Nanorods, Nanowires and Nanotubes. Annu. Rep. Sect. C Phys.Chem. 2013, 109, 211–239. [CrossRef] 42. Guo, Z.; Tan, L. Fundamental and applications of N 43. Zhang, Z.; Zou, R.; Yu, L.; Hu, J. One-Dimensional Silicon-Based Semiconductor Nanomaterials: Synthesis, Structures, Properties and Applications. Crit. Rev. Solid State Mater. Sci. 2011, 36, 148–173. [CrossRef] 44. Gnanamoorthy, G.; Yadav, V.K.; Latha, D.; Karthikeyan, V.; Narayanan, V. Enhanced photocatalytic performance of ZnSnO3/rGO nanocomposite. Chem. Phys. Lett. 2020, 739, 137050. [CrossRef] 45. Gonsalves, K.E.; Rangarajan, S.P.; Wang, J. Chapter 1—Chemical synthesis of nanostructured metals, metal alloys, and semicon- ductors. In Handbook of Nanostructured Materials and Nanotechnology; Nalwa, H.S., Ed.; Academic Press: Burlington, NJ, USA, 2000; pp. 1–56. [CrossRef] pp 46. Klein, E.; Lesyuk, R.; Klinke, C. Insights into the formation mechanism of two-dimensional lead halide nanostructures. Nanoscale 2018, 10, 4442–4451. [CrossRef] 47. Sahoo, P.K.; Kim, K.; Powell, M.A.; Equeenuddin, S.M. Recovery of metals and other beneﬁcial products from coal ﬂy ash: A sustainable approach for ﬂy ash management. Int. J. Coal Sci. Technol. 2016, 3, 267–283. [CrossRef] 48. References Beilstein J. Nanotechnol. 2018, 9, 1050–1074. [CrossRef] y y g 27. Edvinsson, T. Optical quantum conﬁnement and photocatalytic properties in two-, one- and zero-dimensional nanostructures. R. Soc. Open Sci. 2018, 5, 180387. [CrossRef] 27. Edvinsson, T. Optical quantum conﬁnement and photocatalytic properties in two-, one- and zero-di Soc. Open Sci. 2018, 5, 180387. [CrossRef] 28. Brehm, M.; Grydlik, M. Site-controlled and advanced epitaxial Ge/Si quantum dots: Fabrication, properties, and applications. Nanotechnology 2017, 28, 392001. [CrossRef] 29. Patel, K.D.; Singh, R.K.; Kim, H.-W. Carbon-based nanomaterials as an emerging platform for theranostics. Mater. Horiz. 2019, 6, 434–469. [CrossRef] 30. Singh, R.; Singh, K.R. A Review on Nano Materials of Carbon. J. Appl. Phys. 2017, 9, 42–57. 31. Han, N.; Wang, F.; Ho, J. One-Dimensional Nanostructured Materials for Solar Energy Harvesting. Nanomater. Energy 2012, 1, 4–17. [CrossRef] 32. Om, K.; Manjit, K. Single Electron Transistor: Applications & Problems. Int. J. VLSI Des. Commun. Syst. (VLSICS) 2010, 1, 24–29. 33. Cao, H. Synthesis, Characterization, and Applications of Zero-Dimensional (0D) Nanostructures. In Synthesis and Applications of Inorganic Nanostructures; Wiley: Hoboken, NJ, USA, 2017. [CrossRef] j g pp 33. Cao, H. Synthesis, Characterization, and Applications of Zero-Dimension Inorganic Nanostructures; Wiley: Hoboken, NJ, USA, 2017. [CrossRef] j g pp y 33. Cao, H. Synthesis, Characterization, and Applications of Zero-Dimensional (0D) Nanostructures. In Synthesis and Applications of Inorganic Nanostructures; Wiley: Hoboken, NJ, USA, 2017. [CrossRef] g y J [ ] 34. Tiwari, J.N.; Tiwari, R.N.; Kim, K.S. Zero-dimensional, one-dimensional, two-dimensional and three-dimensional nanostructured materials for advanced electrochemical energy devices. Prog. Mater. Sci. 2012, 57, 724–803. [CrossRef] 34. Tiwari, J.N.; Tiwari, R.N.; Kim, K.S. Zero-dimensional, one-dimensional, two-dimensional and three materials for advanced electrochemical energy devices. Prog. Mater. Sci. 2012, 57, 724–803. [CrossR gy g 35. Mostoﬁzadeh, A.; Li, Y.; Song, B.; Huang, Y. Synthesis, Properties, and Applications of Low-D Nanomaterials. J. Nanomater. 2011, 2011, 685081. [CrossRef] 35. Mostoﬁzadeh, A.; Li, Y.; Song, B.; Huang, Y. Synthesis, Properties, and Applications of Low-Dimensional Carbon-Related Nanomaterials. J. Nanomater. 2011, 2011, 685081. [CrossRef] Goyenola, C.; Stafström, S.; Hultman, L.; Gueorguiev, A.G. Structural Patterns Arising during Synthetic Gro 36. Goyenola, C.; Stafström, S.; Hultman, L.; Gueorguiev, A.G. Structural Patterns Arising during Synthetic Growth of Fullerene-Like Sulfocarbide. J. Phys. Chem. C 2012, 116, 21124–21131. [CrossRef] 37 Edelstein A S Nanomaterials In Encyclopedia of Materials: Science and Technology; Buschow K H J Cahn R W Flemings M C 36. References Sensors 2019, 19, 1186. [CrossRef] [PubMed] p g 1186. [CrossRef] [PubMed] 70. Tierno, P.; Goedel, W.A. Using Electroless Deposition for the Preparation of Micron Sized Polymer/Metal Core/Shell Particles 1186. [CrossRef] [PubMed] 70. Tierno, P.; Goedel, W.A. Using Electroless Deposition for the Preparation of Micron Sized Polymer/Metal Core/Shell Particles and Hollow Metal Spheres J Phys Chem B 2006 110 3043 3050 [CrossRef] 70. Tierno, P.; Goedel, W.A. Using Electroless Deposition for the Preparation of Micron Sized Polymer/M and Hollow Metal Spheres. J. Phys. Chem. B 2006, 110, 3043–3050. [CrossRef] p y 71. Hayashi, H.; Hakuta, Y. Hydrothermal Synthesis of Metal Oxide Nanoparticles in Supercritical Water. Materials 2010, 3, 3794–3817. [CrossRef] [ ] 72. Li, J.; Wu, Q.; Wu, J. Synthesis of Nanoparticles via Solvothermal and Hydrothermal Methods. In Handbook of Nanoparticles; Aliofkhazraei, M., Ed.; Springer International Publishing: Cham, Switzerland, 2016; pp. 295–328. [CrossRef] 72. Li, J.; Wu, Q.; Wu, J. Synthesis of Nanoparticles via Solvothermal and Hydrothermal Methods. Aliofkhazraei, M., Ed.; Springer International Publishing: Cham, Switzerland, 2016; pp. 295–328. [C u, Q.; Wu, J. Synthesis of Nanoparticles via Solvothermal and Hydrothermal Methods. In Handbook of Na zraei, M., Ed.; Springer International Publishing: Cham, Switzerland, 2016; pp. 295–328. [CrossRef] p g g pp 73. Ramesh, S. Sol-Gel Synthesis and Characterization of Nanoparticles. J. Nanosci. 2013, 2013, 929321. [Cr ol-Gel Synthesis and Characterization of Nanoparticles. J. Nanosci. 2013, 2013, 929321. [CrossRef] 74. Kumar Yadav, V.; Fulekar, M.H. Green synthesis and characterization of amorphous silica nanoparticles from ﬂy ash. Mater. Today Proc. 2019, 18, 4351–4359. [CrossRef] 75. Essawy, H.; Fathy, N.; Tawﬁk, M.; El-Sabbagh, S.; Ismail, N.; Youssef, H. Fabrication of single-walled carbon nanotubes from vulcanized scrap rubber via thermal chemical vapor deposition. RSC Adv. 2017, 7, 12938–12944. [CrossRef] 76. Piriyawong, V.; Thongpool, V.; Asanithi, P.; Limsuwan, P. Preparation and Characterization of Alumina Nanoparticles in Deionized Water Using Laser Ablation Technique. J. Nanomater. 2012, 2012, 819403. [CrossRef] 77. Yadav, D.A. Synthesis of nanomaterials by physical and chemical methods. Int. Educ. Res. J. 2017, 3, 35 y y p y J 78. Fariq, A.; Khan, T.; Yasmin, A. Microbial synthesis of nanoparticles and their potential applications Biomed. 2017, 15, 241–248. [CrossRef] 78. Fariq, A.; Khan, T.; Yasmin, A. Microbial synthesis of nanoparticles and their potential applications in biomedicine. J. Appl. Biomed. 2017, 15, 241–248. [CrossRef] 79. Peralta-Videa, J.R.; Huang, Y.; Parsons, J.G.; Zhao, L.; Lopez-Moreno, L.; Hernandez-Viezcas, J.A.; Gardea-Torresdey, J.L. References [CrossRef] y p g g 59. Wang, J.; Gu, H. Novel Metal Nanomaterials and Their Catalytic Applications. Molecules 2015, 20, 1707 l d l d l f 59. Wang, J.; Gu, H. Novel Metal Nanomaterials and Their Catalytic Applications. Molecules 2015, 20, 17070–17092. [CrossRef] 59. Wang, J.; Gu, H. Novel Metal Nanomaterials and Their Catalytic Applications. Molecules 2015, 20, 17070–17092. [CrossRef] 60. Julien, M.C.; Mauger, A. Nanostructured MnO2 as Electrode Materials for Energy Storage. Nanomaterials 2017, 7, 396. [CrossRef] 61 Singh P; Jain S Biosynthesis of Nanomaterials: Growth and Properties Rev Adv Sci Eng 2014 3 231–238 [CrossRef] 60. Julien, M.C.; Mauger, A. Nanostructured MnO2 as Electrode Materials for Energy Storage. Nanomaterials 2017, 7, 396. [CrossRef] 61. Singh, P.; Jain, S. Biosynthesis of Nanomaterials: Growth and Properties. Rev. Adv. Sci. Eng. 2014, 3, 231–238. [CrossRef] 60. Julien, M.C.; Mauger, A. Nanostructured MnO2 as Electrode Materials for Energy Storage. Nanomat 61. Singh, P.; Jain, S. Biosynthesis of Nanomaterials: Growth and Properties. Rev. Adv. Sci. Eng. 2014, 3 62. Luther, W. Industrial Application of Nanomaterials—Chances and Risks. Technologiezentrum 2004, 54, 63. Gavagnin, M.; Wanzenboeck, H.D.; Wachter, S.; Shawrav, M.M.; Persson, A.; Gunnarsson, K.; Svedlindh, P.; Stöger-Pollach, M.; Bertagnolli, E. Free-Standing Magnetic Nanopillars for 3D Nanomagnet Logic. ACS Appl. Mater. Interfaces 2014, 6, 20254–20260. [CrossRef] 64. Georgakilas, V.; Perman, J.A.; Tucek, J.; Zboril, R. Broad Family of Carbon Nanoallotropes: Classiﬁcation, Chemistry, and Applications of Fullerenes, Carbon Dots, Nanotubes, Graphene, Nanodiamonds, and Combined Superstructures. Chem. Rev. 2015, 115, 4744–4822. [CrossRef] [ ] 65. Semaltianos, N.G. Nanoparticles by Laser Ablation. Crit. Rev. Solid State Mater. Sci. 2010, 35, 105–124. [CrossRef] G. Nanoparticles by Laser Ablation. Crit. Rev. Solid State Mater. Sci. 2010, 35, 105–124. [CrossRef] 65. Semaltianos, N.G. Nanoparticles by Laser Ablation. Crit. Rev. Solid State Mater. Sci. 2010, 35, 105–1 66. Sarkar, J.K.; Wang, Q. Different Pyrolysis Process Conditions of South Asian Waste Coconut Shell and Characterization of Gas, Bio-Char, and Bio-Oil. Energies 2020, 13, 1970. [CrossRef] 67. Dhand, C.; Dwivedi, N.; Loh, X.J.; Ng, A.; Verma, N.; Beuerman, R.; Lakshminarayanan, R.; Ramakrishna, S. Methods and Strategies for the Synthesis of Diverse Nanoparticles and Their Applications: A Comprehensive Overview. RSC Adv. 2015, 5, 105003–105037. [CrossRef] 68. Umadevi, S.; Umamaheswari, R.; Ganesh, V. Lyotropic liquid crystal-assisted synthesis of micro- and nanoparticles of silver. Liq. Cryst. 2017, 44, 1409–1420. [CrossRef] 69. Tonelli, D.; Scavetta, E.; Gualandi, I. Electrochemical Deposition of Nanomaterials for Electrochemical Sensing. References Yin, P.T.; Shah, S.; Chhowalla, M.; Lee, K.-B. Design, synthesis, and characterization of graphene-nanoparticle hybrid materials for bioapplications. Chem. Rev. 2015, 115, 2483–2531. [CrossRef] [PubMed] 49. Lin, J.; Chen, X.; Huang, P. Graphene-based nanomaterials for bioimaging. Adv. Drug Deliv. Rev. 2016, [PubMed] 50. Shah, M.; Fawcett, D.; Sharma, S.; Tripathy, S.; Poinern, G. Green Synthesis of Metallic Nanoparticles via Biological Entities. Materials 2015, 8, 7278–7308. [CrossRef] [PubMed] 51. Koski, K.J.; Cui, Y. The New Skinny in Two-Dimensional Nanomaterials. ACS Nano 2013, 7, 3739–3743. [CrossRef] [PubMed] 51. Koski, K.J.; Cui, Y. The New Skinny in Two-Dimensional Nanomaterials. ACS Nano 2013, 7, 3739–3743. [CrossRef] [PubMed] 52 Gnanamoorthy, G ; Ramar, K ; Padmanaban, A ; Yadav, VK ; Suresh Babu, K ; Karthikeyan, V; Narayanan, V Implementation 51. Koski, K.J.; Cui, Y. The New Skinny in Two-Dimensional Nanomaterials. ACS Nano 2013, 7, 3739–3743. [CrossRef] [PubMed] 52. Gnanamoorthy, G.; Ramar, K.; Padmanaban, A.; Yadav, V.K.; Suresh Babu, K.; Karthikeyan, V.; Narayanan, V. Implementation of ZnSnO3 nanosheets and their RE (Er, Eu, and Pr) materials: Enhanced photocatalytic activity. Adv. Powder Technol. 2020, 31, 1209–1219. [CrossRef] 53. Boroumand Moghaddam, A.; Namvar, F.; Moniri, M.; Md Tahir, P.; Azizi, S.; Mohamad, R. Nanoparticles Biosynthesized by Fungi and Yeast: A Review of Their Preparation, Properties, and Medical Applications. Molecules 2015, 20, 16540–16565. [CrossRef] 22 of 26 Crystals 2021, 11, 634 54. Miró, P.; Audiffred, M.; Heine, T. An Atlas of Two-Dimensional Materials. Chem. Soc. Rev. 2014, 43, 6537–6554. [CrossRef] 55. Van Gough, D.; Juhl, A.T.; Braun, P.V. Programming structure into 3D nanomaterials. Mater. Today 2009, 12, 28–35. [CrossRef] 56. Yaya, A. Layered Nanomaterials—A Review. Glob. J. Eng. Des. and Technol. 2012, 1, 32–41. 54. Miró, P.; Audiffred, M.; Heine, T. An Atlas of Two-Dimensional Materials. Chem. Soc. Rev. 2014, 43, 6537–6554. [CrossRef] g g g y 56. Yaya, A. Layered Nanomaterials—A Review. Glob. J. Eng. Des. and Technol. 2012, 1, 32–41. 57. Korotcenkov, G. Current Trends in Nanomaterials for Metal Oxide-Based Conductometric Gas Se Limitations. Part 1: 1D and 2D Nanostructures. Nanomaterials 2020, 10, 1392. [CrossRef] [PubMed] Limitations. Part 1: 1D and 2D Nanostructures. Nanomaterials 2020, 10, 1392. [CrossRef] [PubMed] 58. Sharma, S.; Jaiswal, S.; Duffy, B.; Jaiswal, A. Nanostructured Materials for Food Applications: Spectroscopy, Microscopy and Physical Properties. Bioengineering 2019, 6, 26. [CrossRef] 58. Sharma, S.; Jaiswal, S.; Duffy, B.; Jaiswal, A. Nanostructured Materials for Food Applications: Spectr Physical Properties. Bioengineering 2019, 6, 26. References Plant- based green synthesis of metallic nanoparticles: Scientiﬁc curiosity or a realistic alternative to chemical synthesis? Nanotechnol. Environ. Eng. 2016, 1, 4. [CrossRef] g 80. Bansal, V.; Poddar, P.; Ahmad, A.; Sastry, M. Room-Temperature Biosynthesis of Ferroelectric Barium Titanate Nanoparticles. J. Am. Chem. Soc. 2006, 128, 11958–11963. [CrossRef] 81. Castro, L.; Blázquez, M.L.; Muñoz, J.; González, F.; Ballester, A. Biological Synthesis of Metallic Nanoparticles Using Algae. IET Nanobiotechnol. 2013, 7, 109–116. [CrossRef] [PubMed] 82. Iravani, S. Bacteria in Nanoparticle Synthesis: Current Status and Future Prospects. Int. Sch. Res. Not. 2014, 2014, 59316. [CrossRef] [PubMed] 3. Sharma, D.; Kanchi, S.; Bisetty, K. Biogenic synthe 83. Sharma, D.; Kanchi, S.; Bisetty, K. Biogenic synthesis of nanoparticles: A review. Arab. J. Chem. 2019, 12, 3576–3600. [CrossRef] 84. Kim, S.; Kim, K.H.; Bark, C.W. Two-Dimensional Nanomaterials: Their Structures, Synthesis, and Applications. Sci. Adv. Mater. 2017, 9, 1441–1457. [CrossRef] y g y p 84. Kim, S.; Kim, K.H.; Bark, C.W. Two-Dimensional Nanomaterials: Their Structures, Synthesis, and Applications. Sci. Adv. Mater. 2017, 9, 1441–1457. [CrossRef] 85. Zavabeti, A.; Jannat, A.; Zhong, L.; Haidry, A.A.; Yao, Z.; Ou, J.Z. Two-Dimensional Materials in Large-Areas: Synthesis, Properties and Applications. Nano-Micro Lett. 2020, 12, 66. [CrossRef] p pp 86. Colson, P.; Henrist, C.; Cloots, R. Nanosphere Lithography: A Powerful Method for the Controlled Manufacturing of Nanomate- rials. J. Nanomater. 2013, 2013, 948510. [CrossRef] 23 of 26 Crystals 2021, 11, 634 87. Pingali, K.C.; Deng, S.; Rockstraw, D.A. Synthesis of Nanowires by Spray Pyrolysis. J. Sens. 2009, 2009, 683280. [CrossRef] 88. Suryanarayana, C.; Prabhu, B. Synthesis of Nanostructured Materials by Inert-Gas Condensation Methods; Nanostructured materials (second edition); William Andrew Publishing: Norwich, NY, USA, 2007; pp. 47–90. 87. Pingali, K.C.; Deng, S.; Rockstraw, D.A. Synthesis of Nanowires by Spray Pyrolysis. J. Sens. 2009, 2009, 683280. [CrossRef] 88. Suryanarayana, C.; Prabhu, B. Synthesis of Nanostructured Materials by Inert-Gas Condensation Methods; Nanostructured materials 87. Pingali, K.C.; Deng, S.; Rockstraw, D.A. Synthesis of Nanowires by Spray Pyrolysis. J. Sens. 2009, 2009, 683280. [CrossRef] g , ; g, ; , y y p y y y J , , [ ] 88. Suryanarayana, C.; Prabhu, B. Synthesis of Nanostructured Materials by Inert-Gas Condensation Methods; Nanostructured materials (second edition); William Andrew Publishing: Norwich, NY, USA, 2007; pp. 47–90. 89. Habiba, K.; Makarov, V.; Weiner, B.; Morell, G. Fabrication of Nanomaterials by Pulsed Laser Synthesis; In book: Manufac- turing Nanostructures, Chapter: 10. References Available online: https://www.researchgate.net/publication/270583669_Fabrication_of_ Nanomaterials_by_Pulsed_Laser_Synthesis (accessed on 30 May 2021). p p g p als_by_Pulsed_Laser_Synthesis (accessed on 30 May 2021). y y ( y ) .W.; Suslick, K.S. Sonochemical synthesis of nanomaterials. Chem. Soc. Rev. 2013, 42, 2555–2567. [CrossRef] y y y 90. Xu, H.; Zeiger, B.W.; Suslick, K.S. Sonochemical synthesis of nanomaterials. Chem. Soc. Rev. 2013, 4 90. Xu, H.; Zeiger, B.W.; Suslick, K.S. Sonochemical synthesis of nanomaterials. Chem. Soc. Rev. 2013, 42, 2555–2567. [CrossRef] 91. Ray, P.C.; Yu, H.; Fu, P.P. Toxicity and Environmental Risks of Nanomaterials: Challenges and Future Needs. J. Environ. Sci. Health Part C 2009, 27, 1–35. [CrossRef] 91. Ray, P.C.; Yu, H.; Fu, P.P. Toxicity and Environmental Risks of Nanomaterials: Challenges and Future Needs. J. Environ. Sci. Health Part C 2009, 27, 1–35. [CrossRef] 92. Gnanamoorthy, G.; Ali, D.; Yadav, V.K.; Dhinagaran, G.; Venkatachalam, K.; Narayanan, V. New construction of Fe3O4/rGO/ZnSnO3 nanocomposites enhanced photoelectro chemical properties. Opt. Mater. 2020, 109, 110353. [CrossRef] 93 Yang J ; Hou B ; Wang J ; Tian B ; Bi J ; Wang N ; Li X ; Huang X Nanomaterials for the Removal of Heavy Metals from 92. Gnanamoorthy, G.; Ali, D.; Yadav, V.K.; Dhinagaran, G.; Venkatachalam, K.; Narayanan, V. New construction of Fe3O4/rGO/ZnSnO3 nanocomposites enhanced photoelectro chemical properties. Opt. Mater. 2020, 109, 110353. [CrossRef] 93. Yang, J.; Hou, B.; Wang, J.; Tian, B.; Bi, J.; Wang, N.; Li, X.; Huang, X. Nanomaterials for the Removal of Heavy Metals from Wastewater. Nanomaterials 2019, 9, 424. [CrossRef] Fe3O4/rGO/ZnSnO3 nanocomposites enhanced photoelectro chemical properties. Opt. Mater. 2020, 109, 110353. [CrossRef] 93. Yang, J.; Hou, B.; Wang, J.; Tian, B.; Bi, J.; Wang, N.; Li, X.; Huang, X. Nanomaterials for the Removal of Heavy Metals from Wastewater. Nanomaterials 2019, 9, 424. [CrossRef] 93. Yang, J.; Hou, B.; Wang, J.; Tian, B.; Bi, J.; Wang, N.; Li, X.; Huang, X. Nanomaterials for the Rem Wastewater. Nanomaterials 2019, 9, 424. [CrossRef] 94. Dai, Z.R.; Pan, Z.W.; Wang, Z.L. Growth and Structure Evolution of Novel Tin Oxide Diskettes. J. Am. Chem. Soc. 2002, 124, 8673–8680. [CrossRef] [PubMed] Annabi, N.; Dokmeci, M.R.; Khademhosseini, A. Carbon-based nanomaterials: Multifunctional materials for ring. ACS Nano 2013, 7, 2891–2897. [CrossRef] 95. Cha, C.; Shin, S.R.; Annabi, N.; Dokmeci, M.R.; Khademhosseini, A. Carbon-based nanomaterials: Mul biomedical engineering. ACS Nano 2013, 7, 2891–2897. [CrossRef] 96. Savjani, K.T.; Gajjar, A.K.; Savjani, J.K. Drug solubility: Importance and enhancement techniques. ISRN Pharm. References Aerosol synthesis of shape-controlled template particles: A route to Ta3N5 nan photocatalysts. J. Mater. Chem. A 2016, 4, 8451–8457. [CrossRef] p y 107. Swarnavalli, G.C.J.; Joseph, V.; Kannappan, V.; Roopsingh, D. A Simple Approach to the Synthesis of Hexagonal-Shaped Silver Nanoplates. J. Nanomater. 2011, 2011, 825637. [CrossRef] p J , , [ ] 108. He, X.; Zhao, X.; Li, Y.; Sui, X. Shape-Controlled Synthesis for Silver: Triangular/Hexagonal Nanoplates, Chain-Like Nanoplate Assemblies, and Nanobelts. J. Mater. Res. 2009, 24, 2200–2209. [CrossRef] 109. Zeb Gul Sial, M.A.; Ud Din, M.A.; Wang, X. Multimetallic nanosheets: Synthesis and applications in fuel cells. Chem. Soc. Rev. 2018, 47, 6175–6200. [CrossRef] [PubMed] 110. Zhang, Z.; Chen, H.; Xing, C.; Guo, M.; Xu, F.; Wang, X.; Gruber, H.; Zhang, B.; Tang, J. Sodium Citrate: A Universal Reducing Agent for Reduction/Decoration of Graphene Oxide with Au Nanoparticles. Nano Res. 2011, 4, 599–611. [CrossRef] 111. Zhou, Y.; Chen, G.; Yu, Y.; Yan, C.; Sun, J.; He, F. Synthesis of Metal Oxide Nanosheets through a Novel Approach for Energy A li ti J Mater Chem A 2015 4 781 784 [C R f] 110. Zhang, Z.; Chen, H.; Xing, C.; Guo, M.; Xu, F.; Wang, X.; Gruber, H.; Zhang, B.; Tang, J. Sodium Citrate: A Universal Reducing Agent for Reduction/Decoration of Graphene Oxide with Au Nanoparticles. Nano Res. 2011, 4, 599–611. [CrossRef] 111. Zhou, Y.; Chen, G.; Yu, Y.; Yan, C.; Sun, J.; He, F. Synthesis of Metal Oxide Nanosheets through a Novel Approach for Energy Applications. J. Mater. Chem. A 2015, 4, 781–784. [CrossRef] 111. Zhou, Y.; Chen, G.; Yu, Y.; Yan, C.; Sun, J.; He, F. Synthesis of Me Applications. J. Mater. Chem. A 2015, 4, 781–784. [CrossRef] pp 112. Liu, J.; Yang, H.; Xue, X. Preparation of Different Shaped α-Fe2O3 Nanoparticles with Large CrystEngComm 2019, 21, 1097–1101. [CrossRef] Xue, X. Preparation of Different Shaped α-Fe2O3 Nanoparticles with Large Particle of Iron Oxide Red , 21, 1097–1101. [CrossRef] y g 113. Dai, X.-C.; Hou, S.; Huang, M.-H.; Li, Y.-B.; Li, T.; Xiao, F.-X. Electrochemically anodized one-dimensional semiconductors: A fruitful platform for solar energy conversion. J. Phys. Energy 2019, 1, 022002. [CrossRef] 114. Kawde, A.-N.; Aziz, M.A.; El-Zohri, M.; Baig, N.; Odewunmi, N. Cathodized Gold Nanoparticle-Modiﬁed Graphite Pencil Electrode for Non-Enzymatic Sensitive Voltammetric Detection of Glucose. Electroanalysis 2017, 29, 1214–1221. [CrossRef] 115. Ghicov, A.; Schmuki, P. References 2012, 2012, 195727. [CrossRef] [ ] 97. Dubé, M.A.; Salehpour, S. Applying the Principles of Green Chemistry to Polymer Production Technology. Macromol. React. Eng. 2014, 8, 7–28. [CrossRef] ngari, G.; Gambinossi, F.; Passaponti, M.; Salvietti, E.; Di Benedetto, F.; Caporali, S.; Innocenti, M. Electropla Applications: Recent Trends in Research and Development Coatings 2018 8 260 [CrossRef] 98. Giurlani, W.; Zangari, G.; Gambinossi, F.; Passaponti, M.; Salvietti, E.; Di Benedetto, F.; Caporali, S.; Innocenti, M. Electroplating for Decorative Applications: Recent Trends in Research and Development. Coatings 2018, 8, 260. [CrossRef] Ch Y h W C Y Ch h l l l S h 98. Giurlani, W.; Zangari, G.; Gambinossi, F.; Passaponti, M.; Salvietti, E.; Di Benedetto, F.; Caporali, S.; for Decorative Applications: Recent Trends in Research and Development. Coatings 2018, 8, 260. [C 99. Chen, Y.; Fan, Z.; Zhang, Z.; Niu, W.; Li, C.; Yang, N.; Chen, B.; Zhang, H. Two-Dimensional Metal Nanomaterials: Synthesis, Properties, and Applications. Chem. Rev. 2018, 118, 6409–6455. [CrossRef] 100. Yang, F.; Song, P.; Ruan, M.; Xu, W. Recent progress in two-dimensional nanomaterials: Synthesis, engineering, and applications. FlatChem 2019, 18, 100133. [CrossRef] FlatChem 2019, 18, 100133. [CrossRef] 101. Paul, R.; Reifenberger, R.G.; Fisher, T.S.; Zemlyanov, D.Y. Atomic Layer Deposition of FeO on Pt(111) by Ferrocene Adsorption and Oxidation. Chem. Mater. 2015, 27, 5915–5924. [CrossRef] er, R.G.; Fisher, T.S.; Zemlyanov, D.Y. Atomic Layer Deposition of FeO on Pt(111) by Ferrocene Adsorption m. Mater. 2015, 27, 5915–5924. [CrossRef] 101. Paul, R.; Reifenberger, R.G.; Fisher, T.S.; Zemlyanov, D.Y. Atomic Layer Deposition of FeO on Pt(1 and Oxidation. Chem. Mater. 2015, 27, 5915–5924. [CrossRef] 102. Zhang, S.; Liu, H.; Yu, J.; Li, B.; Ding, B. Multi-functional ﬂexible 2D carbon nanostructured networks. Nat. Commun. 2020, 11, 5134. [CrossRef] [PubMed] 103. Rai, M.; Ingle, A.; Birla, S.; Yadav, A.; Santos, C. Strategic Role of Selected Noble Metal Nanoparticles in Medicine. Crit. Rev. Microbiol. 2016, 42, 696–719. [CrossRef] [PubMed] 104. Khodashenas, B.; Ghorbani, H. Synthesis of Silver Nanoparticles with Different Shapes. Arab. J. Chem. 2015, 12, 1823–1838. [CrossRef] [ ] 105. Jiang, X.; Yu, A.B.; Yang, W.R.; Ding, Y.; Xu, C.X.; Lam, S. Synthesis and Growth of Hematite Nanodiscs through a Facile Hydrothermal Approach. J. Nanopart. Res. 2009, 12, 877–893. [CrossRef] p E. Aerosol synthesis of shape-controlled template particles: A route to Ta3N5 nanoplates and octahedra as ater. Chem. A 2016, 4, 8451–8457. [CrossRef] 106. Fu, J.; Skrabalak, S.E. References Self-ordering electrochemistry: A review on growth and functionality of TiO2 nanotubes and other self-aligned MOx structures. Chem. Commun. 2009, 2791–2808. [CrossRef] [PubMed] g 116. Syrek, K.; Kapusta-Kołodziej, J.; Jarosz, M.; Sulka, G.D. Effect of electrolyte agitation on anodic titanium dioxide (ATO) growth and its photoelectrochemical properties. Electrochim. Acta 2015, 180, 801–810. [CrossRef] 24 of 26 24 of 26 Crystals 2021, 11, 634 117. Lawrence, M.J.; Celorrio, V.; Shi, X.; Wang, Q.; Yanson, A.; Adkins, N.J.E.; Gu, M.; Rodríguez-López, J.; Rodriguez, P. Electrochem- ical Synthesis of Nanostructured Metal-Doped Titanates and Investigation of Their Activity as Oxygen Evolution Photoanodes. ACS Appl. Energy Mater. 2018, 1, 5233–5244. [CrossRef] pp gy 118. Yadav, V.K.; Khan, S.H.; Malik, P.; Thappa, A.; Suriyaprabha, R.; Ravi, R.K.; Choudhary, N.; Kalasariya, H.; Gnanamoorthy, G. Microbial Synthesis of Nanoparticles and Their Applications for Wastewater Treatment. In Microbial Biotechnology: Basic Research and Applications; Singh, J., Vyas, A., Wang, S., Prasad, R., Eds.; Springer: Singapore, 2020; pp. 147–187. [CrossRef] pp g y g p g g p pp 119. Tiwari, S.; Kumar, V.; Huczko, A.; Oraon, R.; De Adhikari, A.; Nayak, G. Magical Allotropes of Carbon: Prospects and Applications. Crit. Rev. Solid State Mater. Sci. 2015, 41, 257–317. [CrossRef] 120. Zaytseva, O.; Neumann, G. Carbon nanomaterials: Production, impact on plant development, agricultural and environmental applications. Chem. Biol. Technol. Agric. 2016, 3, 17. [CrossRef] ; Graham, U.; Ward, C.; Kostova, I.; Maroto-Valer, M.; Dai, S. Coal-Derived Unburned Carbons in Fly Ash: A Geol. 2017, 179, 11–27. [CrossRef] 121. Hower, J.; Groppo, J.; Graham, U.; Ward, C.; Kostova, I.; Maroto-Valer, M.; Dai, S. Coal-Derived Unb Review. Int. J. Coal Geol. 2017, 179, 11–27. [CrossRef] 122. Salah, N.; Al-Ghamdi, A.; Memic, A.; Habib, S.S.; Khan, Z.H. Formation of Carbon Nanotubes from Carbon Rich Fly Ash: Growth Parameters and Mechanism. Mater. Manuf. Process. 2016, 31, 146–156. [CrossRef] 123. Loh, Y.R.; Sujan, D.; Rahman, M.E.; Das, C.A. Sugarcane bagasse—The future composite material: A literature review. Resour. Conserv. Recycl. 2013, 75, 14–22. [CrossRef] 124. Xu, Q.; Ji, T.; Gao, S.J.; Yang, Z.; Wu, N. Characteristics and Applications of Sugar Cane Bagasse Ash Waste in Cementitious Materials. Materials 2018, 12, 39. [CrossRef] 125. Kroto, H.W.; Heath, J.R.; O’Brien, S.C.; Curl, R.F 125. Kroto, H.W.; Heath, J.R.; O’Brien, S.C.; Curl, R.F.; Smalley, R.E. buckminsterfullerene. Nature 1985, 318, 162–163. [CrossRef] 126. Iijima, S. Helical Microtubules of Graphitic Carbon. Nature 1991, 354, 56–58. [CrossRef] 125. References Studies on Silica Obtained from Rice Husk. Ceram. Int. 2001, 27, 219–224. [CrossRef] 139. Yalçin, N.; Sevinç, V. Studies on Silica Obtained from Rice Husk. Ceram. Int. 2001, 27, 219–224. [Cro 140. Yalçın, N.; Sevinç, V. Studies of the Surface Area and Porosity of Activated Carbons Prepared from Rice Husks. Carbon 2000, 38, 1943–1945. [CrossRef] 141. Dalling, T. FAO Statistical yearbook 2013: World food and agriculture. In Food and Agriculture Organization of the United Nations; FAO Fiat Fanis: Rome, Italy, 2010; p. 189. y p 142. Osman, A.I.; Farrell, C.; Al-Muhtaseb, A.a.H.; Harrison, J.; Rooney, D.W. The production and applica from high alkali silicate herbaceous biomass. Sci. Rep. 2020, 10, 2563. [CrossRef] [PubMed] sman, A.I.; Farrell, C.; Al-Muhtaseb, A.a.H.; Harrison, J.; Rooney, D.W. The production and application of carb om high alkali silicate herbaceous biomass Sci Rep 2020 10 2563 [CrossRef] [PubMed] rell, C.; Al-Muhtaseb, A.a.H.; Harrison, J.; Rooney, D.W. The production and application of carbon nanomater , ; , ; , ; , J ; y, p pp from high alkali silicate herbaceous biomass. Sci. Rep. 2020, 10, 2563. [CrossRef] [PubMed] silicate herbaceous biomass. Sci. Rep. 2020, 10, 2563. [CrossRef] [PubMed] g p 143. Kumar, D.R.; Singh, D.R.; Singh, D. Natural and Waste Hydrocarbon Precursors for the Synthesis of Carbon Based Nanomaterials: Graphene and CNTs. Renew. Sustain. Energy Rev. 2016, 58, 976–1006. [CrossRef] p gy 144. Chatterjee, A.K.; Sharon, M.; Banerjee, R.; Neumann-Spallart, M. CVD Synthesis of Carbon Nanotubes Using a Finely Dispersed C b lt C t l t d Th i U i D bl L El t h i l C it El t hi A t 2003 48 3439 3446 [C R f] p gy 144. Chatterjee, A.K.; Sharon, M.; Banerjee, R.; Neumann-Spallart, M. CVD Synthesis of Carbon Nanotubes Using a Finely Dispersed Cobalt Catalyst and Their Use in Double Layer Electrochemical Capacitors. Electrochim. Acta 2003, 48, 3439–3446. [CrossRef] 145. Myint, D.; Gilani, S.A.; Kawase, M.; Watanabe, K.N. Sustainable Sesame (Sesamum indicum L.) Production through Improved Technology: An Overview of Production, Challenges, and Opportunities in Myanmar. Sustainability 2020, 12, 3515. [CrossRef] y , ; , ; , ; , ( ) g p Technology: An Overview of Production, Challenges, and Opportunities in Myanmar. Sustainability 2020, 12, 3515. [CrossRef] 146. Janas, D. From Bio to Nano: A Review of Sustainable Methods of Synthesis of Carbon Nanotubes. Sustainability 2020, 12, 4115. [C R f] 146. Janas, D. References Kroto, H.W.; Heath, J.R.; O Brien, S.C.; Curl, R.F.; Smalley, R.E. buckminsterfullerene. Nature 1985, 318, 162 163. [CrossRef] 126. Iijima, S. Helical Microtubules of Graphitic Carbon. Nature 1991, 354, 56–58. [CrossRef] .T.; Gun’ko, Y.K. Recent advances in research on carbon nanotube-polymer composites. Adv. Mater. 2010, 22, f] 127. Byrne, M.T.; Gun’ko, Y.K. Recent advances in research on carbon nanotube-polymer composites. Ad [CrossRef] 128. Arora, N.; Sharma, N.N. Arc Discharge Synthesis of Carbon Nanotubes: Comprehensive Review. Diam. Relat. Mater. 2014, 50, 135–150. [CrossRef] 129. Vallejos, S.; Di Maggio, F.; Shujah, T.; Blackman, C. Chemical Vapour Deposition of Gas Sensitive Metal Oxides. Chemosensors 2016, 4, 4. [CrossRef] 130. Tripathi, P.K.; Durbach, S.; Coville, N.J. Synthesis of Multi-Walled Carbon Nanotubes from Plastic Waste Using a Stainless-Steel CVD Reactor as Catalyst. Nanomaterials 2017, 7, 284. [CrossRef] 131. Nguyen, H.; Jamali Moghadam, M.; Moayedi, H. Agricultural Wastes Preparation, Management, and Applications in Civil Engineering: A Review. J. Mater. Cycles Waste Manag. 2019, 21, 1039–1051. [CrossRef] 132. Zhang, Y.; Williams, P.T. Carbon nanotubes and hydrogen production from the pyrolysis catalysis or catalytic-steam reforming of waste tyres. J. Anal. Appl. Pyrolysis 2016, 122, 490–501. [CrossRef] pp y y Yadav, V.K.; Yadav, K.K. Fabrication of different SnO2 nanorods for enhanced photocatalytic degradation and y. Environ. Sci. Pollut. Res. 2021, 1, 1–11. 133. Gnanamoorthy, G.; Yadav, V.K.; Yadav, K.K. Fabrication of different SnO2 nanorods for enhanced photo antibacterial activity. Environ. Sci. Pollut. Res. 2021, 1, 1–11. y 134. Ramirez, A.; Royo, C.; Latorre, N.; Mallada, R.; Tiggelaar, R.; Monzon, A. Unraveling The Growth of Vertically Aligned Multi-Walled Carbon Nanotubes by Chemical Vapor Deposition. Mater. Res. Express 2014, 1, 045604. [CrossRef] y p p p 135. Nessim, G.D. Properties, synthesis, and growth mechanisms of carbon nanotubes with special focus on thermal chemical vapor deposition. Nanoscale 2010, 2, 1306–1323. [CrossRef] 136. MacKenzie, K.J.; Dunens, O.M.; Harris, A.T. An Updated Review of Synthesis Parameters and Growth Mechanisms for Carbon Nanotubes in Fluidized Beds. Ind. Eng. Chem. Res. 2010, 49, 5323–5338. [CrossRef] 137. Zhuo, C.; Wang, X.; Nowak, W.; Levendis, Y. Oxidative Heat Treatment of 316L Stainless Steel for Effective Catalytic Growth of Carbon Nanotubes. Appl. Surf. Sci. 2014, 313, 227–236. [CrossRef] 138. Azadi, M.; Bahrololoom, M.; Heidari, F. Enhancing the Mechanical Properties of an Epoxy Coating with Product. J. Coat. Technol. Res. 2011, 8, 117–123. [CrossRef] J , , [ ] 139. Yalçin, N.; Sevinç, V. References The Processing of Calcium Rich Agricultural and Industrial Waste for Recovery of Calcium Carbonate and Calcium Oxide and Their Application for Environmental Cleanup: A Review. Appl. Sci. 2021, 11, 4212. [CrossRef] pp p pp 159. Mei, L.; Zhu, S.; Yin, W.; Chen, C.; Nie, G.; Gu, Z.; Zhao, Y. Two-dimensional nanomaterials beyond graphene for antibacterial applications: Current progress and future perspectives. Theranostics 2020, 10, 757–781. [CrossRef] [PubMed] 160. Alberto, N.; Domingues, M.F.; Marques, C.; Andre, P.; Antunes, P. Optical Fiber Magnetic Field Sensors Based on Magnetic Fluid: A Review. Sensors 2018, 18, 4325. [CrossRef] [PubMed] 161. Khan, K.; Tareen, A.K.; Aslam, M.; Sagar, R.U.R.; Zhang, B.; Huang, W.; Mahmood, A.; Mahmood, N.; Khan, K.; Zhang, H.; et al. Recent Progress, Challenges, and Prospects in Two-Dimensional Photo-Catalyst Materials and Environmental Remediation. Nano-Micro Lett. 2020, 12, 167. [CrossRef] 162. Hong, J.W.; Kim, Y.; Wi, D.H.; Lee, S.; Lee, S.-U.; Lee, Y.W.; Choi, S.-I.; Han, S.W. Ultrathin Free-Standing Ternary-Alloy Nanosheets. Angew. Chem. 2016, 128, 2803–2808. [CrossRef] 163. Ud Din, M.A.; Saleem, F.; Ni, B.; Yong, Y.; Wang, X. Porous Tetrametallic PtCuBiMn Nanosheets with a High Catalytic Activity and Methanol Tolerance Limit for Oxygen Reduction Reactions. Adv. Mater. 2017, 29, 1604994. [CrossRef] yg hen, H.; Qu, J.; Dai, L. Recent Advances in Carbon-Based Metal-Free Electrocatalysts. Adv. Mater. 2019 Ref] 164. Paul, R.; Zhu, L.; Chen, H.; Qu, J.; Dai, L. Recent Advances in Carbon-Based Metal-Free Electrocat 31, 1806403. [CrossRef] 165. Paul, R.; Roy, A.K.; Dai, L. Chapter 8—Nanoporous graphitic carbon for efﬁcient supercapacitors and related energy applications. In Hybrid Atomic-Scale Interface Design for Materials Functionality; Roy, A.K., Ed.; Elsevier: Alpharetta, GA, USA, 2021; pp. 143–178. [CrossRef] 166. Zhu, C.; Liu, T.; Qian, F.; Chen, W.; Chandrasekaran, S.; Yao, B.; Song, Y.; Duoss, E.B.; Kuntz, J.D.; Spadaccini, C.M.; et al. 3D printed functional nanomaterials for electrochemical energy storage. Nano Today 2017, 15, 107–120. [CrossRef] 167. Freitas, R.R.Q.; Rivelino, R.; de Brito Mota, F.; de Castilho, C.M.C.; Kakanakova-Georgieva, A.; Gueorguiev, G.K. Topological Insulating Phases in Two-Dimensional Bismuth-Containing Single Layers Preserved by Hydrogenation. J. Phys. Chem. C 2015, 119, 23599–23606. [CrossRef] 168. Prasad, S.; Yadav, K.K.; Kumar, S.; Gupta, N.; Cabral-Pinto, M.M.S.; Rezania, S.; Radwan, N.; Alam, J. Chromium contamination and effect on environmental health and its remediation: A sustainable approaches. J. Environ. Manag. 2021, 285, 112194. [CrossRef] 169. Zhao, F.; Slade Rc Fau-Varcoe, J.R.; Varcoe, J.R. References From Bio to Nano: A Review of Sustainable Methods of Synthesis of Carbon Nanotubes. Sustainability 2020, 12, 4115. [CrossRef] 25 of 26 Crystals 2021, 11, 634 147. Titirici, M.; White, R.; Brun, N.; Budarin, V.; Su, D.; Monte, F.; Clark, J.; MacLachlan, M. Sustainable carbon materials. Chem. Soc. Rev. 2014, 44, 250–290. [CrossRef] , , [ ] 148. Wang, Q.; Liu, X.; Zhang, L.; Lv, Y. Microwave-Assisted Synthesis of Carbon Nanodots through an Eggshell Membrane and Their Fluorescent Application. Analyst 2012, 137, 5392–5397. [CrossRef] [PubMed] pp y 149. Furlan, A.; Gueorguiev, G.K.; Högberg, H.; Stafström, S.; Hultman, L. Fullerene-like CPx: A ﬁrst-principles study of the relative stability of precursors and defect energetics during synthetic growth. Thin Solid Film 2006, 515, 1028–1032. [CrossRef] Furlan, A.; Gueorguiev, G.K.; Högberg, H.; Stafström, S.; Hultman, L. Fullerene-like CPx: A ﬁrst-principle 150. Shi, S.; Chen, F.; Ehlerding, E.B.; Cai, W. Surface Engineering of Graphene-Based Nanomaterials for Biomedical Applications. Bioconjug. Chem. 2014, 25, 1609–1619. [CrossRef] [PubMed] 151. Ali, I.; Basheer, A.A.; Mbianda, X.Y.; Burakov, A.; Galunin, E.; Burakova, I.; Mkrtchyan, E.; Tkachev, A.; Grachev, V. Graphene based adsorbents for remediation of noxious pollutants from wastewater. Environ. Int. 2019, 127, 160–180. [CrossRef] 152. Ghosal, K.; Sarkar, K. Biomedical Applications of Graphene Nanomaterials and Beyond. ACS 2653–2703. [CrossRef] [PubMed] 153. Yadav, V.K.; Gnanamoorthy, G.; Cabral-Pinto, M.M.S.; Alam, J.; Ahamed, M.; Gupta, N.; Singh, B.; Choudhary, N.; Inwati, G.K.; Yadav, K.K. Variations and similarities in structural, chemical, and elemental properties on the ashes derived from the coal due to their combustion in open and controlled manner. Environ. Sci. Pollut. Res. 2021. [CrossRef] [PubMed] 154. Paul, R.; Etacheri, V.; Pol, V.G.; Hu, J.; Fisher, T.S. Highly porous three-dimensional carbon nano anode for a lithium-ion battery. RSC Adv. 2016, 6, 79734–79744. [CrossRef] 155. Paul, R.; Du, F.; Dai, L.; Ding, Y.; Wang, Z.L.; Wei, F.; Roy, A. 3D Heteroatom-Doped Carbon Nanomaterials as Multifunctional Metal-Free Catalysts for Integrated Energy Devices. Adv. Mater. 2019, 31. [CrossRef] 156. Paul, R.; Roy, A.K. BN-codoped CNT based nanoporous brushes for all-solid-state ﬂexible supercapacitors at elevated tempera- tures. Electrochim. Acta 2021, 365, 1805598. [CrossRef] 157. Serrano, M.C.; Gutiérrez, M.C.; del Monte, F. Role of polymers in the design of 3D carbon nanotube-based scaffolds for biomedical applications. Prog. Polym. Sci. 2014, 39, 1448–1471. [CrossRef] pp g y 158. Yadav, V.K.; Yadav, K.K.; Cabral-Pinto, M.M.S.; Choudhary, N.; Gnanamoorthy, G.; Tirth, V.; Prasad, S.; Khan, A.H.; Islam, S.; Khan, N.A. 174. Elsheikh, A.H.; Sharshir, S.W.; Mostafa, M.; Essa, F.; Ahmed Ali, M. Applications of Nanoﬂuids in Solar Energy: A Review of Recent Advances. Renew. Sustain. Energy Rev. 2017, 82, 3483–3502. [CrossRef] 173. Thaker, R.; Patel, J.R. Application of Nano ﬂuids in Solar Energy. J. Altern. Energy Sources Technol. 2015, 6, 1–11. 173. Thaker, R.; Patel, J.R. Application of Nano ﬂuids in Solar Energy. J. Altern. Energy Sources Technol. 2 er, R.; Patel, J.R. Application of Nano ﬂuids in Solar Energy. J. Altern. Energy Sources Technol. 2015, 6, 1–11. R. Application of Nano ﬂuids in Solar Energy. J. Altern. Energy Sources Technol. 2015, 6, 1–11. pp gy gy 174. Elsheikh, A.H.; Sharshir, S.W.; Mostafa, M.; Essa, F.; Ahmed Ali, M. Applications of Nanoﬂu Recent Advances. Renew. Sustain. Energy Rev. 2017, 82, 3483–3502. [CrossRef] References Techniques for the study and development of microbial fuel cells: An electrochem- ical perspective. Chem. Soc. Rev. 2009, 38, 1926–1939. [CrossRef] 168. Prasad, S.; Yadav, K.K.; Kumar, S.; Gupta, N.; Cabral-Pinto, M.M.S.; Rezania, S.; Radwan, N.; Alam, J. Chromium contamination and effect on environmental health and its remediation: A sustainable approaches. J. Environ. Manag. 2021, 285, 112194. [CrossRef] pp g 169. Zhao, F.; Slade Rc Fau-Varcoe, J.R.; Varcoe, J.R. Techniques for the study and development of microb ical perspective. Chem. Soc. Rev. 2009, 38, 1926–1939. [CrossRef] p p 170. Kannan, R.; Kakade, B.A.; Pillai, V.K. Polymer Electrolyte Fuel Cells Using Naﬁon-Based Composite Membranes with Functional- ized Carbon Nanotubes. Angew. Chem. Int. Ed. 2008, 47, 2653–2656. [CrossRef] 171. Huang, X.; Jain, P.; El-Sayed, I.; El-Sayed, M. Plasmonic PTT Therapy (PPTT) Using Gold Nanoparticles. Lasers Med. Sci. 2008, 23, 217–228. [CrossRef] 172. Luo, Z.; Wang, C.; Wei, W.; Xiao, G.; Ni, M. Performance Improvement of a Nanoﬂuid Solar Collector Based on Direct Absorption Collection (DAC) Concepts. Int. J. Heat Mass Transf. 2014, 75, 262–271. [CrossRef] 26 of 26 Crystals 2021, 11, 634 26 of 26
https://openalex.org/W2779241414	http://revistas.udc.es/index.php/reipe/article/download/reipe.2017.0.12.2175/pdf	Portuguese	null	A Ausência de articulação teoria-prática nos componentes curriculares	Revista de estudios e investigación en psicología y educación	2,017	cc-by-sa	3,845	Abstract The paper presents studies carried out in a Brazilian University Center, aiming to confront official documents on the public policies related to theory and practice present in the curricular components of courses of diverse nature. As for the relation theory and practice in the formative curricula, the official discourses and the role of the teacher in this articulation were investigated through a qualitative approach with a dialectical philosophical base. The results point to the fragility between theory and practice, the gap between the locus of formation and the activity of teaching, and gaps in the understanding of dialectical unity (theory and practice), reflected in the curricular components. A escolha da abordagem qualitativa, na ótica da dialética, nesta pesquisa, tomou como ponto de partida “A visão de mundo, entendida como uma percepção organizada da realidade que orienta a produção da pesquisa, se constrói através da prática cotidiana do pesquisador e das condições concretas de sua existência” (GAMBOA, 1997, p. 107). O objetivo desta pesquisa é confrontar o discurso oficial, contido nas Diretrizes Curriculares Nacionais e Instrumento de Avaliação de Cursos de Graduação presencial e a distância, e a prática docente com a finalidade de se verificar a articulação teoria-prática nos componentes curriculares. Keywords: Higher education, articulation theory and practice, teacher training. Correspondencia: Yara Pires Gonçalves, coord.pedagogica@toledoprudente.edu.br Selección y peer-review bajo responsabilidad del Comité Organizador del XIV Congreso Internacional Galego-Portugués de Psicopedagogía Resumo O trabalho apresenta estudos realizados em um Centro Universitário Brasileiro, objetivando confrontar documentos oficiais sobre as políticas públicas relativas à teoria e à prática presentes nos componentes curriculares de cursos de natureza diversa. Quanto à relação teoria e prática nos currículos formativos foram pesquisados os discursos oficiais e o papel do docente nessa articulação, por meio da abordagem qualitativa de base filosófica dialética. Os resultados apontam a fragilidade entre a teoria e a prática, o descompasso entre o lócus de formação e o de atuação docente e lacunas no entendimento da unidade dialética (teoria e prática), refletindo nos componentes curriculares. P l h E i S i ti l ã t i A relevância do objeto de pesquisa: articulação teoria-prática nos componentes curriculares é, portanto, social, institucional e pessoal, pois se coloca como vertebradora da formação de professores e profissionais, possibilitando a geração de conhecimento e desenvolvimento nas sociedades contemporâneas. A pesquisa desenvolveu-se em um Centro Universitário Brasileiro, no interior do Estado de São Paulo, nos cursos de Administração e Ciências Contábeis (bacharelados). Palavras-chave: Ensino Superior, articulação teoria e prática, formação docente. Para estudar a relação teoria e prática nos currículos formativos foram pesquisados os discursos oficiais e o papel do docente na articulação teoria-prática por meio de abordagem qualitativa (BOGDAN,1994, p. 47-51) de base filosófica dialética, com a finalidade de apreendê-lo em seus significados e sentidos. A Ausência de articulação teoría-prática nos componentes curriculares A Absence of theory-practice articulation in the curricular components Yara Pires Gonçalves,* Rita de Cássia M. Trindade Stano** Centro Universitário Toledo; Universidade Federal de Itajubá. Yara Pires Gonçalves, Rita de Cássia M. Trindade Stano** Centro Universitário Toledo; *Universidade Federal de Itajubá. vivenciadas. Formam uma unidade, integram-se e são integradores dos componentes curriculares. A articulação entre eles, no ato educativo, supera a singularidade que os identifica. Correspondencia: Yara Pires Gonçalves, coord.pedagogica@toledoprudente.edu.br rrespondencia: Yara Pires Gonçalves, coord.pedagogica@toledoprudente.edu.br ección y peer-review bajo responsabilidad del Comité Organizador del XIV Congreso Internacional Galego-Portugués de Psicopeda REVISTA DE ESTUDIOS E INVESTIGACIÓN EN PSICOLOGÍA Y EDUCACIÓN eISSN: 2386-7418, 2017, Vol. Extr., No. 12 DOI: https://doi.org/10.17979/reipe.2017.0.12.2175 REVISTA DE ESTUDIOS E INVESTIGACIÓN EN PSICOLOGÍA Y EDUCACIÓN eISSN: 2386-7418, 2017, Vol. Extr., No. 12 DOI: https://doi.org/10.17979/reipe.2017.0.12.2175 Selección y peer-review bajo responsabilidad del Comité Organizador del XIV Congreso Internacion Introducão Mudanças sociais e avanços tecnológicos compõem um cenário contextual marcado por mudanças e transformações diversas. Educar para a incerteza, para uma sociedade em movimento, neste momento de interregno entre o que existe e o que ainda não se definiu (BAUMAN, 2011), dá ao professor, no âmbito educacional, papel social estratégico e exige que ele tenha uma formação adequada aos novos tempos. Deve-se ressaltar que esses documentos se constituem em parâmetros de qualidade em educação para o Ministério da Educação-MEC e nos permite inferir o que se considera qualidade do ponto de vista oficial, isto é, o que é qualidade em educação na ótica dos órgãos governamentais responsáveis e representativos da educação brasileira superior. A título de clareza, alguns conceitos pertinentes ao tema devem ser considerados como: teoria- prática e prática e práxis. Teoria e prática são unidades de movimento, uma está contida na outra, em constante processo: “[...] contexto de que fazer, de práxis, quer dizer, de prática e de teoria” (FREIRE, 2006, p.106 “[...] A teoria e a prática têm, neste contexto, lugar privilegiado nos componentes curriculares que compõem o percurso curricular formativo dos futuros professores e profissionais de outras áreas. Teoria e prática são elementos indissociáveis, permeiam e significam os saberes oriundos das experiências sociais GONÇALVES, STANO toda prática tem um fundamento teórico e vice-versa [..] (FREIRE; SHÖR, 2006, p.12). Para Freire, teoria e prática se constituem em uma unidade dialética, indissociável. cursos, e nos Planos de Ensino das disciplinas/unidades curriculares, elaborados por seus respectivos professores, em especial os componentes curriculares de caráter integrador como estágios supervisionados, trabalhos de conclusão de curso, produção intelectual e atividades de extensão comunitária e educacional. Dentro do contexto atual, é necessário se repensar a teoria e a prática docente como práxis, como prática docente consciente, quando se almeja a transformação social, com vistas à consecução de um projeto educacional emancipatório que pressupõe a formação de sujeitos autônomos, capazes de produzir seu próprio conhecimento, de posicionar-se diante da realidade, de ter postura crítica e refletir sobre suas ações e as dos outros (GIROUX, 1997, SANTOS, 2003, FREIRE, 2004) As Diretrizes Curriculares Nacionais dos Cursos de Graduação estão expressas no Parecer CNE/CES 67/2003, aprovado em 11/03/2003, baseado na Lei de Diretrizes e Bases da Educação Nacional- LDBEN (Lei 9394/96) e no Plano Nacional de Educação (Lei 10.172/2001), dentre outros. Foram instituídas para substituir os currículos mínimos. A teoría e prática nos discursos oficiais A articulação da teoria-prática nos componentes curriculares tem sido foco de pesquisas e discussões no ensino superior, nos cursos de bacharelados e ou licenciaturas, no que diz respeito à sua efetiva concretização ao longo do percurso formativo dos futuros professores e profissionais de outras áreas. Esses cursos são concebidos à luz de algumas variáveis como fundamentos epistemológicos, demandas sociais, políticas governamentais e institucionais, mercado de trabalho, mas sempre regidos por documentos oficiais, conforme exigência legal. De acordo com o objeto de pesquisa, análise da relação teoria-prática nos cursos mencionados, optou-se por confrontar os discursos oficiais expressos, dentre eles, nas Diretrizes Curriculares Nacionais, gerais e específicas por curso, e Instrumento de Avaliação de Cursos de Graduação presencial e a distância, afeto ao Sistema Nacional de Avaliação da Educação Superior-SINAES (2015), considerados parâmetros de qualidade oficial para as políticas públicas vigentes e seu impacto na prática docente, expressos nos Projetos Pedagógicos dos Cursos e Planos de Ensino. A Resolução no. 04, de 13 de julho de 2005, instituiu as Diretrizes Curriculares de Graduação em Administração, bacharelado. No que concerne ao objeto de estudo, articulação teoria-prática nos componentes curriculares, nos eixos ensino, pesquisa e extensão, essa resolução apresenta como um dos elementos obrigatórios no Projeto Pedagógico de Curso, no art. 2º., inciso V: a exigência de previsão dos modos de integração entre teoria e prática. Os conteúdos curriculares se distribuem em quatro campos de formação: básica, profissional, estudos quantitativos e suas tecnologias e complementar, não fazendo menção específica ao objeto em questão. O curso caracteriza-se por formar profissionais generalistas. O posicionamento da teoria-prática nesses documentos, frequência dos indicadores e sua forma de operacionalização apontam o seu grau de importância para a formação do aluno e refletem a formação docente no processo de articulação desses elementos nos componentes curriculares, na ótica oficial. p p g A Resolução no. 10, de 16 de dezembro de 2004, instituiu as Diretrizes Curriculares para o curso de graduação em Ciências Contábeis, bacharelado. Seguindo o mesmo raciocínio, observou-se que o Projeto pedagógico deverá abranger, dentre os elementos estruturais, no art. 2º. incisos V: modos de integração entre teoria e prática e no inciso XI: inclusão opcional de trabalho de curso, que deverão estar centrados em áreas teórico-práticas. Quanto às competências e habilidades exigidas, a resolução não se detém explicita e particularmente à relação teoria-prática. Introducão As diretrizes estabeleceram seis princípios básicos, dentre eles, fortalecer a articulação da teoria com a prática (grifo nosso). A partir desses princípios foram criadas as diretrizes curriculares de cada curso, observando-se múltiplos perfis profissionais, diversidade de carreiras, integração do ensino de graduação com a pós-graduação e privilégio de competências intelectuais que refletissem heterogeneidade das demandas sociais. Em resumo, as Diretrizes Curriculares Nacionais, no referido parecer, “concebem a formação de nível superior como um processo contínuo, autônomo e permanente, com sólida formação básica e uma formação profissional fundamentada na competência teórico-prática (grifo nosso), de acordo com o perfil de um formando adaptável às novas e emergentes demandas” (p.6). A teoría e prática nos discursos oficiais Entretanto, ao tratar dos conteúdos de formação refere-se aos campos de Formação Básica, Formação Profissional e Formação Teórico-Prática, especificamente referindo-se ao Estágio Curricular Supervisionado, Atividades Complementares, Estudos Independentes, Conteúdos Optativos, Prática em Laboratório de Informática utilizando softwares atualizados para a Contabilidade. Neste item, dois aspectos merecem destaque: os estágios poderão ser Os bacharelados formam profissionais para diferentes áreas de trabalho. As licenciaturas pretendem a formação docente, em especial para o ensino fundamental e médio, mas também para no nível superior. Entretanto, no ensino superior brasileiro, temos um percentual alto de bacharéis exercendo a docência, sem formação específica para essa finalidade, o que se reflete na atuação docente. Na instituição, centro universitário campo de pesquisa, temos 114 docentes, sendo 101 bacharéis (88,60%) e 13 com licenciatura (11,40%). Para esta pesquisa, foram realizados levantamentos nos documentos mencionados e verificada a operacionalização desses elementos, teoria-prática, nos componentes curriculares dos cursos de Administração e Ciências Contábeis, bacharelado presenciais, confrontados/verificados nos discursos expressos nos Projetos Pedagógicos dos referidos R Est Inv Psico y Educ, 2017, Extr.(12), A12-07 A AUSÊNCIA DE ARTICULAÇÃO TEORÍA-PRÁTICA NOS COMPONENTES CURRICULARES Administração em que a articulação teoria prática se evidencia: Estágio Supervisionado I e II (5º e 6º termo); Jogos de Empresa (7º termo); Desenvolvimento de Novos Negócios I e II (7º e 8º termo); Consultoria Empresarial (8º termo). No curso de Ciências Contábeis temos: as Práticas Contábeis I até VI (3º termo até 8º termo); Consultoria Contábil II (8º termo); Estágio Supervisionado I e II (5º e 6º termo); Análise das Demonstrações contábeis II (8º termo); Auditoria Contábil II (8º termo). realizados na própria instituição de ensino mediante laboratórios e as atividades de estágio poderão ser reprogramadas e reorientadas, de acordo com os resultados teórico-práticos até que os resultados sejam considerados satisfatórios. As atividades Complementares abrangem a prática de estudos e atividades independentes, transversais e opcionais, de interdisciplinaridade e ações de extensão junto à comunidade. O Trabalho de Conclusão de Curso é, entretanto, componente curricular opcional da instituição o que fragiliza a própria exigência legal. O que se constata é que mesmo apontando lacunas na articulação teoria-prática nos componentes curriculares, o curso de Ciências Contábeis aponta indicadores dessa relação em grau mais acentuado, embora deficitário, do que o curso de Administração. A teoría e prática nos discursos oficiais Quanto ao outro documento legal, utilizado para o confronto entre discurso oficial e articulação teoria-prática, acrescenta-se que o Instrumento de Avaliação de Cursos de Graduação presencial e a distância subsidia os atos autorizativos de cursos e são utilizados pelos avaliadores de cursos como referencial de oferta nas dimensões 1.Organização didático-pedagógica, 2.Corpo docente e 3.Infraestrutura. Na dimensão 1. Organização Didático-Pedagógica, pertinente a este estudo, observou-se que dos 27 itens, apenas 8 são relativos à teoria-prática, mas somente um faz menção à sua articulação, em diferentes graus e significados, nos bacharelados. É o item 1,5 que se refere ao indicador estrutura curricular, incluindo: flexibilidade, interdisciplinaridade, acessibilidade pedagógica e atitudinal, compatibilidade da carga horária, articulação teoria e prática. Ao verificar-se a presença desses indicadores nos respectivos Projetos Pedagógicos de curso e Planos de ensino das disciplinas de caráter integrador, constata-se que, apenas o Projeto Pedagógico do curso de Ciências Contábeis contempla a articulação teoria e prática, no que diz respeito aos conteúdos curriculares e sua abordagem reflete a formação teórico-prática expressa nas Diretrizes Curriculares do curso. O que não ocorre com o Projeto Pedagógico do curso de Administração, em que não há uma preocupação expressa da articulação teoria-prática. Em especial, no caso dos Estágios Supervisionados, observa-se que o curso de Administração propõe situação simulada, como pode-se verificar nas ementas de Estágio Supervisionado I (5º.termo):” Descrição detalhada da empresa e dos principais processos administrativos. Elaboração e análise de fluxogramas operacionais do negócio. Elaboração dos layouts correspondentes aos fluxogramas” e Estágio Supervisionado II (6º. Termo): “Tipos de pesquisa de mercado. Escolha de empresa. Enunciado do problema. Objetivo Geral. Objetivos Específicos. Metodologia. Técnicas de coleta de informações e amostra. Pesquisa de Campo. Tabulação dos resultados. Geração de tabelas e gráficos. Relatório de análise dos dados. Apresentação dos resultados”, o que não ocorre com o curso de Ciências Contábeis. Deve-se observar que o item 1.7 sobre Metodologia não explicita a relação teoria-prática. p p Os itens 1.8, 1.9, 1.10 e 1.11 dizem respeito ao Estágio. O item 1.8 , sobre Estágio Supervisionado Curricular (obrigatório), o foco não é a relação teoria e prática, mas a existência de convênios, formas de apresentação, orientação, supervisão e coordenação. Portanto, muito mais voltado para atendimentos burocráticos do que aprendizagem do aluno, na ótica da articulação teoria-prática. O item 1.9 refere-se ao Estágio Curricular para a Educação Básica, obrigatório para as licenciaturas, e não bacharelado. A teoría e prática nos discursos oficiais O item 1.10, que se refere ao Estágio nas Licenciaturas, o foco são as parcerias, acompanhamento, planejamento, desenvolvimento e avaliação. O item 1.11 Estágio Curricular Supervisionado trata da relação teoria e prática (obrigatório para as licenciaturas e não se aplica aos demais cursos). Este é o item em que a articulação entre teoria-prática é mais evidenciada e complementa o item anterior. No curso de Ciências Contábeis, embora o estágio se utilize de laboratório de informática, os alunos vivenciam situações concretas, utilizam dados reais fornecidos por outras disciplinas e os alunos executam atividades propostas, conforme ementas de Estágio Supervisionado I e II (5º. e 6º. Termo): “Atividades pertinentes ao curso, intercursos e interdisciplinares, ligadas às organizações, integradoras do processo de formação do profissional em Ciências Contábeis, realizadas em empresas públicas e/ou privadas, na Empresa Júnior Toledo-EJT, de acordo com o regulamento do estágio supervisionado obrigatório”. Os dois itens subsequentes, Atividades Complementares e Trabalho de Conclusão de Curso-TCC, são relativos a atividades integradoras da teoria e da prática, mas não contemplam essa preocupação. Por último, complementando a análise nos eixos pesquisa e extensão, observou-se que na pesquisa os cursos da área de negócios, onde se inclui os cursos estudados, fazem parte de um grupo de iniciação científica da IES intitulado: Relações Empresariais, Inclusão e Desenvolvimento Sustentável, supervisionado pelo Núcleo de Estudos e Pesquisa- NEPE. Entretanto, vale destacar a dificuldade que Ainda foram analisados os Planos de Ensino de outras disciplinas também com caráter integrador, em que a relação teoria-prática está presente, sem contudo, corresponder aos conceitos pressupostos de unidade dialética e práxis. São elas: disciplinas do curso de R Est Inv Psico y Educ, 2017, Extr.(12), A12-08 GONÇALVES, STANO professores e alunos têm para produção intelectual. Nos Encontros de Iniciação Científica, anuais, a produção técnica é mais relevante em ambos os cursos. prática ocupam espaço relevante nos componentes curriculares dialógicos e relacionais, de caráter integrador. O processo se coloca como elemento vertebrador nos currículos atuais. Esses cursos apoiados pela Coordenadoria de Extensão e Assuntos Comunitários-CEAC, com o objetivo de promover e incentivar a integração da comunidade acadêmica com a comunidade regional, apresentam experiências, dados e informações que contribuem para a melhoria das atividades acadêmicas. A teoría e prática nos discursos oficiais Os alunos dos dois cursos participam de Programas de Extensão que abrangem: a) Projetos de extensão: Coleta de Preços e Índice de Preços Toledo (Empresa Junior); b) Educação Continuada; c) Atividades Acadêmicas e Prestação de Serviços (Jornada de Cinema); d) Assuntos Comunitários (Coleta Seletiva de Lixo, Doação de Sangue, Cadastro de doadores de medula). A extensão permite a integração intercursos e amplia a relação da comunidade acadêmica com a local e regional. Ao se confrontar a presença da teoria e da prática nos discursos oficiais, Diretrizes Curriculares Nacionais e Instrumento de Avaliação de Cursos de Graduação, com a prática docente, verifica-se que poucos são os itens desses documentos que se referem à teoria e à prática, e a articulação entre eles está explicita em apenas um item. Essa articulação aparece sugerida, apenas, nos Projetos Pedagógicos e ou Planos de Ensino, documentos institucionais, quando se referem a determinados componentes curriculares. Os documentos relativos, tanto aos cursos estudados como aos de licenciatura, prescrevem muito mais conteúdos teóricos do que prática, como legado do pensamento que predominou durante tempos, de que a ciência determinava a sociedade e não o inverso como se constata hoje (CUNHA, 2003, p.68-70). Os componentes curriculares das matrizes estudadas demonstram os estágios curriculares supervisionados, bem como projetos integradores e práticas profissionais colocadas nas séries finais dos cursos, resquícios de uma visão tradicional, positivista, resultando em componentes curriculares isolados, fragmentados sem a devida articulação neles e entre eles. Embora exista, muitas vezes, busca pela articulação teoria-prática, isso ainda não ocorre em conformidade com os pressupostos conceituais tomados como referência, a formação docente nessa direção e as exigências legais, pois os documentos oficiais não se preocupam com essa articulação. Para que se efetive a articulação teoria-prática nos componentes curriculares pelos professores, não basta formação profissional de qualidade, deve-se considerar a intencionalidade docente nessa direção e sua relação com a conscientização. Para Coll (1996, p.319), a intencionalidade está na raiz do processo educativo, determina as mudanças que se quer provocar no aluno e o rumo do seu desenvolvimento. A teoría e prática nos discursos oficiais Essa interlocução é muitas vezes tentada por meio de atividades interdisciplinares, o que nem sempre supera essa fragmentação, pois as causas também podem estar na formação docente pessoal e profissional, nível de intencionalidade do professor no ato educacional direcionado para esse fim, bem como variar de acordo com as possibilidades garantidas pelo contexto institucional em que atuam, por meio de cursos de capacitação e ou apoio financeiro para essa finalidade. Freire vai além “A conscientização não é somente conhecimento e reconhecimento do objeto cognoscível, é opção, decisão, compromisso” (FREIRE, 2003, p.10). Pode-se afirmar que, segundo Heller (2000, p.20) o desenvolvimento da consciência permite ao sujeito utilizá-la intencionalmente, possibilitando a superação de ações cotidianas, do senso comum em direção às não cotidianas, pertinentes à ciência, política, arte, filosofia, afetas às necessidades exigidas nas aprendizagens mais complexas, no ensino superior. Outra questão a ser destacada é em relação à concepção de prática e práxis. O que se constata é que predomina a prática pela prática e não a práxis, prática consciente que se espera do professor, o que pode se colocar como entrave para a articulação entre teoria-prática. Verifica-se, por último, que há uma fragilidade pronunciada, quase ausência, em relação ao que se espera dos processos relacionais e de articulação, intrínsecos aos componentes curriculares, observados no descompasso entre o lócus de formação desses profissionais (matrizes curriculares de acordo com as DCN) e o lócus de atuação docente (escola); em lacunas no entendimento desses professores quanto à concepção de teoria-prática como unidade dialética; presença de resquícios positivistas, em especial nos componentes integradores, estágios, produção intelectual e nas atividades de extensão. Indicadores e Possibilidades - Considerações finais Nas sociedades contemporâneas, caracterizadas pelo movimento, mudanças e indefinições, ganham espaço as relações, as articulações, a dialogicidade e as interlocuções. É nesse contexto que se discute a análise de fundamentos epistemológicos da articulação teoria-prática nos componentes curriculares, à luz dos documentos legais pertinentes e sua relação com a formação docente, considerando-se a posição estratégica do professor para que se efetive essa articulação. A ausência de articulação teoria-prática nos componentes curriculares evidencia a distância entre as demandas sociais, currículos responsivos e formação docente. A intencionalidade docente se coloca como possibilidade de superação dessa dicotomia existente entre teoria e prática, pois está relacionada ao nível de conscientização do professor para além do senso comum (HELLER, 2000) e uma sólida formação do professor, o que supõe desenvolvimento de competências e habilidades voltadas para mudança, em especial senso crítico-reflexivo, flexibilidade e A pesquisa e a análise sobre a articulação teoria-prática nos componentes curriculares supõem a relação teoria e prática como uma unidade dialética. Portanto, a articulação e a integração entre teoria e R Est Inv Psico y Educ, 2017, Extr.(12), A12-09 A AUSÊNCIA DE ARTICULAÇÃO TEORÍA-PRÁTICA NOS COMPONENTES CURRICULARES criatividade para inovar e que corresponda às demandas sociais que exigem aprendizagem permanentemente responsiva. Referências Bauman, Z. A face humana da sociologia. O Estado de São Paulo, 30 abril 2011, disponível em http://cultura.estadao.com.br/notícia s/geral, a face-humana-da-sociologia-imp.712848 Bogdan, R. C. Investigação qualitativa em educação. Poro: Porto Editora, 1994. Coll, C., Palacios, J., Marchesi A. (Org.) Desenvolvimento psicológico e educação. Porto Alegre: Artes Médicas, 1996. v 2. g Cunha, M.I. Formação de professores e currículo no ensino superior. Reflexões sobre o campo político-epistemológico. In: Moraes, C., PACHECO, J.A., Evangelista, M.O. Formação de professores. Perspectivas educacionais curriculares. Coleção Currículo, Políticas e Práticas. Porto: Porto Editora, 2003. Diretrizes Curriculares Nacionais-DCN. Parecer CES/CNE 67/2003, aprovado em 11/03/2003. Diretrizes Curriculares Nacionais do Curso de Graduação EM Administração. Resolução CES/CNE, no. 04, de 13/07/2003. Diretrizes Curriculares Nacionais DO Curso de Graduação em Ciências Contábeis. Resolução CES/CNE no. 10, DE 16/12/2004. Freire, P. Pedagogia do oprimido. 37. ed. São Paulo: Paz e Terra, 2003. Freire, P. Pedagogia da Autonomia: saberes necessários à autonomia. 29ed. São Paulo: Paz e Terra, 2004. Freire, P. Professora sim, tia não: cartas a quem ousa ensinar.16. ed. São Paulo: Olho d’água, 2006. Freire, P.; SHÖR, Ira. Medo e ousadia: o cotidiano do professor.11. ed. Rio de Janeiro: Paz e Terra, 2006. Gamboa, S. A dialética na pesquisa em educação: elementos de contexto. In: FAZENDA, I. (Org.). Metodologia da pesquisa educacional. 4. ed. São Paulo: Cortez, 1997. p. 91-115. Giroux, H.A. Os professores como intelectuais: rumo a uma pedagogia crítica da aprendizagem. Porto Alegra: Artes Médicas, 1997. Gonçalves, Y.P. Qualidade oficial, indicadores do MEC. In: Coimbra, C.L. et al (orgs). Qualidade em Educação. Curitiba/PR: Editora CRV, Série Currículo: Questões Atuais, vol 4, 2011. Heller, A. O cotidiano e a história. 6. ed. São Paulo: Paz e Terra, 2000. Instrumento De Avaliação De Cursos De Graduação Instrumento De Avaliação De Cursos De Graduação Presencial E A Distância. Ministério da Educação. Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira –INEP. Brasília, março de 2015. Instrumento De Avaliação De Cursos De Graduação Presencial E A Distância. Ministério da Educação. Instituto Nacional de Estudos e Pesquisas Educacionais Anísio Teixeira –INEP. Brasília, março de 2015. SANTOS, B. de S. Um discurso sobre as ciências. São Paulo: Cortez, 2003. SANTOS, B. de S. Um discurso sobre as ciências. São Paulo: Cortez, 2003. R Est Inv Psico y Educ, 2017, Extr.(12), A12-10
https://openalex.org/W4312586604	http://ojs.stiudq.ac.id/JUQDQ/article/download/106/48	Indonesian	null	Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an	Ulumul Qur'an	2,022	cc-by	9,582	Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an Rahmah Muharromah Yasin Institut Ilmu Al-Qur’an (IIQ) Jakarta Email: rahmahmuharromah94@gmail.com Abstract: This paper aims to describe how the North Dullah community's reception of the concept of religious moderation from the perspective of the Qur'an. This scientific research aims to find out how the wasathiyah verses are based on the viewpoints and assessments of the community in the field. In this study, the area studied has distinctive local wisdom, namely in the North Dullah sub-district, Tual city, which is known as the Larvul Ngabal Customary Law and the Ain ni ain philosophy. The method used in this research is field research with a qualitative research type that produces descriptive data and uses a phenomenological approach. The results in this study are that religious moderation in the reception of the North Dullah community is a moderate religious way, or an inclusive way of Islam or an open religious attitude, not exchanging beliefs, respecting each other in tolerance, peace and justice. In its implementation in the North Dullah sub-district, it is adjusted to local customs, namely the customary law of Larvul Ngabal and the philosophy of Ain ni ain which is the basis. From the results of the interview, the researcher can conclude that in the legal articles of Larvul Ngabal and the philosophy of Ain ni ain it is clear that the customary law and philosophy are in accordance with Islamic Shari'a law, especially the verses regarding moderation. p y g g Keywords: Reception; Community; Religious Moderation Keywords: Reception; Community; Religious Moderation Abstrak: Tulisan ini bertujuan untuk mendeskripsikan bagaimana resepsi masyarakat Dullah Utara terhadap konsep moderasi beragama perspektif Al-Qur’an. Penelitian karya ilmiah ini bertujuan untuk mendapatkan bagaimana tentang ayat-ayat wasathiyah berdasarkan sudut pandang dan penilaian masyarakat di lapangan. Dalam penelitian ini, daerah yang diteliti memiliki kearifan lokal yang khas yaitu di kecamatan Dullah Utara kota Tual yang dikenal dengan nama Hukum Adat Larvul Ngabal dan filosofi Ain ni ain. Metode yang digunakan dalam penelitian ini adalah penelitian lapangan (field research) dengan jenis penelitian kualitatif yang menghasilkan data dan deskriptif serta melalui pendekatan fenomenologi. Adapun hasil dalam penelitian ini adalah bahwa moderasi beragama dalam resepsi masyarakat Dullah Utara yakni cara beragama yang moderat, atau cara ber-Islam yang inklusif atau sikap beragama yang terbuka, tidak bertukar-menukar keyakinan, saling menghargai dalam toleransi, perdamaian dan keadilan. Dalam pelaksanaannya di kecamatan Dullah Utara disesuaikan dengan adat istiadat setempat yakni hukum adat Larvul Ngabal dan filosofi Ain ni ain yang menjadi landasannya. Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an Rahmah Muharromah Yasin Institut Ilmu Al-Qur’an (IIQ) Jakarta Email: rahmahmuharromah94@gmail.com Dari hasil wawancara, peneliti dapat menyimpulkan bahwa di dalam pasal-pasal hukum Larvul Ngabal dan filosofi Ain ni ain tersebut jelas bahwa hukum adat dan filosofi tersebut bersesuaian dengan hukum syariat Islam khususnya ayat-ayat mengenai moderasi. Kata Kunci: Resepsi, Masyarakat, Moderasi Beragama Ulumul Qur’an: Jurnal Ilmu Al-Qur’an dan Tafsir Volume 2, Nomor 2, September 2022\| p-ISSN: 2774-5169; e-ISSN: 2774-6496\| 261-278 Pendahuluan Rahmah Muharromah Yasin Indonesia negara yang kaya akan keanekaragaman budaya/multikultural. Keragaman merupakan sunnatullah, bahkan sudah menjadi ketentuan Allah, jadi hal ini tidak dapat di tawar-menawar, melainkan di lestarikan perbedaannya. Selain kepercayaan dan agama yang beragam, di dalamnya ada penganut yang memiliki perbedaan sudut pandang atas ajaran agamanya masing-masing. Misalnya dalam agama Islam terdapat berbagai madzhab fiqih yang secara berbeda-beda memberikan fatwa atas hukum dan tertib pelaksanaan suatu ritual ibadah. Keragaman madzhab ini muncul seiring waktu berkembangnya zaman dalam konteks yang berbeda-beda. Oleh karenanya dalam tradisi Islam dikenal dengan ajaran yang bersifat qathi’ (pasti), tsawabit (tidak berubah-ubah/konsisten, dzanni (berubah-ubah), dan ada yang fleksibel. Selain agama Islam, agama lain pun juga memiliki perbedaan dalam tradisi dan ajarannya. (Gunawan, 2015, h. 35). Pengetahuan akan ajaran agama sangatlah penting bagi pemeluk agama karena dengan adanya pengetahuan tersebut maka memungkinkan seseorang dapat mengambil jalan tengah (moderat) jika ada salah satu ajaran yang tidak memungkinkan dijalankan. Sikap ekstrem biasanya muncul adakala seorang pemeluk agama tidak mengetahui jalan alternatif dari suatu kebenaran ajaran yang ia jalani. Disini lah pentingnya peran moderasi beragama untuk dijadikan sudut pandang seseorang dalam beragama. Paham-paham keyakinan yang bersifat fundamentalistik, integralistik-total, atau mengklaim diri sebagai satu-satunya kebenaran, yang pada akhirnya mereka menganggap dirinya lebih benar dan mengkafirkan kelompok yang lain. (Bagir, 2019, h. 44). Upaya moderasi beragama dalam pencegahan sikap diskriminatif, ekstremisme, dan intoleransi tak luput dari peran pemerintah. Pada tanggal 7 Januari 2021, presiden Joko Widodo menandatangani Perpres no. 7 tahun 2021 tentang Rencana Aksi Nasional Pencegahan dan Penanggulangan Ekstremisme (RAN PE). Program tersebut melibatkan sejumlah beberapa lembaga/kementerian di antaranya Kemensos, Badan Nasional Penanggulangan Terorisme, Kemendagri, Kemenag, Kementerian Koperasi UKM, dan Kementerian Pariwisata dan Ekonomi Kreatif. Hal ini dapat diharapkan meningkatkan kesadaran masyarakat untuk mencegah munculnya aksi terorisme melalui pendekatan budaya, ekonomi, dan agama. (Kompas, 2021). Dengan diterapkannya moderasi beragama di Indonesia sebenarnya sudah mapan dengan adanya wasaṭiyyah Islam. Konsep ini sudah relatif mapan yang sudah diterapkan pada ormas-ormas besar Nusantara seperti NU dan Muhammadiyah. NU dengan Islam Nusantaranya sejalan dengan wasaṭiyyah Islam, begitupun juga dengan Muhammadiyah dengan Islam berkemajuannya memiliki konsep yang sama dengan wasaṭiyyah Islam. (Bisri, 2019, h. 8). Masalahnya sekarang ini, ormas-ormas di Indonesia sudah banyak dimasuki paham-paham ekstrem, tidak moderat, mudah menyalahkan kelompok lain, menganggap kelompok yang lain kafir, hingga menggunakan kekerasan dalam merespon kelompok yang berbeda paham. Jadi disini sangat diperlukan adanya moderasi beragama, yang dalam Islam dikenal dengan istilah wasaṭiyyah (وَسَطِيَّة). Pendahuluan Quraish Shihab berpendapat “Moderasi atau wasaṭiyyah bukanlah sikap yang bersifat tidak jelas atau tidak tegas dalam sesuatu bagaikan sikap netral dan pasif, bukan juga pertengahan matematis sebagaimana yang dipahami sementara orang dari hasil pemikiran filsuf yunani”. (Shihab, 2019, h. ix). Disini dapat dijelaskan bahwa kata moderat merupakan kata sifat yang aslinya dari kata moderation, yang artinya tidak berlebihan ataupun sedang. Setelah diserap kata moderat berubah menjadi kata moderasi, yang didefinisikan sebagai minimnya ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 262 262 Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an kekerasan atau menghindari dari keesktreman. Yang dapat disimpulkan dari pakar bahasa adalah sesuatu yang harus keluar dari kedua sisinya. Moderasi dalam bahasa arab berarti wasaṭ atau wasaṭiyyah. Kata wasaṭ dalam derifasinya diulang sebanyak lima kali dalam Al-Qur’an dan kesemuanya mengandung makna “berada di antara dua ujung”. Wasaṭiyyah sudah menjadi konsep pembeda antara dua hal yang berbeda. Dalam hal ini konsep wasaṭiyyah tidak menyetujui adanya radikalisme, begitu sebaliknya konsep ini tidak akan mengurangi pemahaman isi kandungan Al-Qur’an yang menjadi dasar pokok syariat Islam. Dengan demikian konsep wasaṭiyyah lebih memaknai ke arah toleransi dan mengikuti asas-asas hukum syariat. Pemahaman moderat atau yang disebut wasaṭiyyah ini merupakan salah satu ideologi Islam yang tidak dimiliki oleh penganut/agama yang lain. ُ ْ ُ َ َ ََ “Dan demikian itu pula Kami telah menjadikan kamu (umat muslim) ”umat pertengahan....” (QS. Al-Baqarah [2]: 143) ََ “Dan demikian itu pula Kami telah menjadikan kamu (umat muslim) ”umat pertengahan....” (QS. Al-Baqarah [2]: 143) Keadilan merupakan landasan utama dalam sebuah organisasi ataupun struktur masyarakat. Karena dengan adanya keadilan pasti akan terjamin semua hak bagi seorang individu sampai dengan lingkungan masyarakat. Dan berakibat dengan terciptanya kesejahteraan bersama, yang pastinya tentu dibarengi dengan perilaku yang sesuai dengan asas-asas hukum yang berlaku. (Syafrudin, 2009, h. 105). Dengan adanya syariat Islam yang sudah cukup moderat patutnya telah menjadi tameng dan penjagaan konsistensi asas Islam yang telah disiarkan oleh Nabi SAW, oleh karena itulah diperlukan moderasi ini sudah dapat mengembalikan citra Islam yang selayaknya, agar agama lain dapat merasakan keberkahan akan ajaran Islam yang rahmatan lil ‘alamin. Berdasarkan indeks Kerukunan Umat Beragama (KUB) di Indonesia tahun 2019 yang dirilis oleh Kementerian Agama mencapai angka 73,83%. Dengan peringkat 5 besar yaitu provinsi Papua Barat (82,1%), NTT (81,1%), Bali (80,1%), Sulut (79,9%), dan Maluku (79,4%). Dan yang terbawah dimulai dari 5 provinsi Aceh (60,2%), Sumba (64,4%), Jabar (68,5%), Banten (68,9%), dan Riau (69,3%). Pendahuluan Angka ini mengalami peningkatan dibanding tahun lalu yaitu mencapai 70,90%. Survei ini melibatkan 400 responden secara acak di setiap provinsi di Indonesia. Rata-rata indeks KUB di Indonesia selalu di atas 70% sejak tahun 2015. (Tirto, 2020). Salah satu daerah yang toleran di Indonesia yaitu di kota Tual provinsi Maluku tepatnya di Kecamatan Dullah Utara. Secara astronomis Kecamatan Dullah Utara terletak di 1320 41` – 1320 49` BT dan 50 26` – 50 38` LS. Menurut geografis daerah ini sebelah selatan dibatasi oleh kecamatan Dullah Selatan, sebelah utara dibatasi oleh Laut Banda, sebelah timur dibatasi oleh kab. Maluku Tenggara yaitu selat Nerong, dan sebelah barat dibatasi oleh Laut Banda. Memiliki luas wilayah total 91,57 km2 terdiri dari 8 pulau diantaranya 3 pulau yang berpenghuni dan 5 pulau yang tidak didiami. Dengan total populasi sebanyak 23.281 jiwa. Desa ini berpenghuni dengan perbedaan heterogen suku, budaya dan agama, diantaranya suku bangsa Kei, Ambon, Tobelo, Banda, dan suku pendatang seperti Jawa, Bugis, Buton, dan lainnya. Agama yang dianut penduduk pun beragam, seperti Islam (25 masjid, 6 musholla), kristen protestan (2 gereja), kristen katolik (5 gereja). (BPS Kab. Malra, 2020, h. 4). Dalam dekade 5 tahun terakhir, di wilayah Kecamatan Dullah Utara sering terjadi bentrokan dan pembunuhan antar suku/marga yang disebabkan oleh sengketa tanah adat atau masalah harga diri perempuan, untuk konflik antar umat beragama sudah jarang terjadi sejak kerusuhan besar tahun 1999 di Maluku dan Maluku Utara. Volume 2, nomor 2, September 2022 263 Rahmah Muharromah Yasin Walaupun masih ada sedikit bentrokan-bentrokan kecil antara warga muslim dan nonmuslim. Ini sebagai bukti bahwa masyarakat belum sepenuhnya menerapkan nilai-nilai moderasi walaupun sudah mulai sedikit perubahan akan terciptanya lingkungan yang toleran terhadap pemeluk agama lain. (Persbhayangkara.id, 2021). Dari penjelasan latar belakang di atas peneliti ingin menarik sebuah tema untuk diteliti lebih mendalam yaitu resepsi masyarakat terhadap konsep moderasi beragama menurut Al-Qur’an dan implikasinya pada daerah heterogen di Kecamatan Dullah Utara. Penelitian ini berjenis studi living Qur’an yang akan mengaplikasikan nilai-nilai maupun upaya dalam meningkatkan lingkungan yang bermoderasi agama. ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 264 A. Diskursus Moderasi Beragama A. Diskursus Moderasi Beragama Dalam Islam moderasi dikenal dengan sebutan wasaṭiyyah. Kata moderasi berasal dari bahasa Latin yaitu moderâtio artinya ke-sedang-an (tidak kelebihan dan tidak kekurangan). Ini bisa diartikan sebagai penguasaan diri (dari sikap kelebihan dan kekurangan). Menurut KBBI moderasi diartikan sebagai pengurangan kekerasan dan penghindaran ekstremisme. (KBBI, 2002, h. 354). Dalam bahasa arab, moderasi dikenal dengan kata wasaṭ atau wasaṭiyyah Al-Asfahani (w. 502 H) mengartikan wasaṭan dengan sawa’un yang artinya tengah-tengah atau diantara dua batas, atau diartikan dengan keadilan, standar, atau biasa-biasa saja. (Al-Asfahani, 2009, h. 869). Moderate tidak terlepas dari dua kata kunci, yakni balance (keseimbangan) dan justice (keadilan). Tanpa adanya dua nilai tersebut dalam moderasi beragama akan tidak menjadi efektif. (Kamali, 2015, p. 14). Adapun lawan kata dari moderasi yaitu berlebihan (tatharruf) yang mengandung makna extreme, radical, dan excessive. Kata extreme juga berarti ‘berbuat keterlaluan, pergi dari ujung ke ujung, berbalik memutar, mengambil tindakan/jalan sebaliknya’. Dalam KBBI, kata ekstrem didefinisikan sebagai ‘paling ujung, paling tinggi, dan paling keras atau sangat keras dan teguh, fanatik’. Kesemuanya mempunyai makna yang berlebihan dan itu sangat tidak baik bagi sikap seseorang dalam beragama. Dalam bahasa arab, kata ekstrem yang mempunyai makna sama seperti ghuluw dan tasyaddud yang merujuk pada makna ‘berlebihan’ ini dapat diterapkan untuk seseorang yang bersikap ekstrem, serta melebihi batas dan ketentuan syariat agama. Dipersamakan dengan kata tatharruf yang berasal dari kata tharaf yang bermakna pinggir atau ujung sesuatu. Kata ini asalnya digunakan pada hal-hal yang material, lambat laun berkembang kata ini digunakan untuk hal yang immaterial seperti pemikiran, keagamaan, ataupun perilaku/tingkah laku. Makna ghuluw digunakan sebagai tipe seseorang yang beragama secara berlebihan dan akhirnya melenceng dari esensi agamanya. (Munzhur, h. 141). Atau bersikap berlebihan (ekstrim) pada suatu masalah dengan melampaui batas yang telah ditentukan. (Asqalani, 1990, h. 241). Derivasi dari kata ghuluw yaitu tanattu’ (sikap yang keras), tashaddud (menyusah sesuatu), ifrât (mempersempit), atau takalluf (memaksakan diri). Dalam bahasa inggris kata ekstremisme terambil dari kata ekstrem yaitu extreme diartikan sebagai very great in degree; the greatest degree; not ordinary or usual; dan serious or severe. Yang mempunyai makna gelar yang berlebihan, tidak biasa, terlalu serius, dan tidak biasanya. Kalau diibaratkan, moderasi adalah ibarat gerak dari pinggir yang selalu cenderung menuju ke pusat atau sumbu, sedangkan ekstremisme adalah gerak sebaliknya menjauhi pusat menuju sisi terluar dan entrifugal. b. Al-wusṭa Kata ini muncul pada QS. Al-Baqarah [2]: 238, حَافِظُوْ اٰ ٰعَلَىٰ ٰ الصَّلَوِٰتٰ ٰ وَ الصَّلِٰوةٰٰ ٰ الْوُسْط ىٰوَ قُوْ مُوْ اٰ ِٰ ٰ ِلِلٰٰ قَٰنِتِيْنٰ 23٨ٰ(البقرة/ 23٨: 2 ) “Peliharalah semua salat dan salat wusṭa. Dan laksanakanlah (salat) karena Allah dengan khusyuk.” (QS. Al-Baqarah [2]: 238 A. Diskursus Moderasi Beragama Meminjam dalam ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 264 Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an analogi ini dalam konteks moderasi beragama, sikap moderat merupakan pilihan cara pandang, sikap, perilaku di antara pilihan ekstrem yang ada. Sedangkan sikap ekstremisme beragama adalah cara pandang, sikap, dan perilaku yang melebihi batas moderasi dalam pemahaman dan praktik beragama. Karenanya moderasi beragama dapat dipahami sebagai cara pandang, sikap, dan perilaku selalu mengambil posisi di tengah, selalu bertindak adil, dan tidak ekstrem dalam beragama. B. Term Moderasi (Wasaṭiyyah) dalam Al-Qur’an Moderasi dalam bahasa arab berarti wasaṭ atau wasaṭiyyah. Kata wasaṭ dalam derifasinya diulang sebanyak lima kali dalam Al-Qur’an yaitu wasaṭa, al-wusṭa, awsaṭ, awsaṭuhum, dan wasaṭna. Dan term-term lain yang mempunyai makna yang sama seperti al-‘adl, as-sadâd, al-qashd, dan al-istiqâmah. a. Wasaṭa Kata ini tercantum di QS. Al-Baqarah [2]: 143, ٰ وَ كَذَٰلِكٰ ٰ جَعَلْنْٰكُمٰ ٰ اُمَّةَّٰٰو سَط اٰ ٰل ِتَكُوْ نُوْ اٰ ٰ شُهَدَاَٰءٰعَلَىٰٰ ِٰالنَّاسٰ ُٰوَ يَكَٰوْ نٰ ُٰالرَّسُوْ لٰ ْٰعَلَيْكُمٰشَهِيْد اٰٰۗ ... ٰ 143 ٰ ٰ( ٰالبقرة/ 2 ٰ: 143 ٰ) “Dan demikian pula Kami telah menjadikan kamu (umat Islam) ”umat pertengahan” agar kamu menjadi saksi atas (perbuatan) manusia dan agar Rasul (Muhammad) menjadi saksi atas (perbuatan) kamu ....” (QS. Al-Baqarah [2]: 143) Kata ini tercantum di QS. Al-Baqarah [2]: 143, ٰ وَ كَذَٰلِكٰ ٰ جَعَلْنْٰكُمٰ ٰ اُمَّةَّٰٰو سَط اٰ ٰل ِتَكُوْ نُوْ اٰ ٰ شُهَدَاَٰءٰعَلَىٰٰ ِٰالنَّاسٰ ُٰوَ يَكَٰوْ نٰ ُٰالرَّسُوْ لٰ ْٰعَلَيْكُمٰشَهِيْد اٰٰۗ ... ٰ 143 ٰ ٰ( ٰالبقرة/ 2 ٰ: 143 ٰ) “Dan demikian pula Kami telah menjadikan kamu (umat Islam) ”umat pertengahan” agar kamu menjadi saksi atas (perbuatan) manusia dan agar Rasul (Muhammad) menjadi saksi atas (perbuatan) kamu ....” (QS. Al-Baqarah [2]: 143) Rasyid Ridha (w. 1354 H) menafsirkan ayat ini dengan QS. Al-Baqarah [2]: 213, yakni dengan cara hidayah inilah mereka menjadi umat yang adil (wasaṭan). Yang dimaksud wasaṭ adalah adil dan pilihan. Karena meminta tambahan dari apa yang dituntut dalam suatu perkara adalah berlebihan (ekstrem), sedangkan menguranginya adalah kelalaian. Sikap berlebihan dan lalai adalah tindakan yang menyimpang dari jalur yang benar dan lurus. Dan demikian itu merupakan perbutaan tercela. Karenanya perbutan yang terbaik adalah yang di tengah-tengah di antara dua perkara. (Ridha, h. 126). Sayyid Quthb (w. 1386 H) mengatakan dalam tafsirnya bahwa umat Islam memiliki sikap wasaṭ dalam seluruh aspeknya, baik dalam makna baik (hasan), atau utama (fadhl), atau wasaṭ dalam makna pertengahan dan kesederhanaan (qashd), atau wasaṭ dalam maknanya yang bersifat materi. Dan menjadi saksi bagi seluruh umat manusia karena mereka sebagai pemegang hukum yang adil di antara umat yang lain. (Quthb, 1992, h. 468). Umat ini merupakan umat yang mempunyai cara pandang yang moderat terhadap keyakinan (i’tiqad), dalam cara berpikir dan rasa, dalam organisasi dan koordinasi, dalam berinteraksi dan menjalin koneksi, umat pertengahan pada zamannya dan pertengahan dalam posisinya. Ummatan wasaṭan dalam tashawwur (gambaran) pemikiran, pandangan, persepsi dan keyakinan. Dan semata-mata sebagai umat yang pemenuhan nalurinya seimbang dan persesuain dengan pemenuhan jasmani. Dengan keseimbangan ini akan meningkatkan mutu kehidupan. Pada waktu yang sama, ia memelihara kehidupan dan mengembangkannya, menjalankan semua aktivitas di dunia spritual dengan tidak berlebih-lebihan dan tidak mengurang- ngurangkan, melainkan dengan sederhana, teratur dan seimbang. a. Wasaṭa Volume 2, nomor 2, September 2022 265 Rahmah Muharromah Yasin Diantara para mufassir dalam ayat ini menunjukkan kata wusṭa memiliki hubungan dengan makna wasaṭ, di antaranya, Ibnu Jauzi berpendapat tentang shalat wusṭa itu ada tiga, (1) Shalat yang paling tengah posisinya, (2) Shalat yang paling tengah kadarnya, dan (3) Shalat yang paling utama. Adapun yang dimaksud dengan tengahnya sesuatu adalah yang paling baik atau yang paling adil. (Al-Jauzi, h, 283). Al-Qasimi (w. 1332 H) juga berpendapat di dalam kosakata wasaṭa dengan ayat ini, yaitu pertengahan antara panjang dan pendek, tapi kemungkinan dari sisi lain, yakni ‘dan shalat wusṭa’ yang dimana maksudnya sifat shalat yang diperintahkan untuk senantiasa dijaga dan merupakan shalat yang terbaik karena memiliki keutamaan yang agung di sisi Allah SWT.(Qasimi, 1978, h. 623). Sementara itu Ibnu ‘Asyur (w. 1393 H) berkata adapun dalil yang mengkaitkan dengan saat menyifati shalat wusṭa, dimaknai sebagai al-khiyar (yang paling baik) dan al-fadhl (yang utama). Maka mereka kemudian menilisik apa yang tertera tentang keutamaan sebagian shalat. Dan ada juga menyebutkan sifat wasaṭ sebagai pertengahan dari sisi jumlah, maka diantaara mereka ada yang berpendapat yang mencari shalat diantara dua semua sisinya. (‘Asyur, 1984, h. 467). Dari penjelasan di atas dapat disimpulkan bahwa, ada kalimat setelah ‘dan shalat wusṭa yaitu “peliharalah semua shalatmu” yang merupakan petunjuk dan arahan serta perintah untuk melaksanakan shalat dengan cara pertengahan, tidak terlalu panjang yang membosankan, dan tidak perlu terlalu pendek. Maksudnya hendaklah shalat dilakukan dengan cara pertengahan; antara panjang dan pendek. c. Awsaṭ Kata ini terdapat dalam QS. Al-Maidah [5]: 89, ... ٰٰ ٰ فَكَفَّارَتُهٰٰ ُٰاِطْعَامٰٰ ِٰعَشَرَةٰٰ ٰ مَسَٰكِيْنٰٰ ْٰمِنٰٰ ِٰاَوْ سَطٰٰمَاٰٰ َٰتُطْعِمُوْ نٰٰ ْٰاَهْلِيْكُمٰٰ ْٰاَوٰٰ ْٰكِسْوَ تُهُمٰٰ ْٰاَوٰٰ ُٰتَحْرِ يْرٰٰ ٰ رَقَبَةٰٰ ۗ ... ٰٰ٩٨ٰٰ ٰٰ( ٰ الما ئدة/5 ٰٰ: ٩٨) c. Awsaṭ Kata ini terdapat dalam QS. Al-Maidah [5]: 89, ... ٰٰ ٰ فَكَفَّارَتُهٰٰ ُٰاِطْعَامٰٰ ِٰعَشَرَةٰٰ ٰ مَسَٰكِيْنٰٰ ْٰمِنٰٰ ِٰاَوْ سَطٰٰمَاٰٰ َٰتُطْعِمُوْ نٰٰ ْٰاَهْلِيْكُمٰٰ ْٰاَوٰٰ ْٰكِسْوَ تُهُمٰٰ ْٰاَوٰٰ ُٰتَحْرِ يْرٰٰ ٰ رَقَبَةٰٰ ۗ ... ٰٰ٩٨ٰٰ ٰٰ( ٰ الما ئدة/5 ٰٰ: ٩٨ٰ) “... maka kafaratnya (denda pelanggaran sumpah) ialah memberi makan sepuluh orang miskin, yaitu dari makanan yang biasa kamu beri kan kepada keluargamu, atau b i k k i d k k h b h ” (QS Al c. Awsaṭ Kata ini terdapat dalam QS. Al-Maidah [5]: 89, ... ٰٰ ٰ فَكَفَّارَتُهٰٰ ُٰاِطْعَامٰٰ ِٰعَشَرَةٰٰ ٰ مَسَٰكِيْنٰٰ ْٰمِنٰٰ ِٰاَوْ سَطٰٰمَاٰٰ َٰتُطْعِمُوْ نٰٰ ْٰاَهْلِيْكُمٰٰ ْٰاَوٰٰ ْٰكِسْوَ تُهُمٰٰ ْٰاَوٰٰ ُٰتَحْرِ يْرٰٰ ٰ رَقَبَةٰٰ ۗ ... a. Wasaṭa ٰٰ٩٨ٰٰ ٰٰ( ٰ الما ئدة/5 ٰٰ: ٩٨ٰ) “... maka kafaratnya (denda pelanggaran sumpah) ialah memberi makan sepuluh orang miskin, yaitu dari makanan yang biasa kamu beri kan kepada keluargamu, atau memberi mereka pakaian atau memerdekakan seorang hamba sahaya ....” (QS. Al- Mâidah [5]: 89) c. Awsaṭ ) “... maka kafaratnya (denda pelanggaran sumpah) ialah memberi makan sepuluh orang miskin, yaitu dari makanan yang biasa kamu beri kan kepada keluargamu, atau memberi mereka pakaian atau memerdekakan seorang hamba sahaya ....” (QS. Al- Mâidah [5]: 89) “... maka kafaratnya (denda pelanggaran sumpah) ialah memberi makan sepuluh orang miskin, yaitu dari makanan yang biasa kamu beri kan kepada keluargamu, atau memberi mereka pakaian atau memerdekakan seorang hamba sahaya ....” (QS. Al- Mâidah [5]: 89) Para mufassir ada yang berpendapat maknanya sama dengan awsaṭuhum dan ada juga berpendapat tidak sama. Imam Zamakhsyari (w. 1143 H) mengatakan ayat ini maksudnya adalah dari yang paling tengah dan sederhana. Sebab di antara mereka ada yang boros dalam memberi makan keluarganya namun ada pula yang kikir atau pelit. (Zamakhsyari, h. 640). Sayyid Quthb (w. 1386 H) juga berpendapat bahwa kata wasaṭ bisa bermakna terbaik atau yang pertengahan. Keduanya mempunyai makna yang sama dengan kata awsaṭ. Dan penggabungannya telah lepas dari makna sederhana (qashd), sebab yang di tengah-tengah itu adalah yang terbaik, dan di tengah itu adalah yang paling baik dalam ajaran Islam. Dari berbagai penafsiran ada sedikit beragam makna seperti paling utama (afdhal), antara sedikit dan banyak (tengah-tengah), antara yang baik dan yang jelek (pertengahan), antara sempit dan lapang. Dilihat dari tema konteks ayat ini, bahwa semuanya sepakat memberi makan orang miskin yang terbaik dari apa yang sehari- hari kita makan serta menyesuaikan dengan kondisi ekonomi, jadi tidak perlu memaksakan diri untuk memberi sesuatu yang mahal, tetapi makanan yang halālan ṭayyibah walaupun itu kelihatan murah. ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 266 Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an d. Awsaṭuhum Kata awsaṭuhum terdapat dalam QS. Al-Qalam [68]: 28, kata ini memiliki makna yang hampir sama dengan kata awsaṭ, َقَالْاَوْسَطُهُمْاَلَمْاَقُلْلَّكُمَلَوْ لَتُسَب حُوْ ن2٨(القلم/ 6٨: 2٨) “Berkatalah seorang yang paling bijak di antara mereka, “Bukankah aku telah mengatakan kepadamu, mengapa kamu tidak bertasbih (kepada Tuhanmu).” (QS. Al- Qalam [68]: 28) “Berkatalah seorang yang paling bijak di antara mereka, “Bukankah aku telah mengatakan kepadamu, mengapa kamu tidak bertasbih (kepada Tuhanmu).” (QS. Al- Qalam [68]: 28) Menurut penjelasan dari Ibnu ‘Abbas ra (w. 68 H) kata awsaṭuhum artinya adalah ‘adaluhum (yang paling adil). Pendapat ini senada dengan imam Qatadah (w. 117 H), ia berkata maksud ayat ini yaitu yang paling adil dalam ucapan. Dan mereka adalah kaum yang sangat takut dan paling baik untuk kembali baik. Imam Al-Qurṭubi (w. 671 H) berkata maksudnya yang paling ideal, paling adil, dan paling cerdas. (Qurthubi, 1987, h. 244). Ibnu Katsir (w. 774 H) juga berkata, Ibnu ‘Abbas (w. 68 H), Mujahid (w. 324 H), Said bin Jubair (w. 258 H), Qatadah (w. 117 H) dan Ikrimah (w. 107 H) mengatakan bahwa maksudnya adalah yang paling adil dan paling baik. (Katsir, h. 406). Dari apa yang sudah dijelaskan, jadi jelaslah bahwa kata awsaṭuhum atau awsaṭ hampir memiliki makna yang sama, dari berbagai sisi dan beragam makna, diantaranya memiliki makna yang paling utama (afdhal), paling baik (khiyar), dan yang paling adil. e. Wasaṭna Kata ini terdapat dalam firman-Nya, Kata ini terdapat dalam firman-Nya, ) “Lalu menyerbu ke tengah-tengah kumpulan musuh,” (QS. Al-‘Âdiyat [100]: 5) َ ُّ َّ ُ َُ ُ َِٰل ُ ََّْ ُ َُ ََّ ُۗ ُۗ ُ َ ْ ٰ ي اَيُّهَاٰٰ َٰالَّذِيْنٰٰٰ ا مَنُوْ اٰٰكُوْ نُوْ اٰٰ َٰقَوَّ امِيْنٰٰ ِٰ ٰ ِلِلٰٰ ٰ شُهَدَاَٰءِٰٰٰبٰ ِالْقِسْطٰٰ َٰوَ لٰٰ ْٰيَجْرِ مَنَّكُمٰٰ ٰ شَنَاُٰنٰٰ ٰ قَوْ مٰٰ ٰ ى عَلٰٰ َّٰاَلٰٰتَعْدِلُوْ اٰۗ ٰۗاِعْدِلُوْ اٰٰ َٰهُوٰٰ ُٰاَقْرَبٰ ٰ لِلتَّقْوٰ ىٰوَ اتَّقُواٰ َٰ ٰاللّٰۗ َّٰاِنٰ َٰ ٰاللّٰ ٰ خَبِيْرٰ ٰبِمَاٰ َٰتَعْمَلُوْ نٰ٨ٰ ٰ( ٰ الما ئدة/ 5 ٰٰ:٨) “Wahai orang-orang yang beriman! Jadilah kamu sebagai penegak keadilan karena Allah, (ketika) menjadi saksi dengan adil. Dan janganlah kebencianmu terhadap suatu kaum mendorong kamu untuk berlaku tidak adil. Berlaku adillah. Karena (adil) itu lebih dekat kepada takwa. Dan bertakwalah kepada Allah, sungguh, Allah Mahateliti terhadap apa yang kamu kerjakan.” (QS. Al-Mâidah [5]: 8) ( ) “Wahai orang-orang yang beriman! Jadilah kamu sebagai penegak keadilan karena Allah, (ketika) menjadi saksi dengan adil. Dan janganlah kebencianmu terhadap suatu kaum mendorong kamu untuk berlaku tidak adil. Berlaku adillah. Karena (adil) itu lebih dekat kepada takwa. Dan bertakwalah kepada Allah, sungguh, Allah Mahateliti terhadap apa yang kamu kerjakan.” (QS. Al-Mâidah [5]: 8) Aṭ- Ṭabari (w. 310 H) menafsirkan ayat ini dengan makna hendaklah berlaku adil menjadi sikap kalian dan akhlak kalian, dengan senantiasa menegakkan kebenaran karena Allah dan menjadi saksi dengan adil, baik terhadap musuh, teman, ataupun kerabat. Disini dijelaskan wajib berlaku adil kepada siapapun walaupun terhadap musuh, dan jangan berlaku ekstrem atau melampaui batas terhadap syariat agama yang sudah ditetapkan oleh Allah SWT. (Aṭ- Ṭabari, h. 95). Hal senada seperti yang disampaikan oleh Ibnu Katsir (w. 774 H), bahwa hendaklah berperilaku adil tidak boleh terseret oleh kebencian, menjadi penegak keadilan bagi setiap umat manusia disertai dengan cara yang ma’ruf walaupun itu berlaku kepada musuh. (Ibnu Katsir, h. 95). Adil yang diperintahkan pada umat ini adalah hak umum bagi setiap orang dari seluruh umat manusia. Tidak terhalang oleh perbedaan warna kulit, suku, bangsa, atau agama. Adil itu adalah hak bagi setiap manusia, bukan hanya bersikap adil di antara sesama kaum muslimin saja. Keadilan itu harus berlaku bagi setiap manusia karena kedudukannya sebagai sesama manusia, dan sebab keadilan adalah sifat manusia. ٰ يَٰيُّهَا اٰ َٰالَّذِيْنٰٰ ا مَنُواٰاتَّقُواٰ َٰ ّٰللاٰوَ قُوْ لُوْ اٰ ٰ قَوْ لٰ ٰ سَدِيْد اٰ٧0 ٰ ٰٰ(الحزاب/ 33 ٰٰ:٧0 ٰ) “Wahai orang-orang yang beriman! Bertakwalah kamu kepada Allah dan ucapkanlah perkataan yang benar,” (QS Al-Ahzâb [33]: 70) ) “Lalu menyerbu ke tengah-tengah kumpulan musuh,” (QS. Al-‘Âdiyat [100]: 5) Para mufassir menyebut maksud ayat ini adalah berada di tengah-tengah di suatu tempat. Aṭ- Ṭabari (w. 310 H) menyebut para musuh dengan kendaraanya tersebut menyerbu ke tengah-tengah kumpulan suatu kaum. Baik wasaṭtu al-qaum ( ُ وسطتُُالقوم) atau wassaṭtu (ُ وسَّطت) dengan tasydid atau tawassaṭtuhu (توسَّطته) adalah satu makna. (Aṭ- Ṭabari, h. 286). Ibnul Jauzi (w. 597 H) mengatakan, Ibnu Mas’ud (w. 32 H) berkata mengenai ayat ini maksudnya adalah Muzdalifah, wilayah terbuka yang terletak di antara kota Mekkah dan Mina di Arab Saudi. (Al-Jauzi, h. 209). Sedangkan imam Al-Qurṭubi (w. 671 H) berkata, ُ َجَْعًا adalah maf’ul bih, َُف َوَسَطْن maksudnya dengan kendaraannya menyerbu ke tengah musuh. Dikatakan ُ وَسَطْتُُ َُالقَوْمُُ ُْاَوْسَط ه مُُ َُوَسْطُُ ُ وَسَط ه , artinya adalah saya berada di tengah-tengah. ُ وَسَطْتُُ َُالقَوْم walaupun dengan tasydid ataupun tidak tetap bermakna sama. Ada juga berpendapat kalau huruf sinnya bertasydid maka artinya menjadi kumpulan yang terbelah dua, tetapi kalau huruf sinnya tanpa tasydid berarti di tengah-tengah kerumunan. Sayyid Quthb (w. 1386 H) berkata, maksudnya kuda-kuda itu menyeruak di tengah-tengah barisan musuh dengan tiba-tiba membuat kekacauan dan keguncangan di tengah-tengah mereka. Al-Qasimi (w. 1332 H) pun berpendapat, senada dengan pendapat Sayyid Quthb (w. 1386 H), dan sedikit menambahkan bahwa huruf سُ dengan tasydid atau takhfif maknanya tetap sama. Dari beberapa penjelasan di atas, jelaslah hampir ada sedikit kesamaan dalam kata وسط dan التوسَّط. f. Al-‘adl Volume 2, nomor 2, September 2022 267 Rahmah Muharromah Yasin Kata ‘adl diulang sebanyak 28 kali dalam Al-Qur’an. Secara istilah ada term- term di dalam Al-Qur’an yang mempunyai makna yang sama, seperti al-qisṭ (القسط), dan al-mizan (ُامليزان), dan mengandung yang terbaik atau pertengahan. م Menurut Aṭ- Ṭabari (w. 310 H), bahwa adil adalah takwil dari kata wasaṭ dan bermakna khiyar (yang paling terbaik), karena orang terbaik di antara manusia yaitu yang paling adil. (Aṭ- Ṭabari, h. 155). Imam Al-Qurṭubi (w. 671 H) menyebutkan wasaṭ (pertengahan) maknanya adalah al-‘adl (adil). Sebab bahwa yang paling terpujinya sesuatu adalah yang di tengah-tengah. Al-Qurṭubi (w. 671 H) menambahkan bahwa umat Islam dijadikan sebagai umat yang pertama (utama) dalam memberikan kewenangan dan kesaksian bagi seluruh makhluk-Nya, terkecuali bagi orang-orang yang adil. (Al-Qurṭubi, h. 155). h. Al-qashd Kata al-qashd (القصد) mempunyai arti makna moderasi, juga konsistensi, serta mengandung makna arah dan tekad, baik tekad itu menyangkut sesuatu yang baik dan buruk. Ada juga memaknai dengan arti lurus. (Ibnu Faris, h. 95). Penggunaan dalam bentuk masdar dalam kata al-qashd mengisyaratkan tentang betapa sempurna jalan dan penjelasan itu. Bisa juga dalam arti ‘penjelasan tentang jalan yang mengantar kepada kebenaran atau jalan yang lurus’. Atau dalam arti ‘sesuatu bagian yang dibelah dua’. Jika kata tersebut dipakai kepada seseorang maka artinya tidak gemuk, tidak kurus, dan sesuatu itu tidak besar ataupun kecil, tidak sedikit atau tidak banyak. (Shihab, 2019, h. 20). ۗ (Shihab, 2019, h. 20). ْٰوَ اقْصِ دٰ ْٰفِيٰ َٰمَشْيِكَٰٰوْٰاغْضُضٰ ْٰمِنٰ َٰۗصَوْ تِكٰ َّٰاِنٰ َٰاَنْكَرٰ ِٰالْ َصْوَ اتٰ ُٰلَصَوْ تٰ ِٰالْحَمِيْرٰٰࣖ 1٩ٰ ٰ( ٰ ن لقم/ 31 ٰ: 1٩) “Dan sederhanakanlah dalam berjalan dan lunakkanlah suaramu. Sesungguhnya seburuk-buruk suara ialah suara keledai.” (QS. Al-Luqmân [31]: 19) وَ عَلَىٰ ِٰ ٰاللّٰ ُٰقَصْدٰ ِٰالسَّبِيْلٰوَ مِنْهَاٰ ٰ جَاٰ ىٰ رٰۗ ْٰوَ لَوٰ ٰ شَاَٰءٰ ٰ لَهَدْٰىكُمٰ َٰاَجْمَعِيْنٰٰࣖ٩ٰ(النحل/ 16 ٰ:٩) “Dan hak Allah menerangkan jalan yang lurus, dan di antaranya ada (jalan) yang menyimpang. Dan jika Dia menghendaki, tentu Dia memberi petunjuk kamu semua (ke jalan yang benar).” (QS. An-Nahl [16]: 9) للَِِّ م( ) “Dan hak Allah menerangkan jalan yang lurus, dan di antaranya ada (jalan) yang menyimpang. Dan jika Dia menghendaki, tentu Dia memberi petunjuk kamu semua (ke jalan yang benar).” (QS. An-Nahl [16]: 9) Ayat di atas ُْوَاقْصِدُُ ُِْفُُ َُمَشْيِك dapat dipahami dalam arti jangan terlalu cepat ketika berjalan sehingga mengesankan kelemahan dan rendah diri. Dari sini kata al-qashd dipahami dalam arti seimbang dan moderasi. Dalam ُ قَصْدُُ ُِالسَّبِيْل menjelaskan bahwa jalan yang mudah dan dekat akan mencapai keridhaan-Nya, yakni jalan moderasi, tidak berlebihan dalam melakukan sesuatu seperti dalam beribadah atau bermuamalah. g. As-sadâd Kata as-sadâd (السداد) ini terambil dari kata sadada (سدد) yang terdiri dari huruf ُس dan د. Menurut pakar bahasa Ibnu Faris, rangkaian dua huruf itu menunjuk pada makna meruntuhkan sesuatu kemudian memperbaikinya. (Ibnu Faris, h. 148). Maknanya sama dengan istiqâmah (konsistensi). Kata ini menunjukkan ketepatan sasaran. Seseorang yang menyampaikan sesuatu/ucapan yang benar dan mengena tepat pada sasarannya dilukiskan dengan kata ini. Dengan demikian kata tersebut tidak sekedar berarti benar, tetapi ia juga harus tepat sasaran. Pada ayat QS. Al-Ahzâb [33]: 70, Allah SWT memerintahkan orang beriman untuk mengucapkan qaulan sadida. ُ َّ َللّ ُ “Wahai orang-orang yang beriman! Bertakwalah kamu kepada Allah dan ucapkanlah perkataan yang benar,” (QS Al-Ahzâb [33]: 70) ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 268 Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an Dari kata سَدِيْدًا yang mengandung makna meruntuhkan sesuatu kemudian memperbaikinya diperoleh pula petunjuk bahwa ucapan yang meruntuhkan pada saat yang sama juga harus memperbaikinya. Dalam arti, kritik yang disampaikan hendaknya berupa kritik yang membangun atau informasi yang disampaikan haruslah baik, benar, dan mendidik. Dengan perilaku as-sadâd maka si penerima akan menjadi luluh hatinya ketika kita berbicara kepada mereka. Hal ini dapat diterapkan kepada orang-orang berpikiran ekstrem atau liberal, perkatan yang lemah lembut dan lugas serta dengan argumentasi yang kuat dapat merubah cara pandang mereka dalam berperilaku. (Shihab, 2019, h. 19). i. Al-istiqâmah Salah satu derivasi dari makna moderasi yaitu kata istiqâmah (اِستقامة), dan agar seseorang bisa berjalan di atas shirâtal mustaqîm, hendaknya selalu istikamah di atas jalan Allah dan syariat-Nya. Inilah sebenar-benarnya wasaṭiyyah dan inti dari prinsip yang wasaṭi. Kata اِستقم ini berulang sebanyak dua kali, yakni ْٰفَاسْتَقِمٰ ٰ كَمَاٰ َٰاُمِرْ تٰ ْٰوَ مَنٰ َٰتَابٰٰ َٰمَعَكٰ َٰوَ لٰ ٰۗتَطْغَوْ اٰ ٰ اِنَّهٰبِمَاٰ َٰتَعْمَلُوْ نٰ ٰ بَصِ يْرٰ 112 ٰ(هود/ 11 ٰ: 112 ٰ) “Maka tetaplah engkau (Muhammad) (di jalan yang benar), sebagaimana telah diperintahkan kepadamu dan (juga) orang yang bertobat bersamamu, dan janganlah kamu melampaui batas. Sungguh, Dia Maha Melihat apa yang kamu kerjakan.” (QS. Hûd [11]: 112) ٰ فَلِذَٰلِكٰ ُٰفَادْعٰۚ ْٰوَ اسْتَقِمٰ ٰ كَمَاٰ َٰۚاُمِرْ تٰ َٰوَ لٰ ْٰتَتَّبِعٰ ٰ اَهْوَ اْٰۚءَهُمٰ ... ٰ 15 ٰ ٰ( ٰالشورى/ 42 ٰٰ: 15 ٰ) Volume 2, nomor 2, September 2022 269 Rahmah Muharromah Yasin “Karena itu, serulah (mereka beriman) dan tetaplah (beriman dan berdakwah) sebagaimana diperintahkan kepadamu (Muhammad) dan janganlah mengikuti keinginan mereka ....” (QS. Asy-Syûra [42]: 15) Di ayat lain juga juga disebutkan makna istiqamah dan kedudukannya seperti QS. At-Taubah [9]: 7, QS. Yunus [10]: 89, QS. Fushshilat [41]: 6 dan 30, QS. Al- Ahqaf [46]: 13, QS. Al-Jin [72]: 16, dan QS. At-Takwir [81]: 28. Sayyid Quthb (w. 1386 H) juga menambahkan bahwa larangan yang datang sesudah perintah istiqâmah itu (QS. Hud [11]: 112) bukannya larangan pengabaian atau pengurangan, melainkan larangan pelampauan batas. Ini karena perintah istaqim serta apa yang diakibatkannya dalam jiwa manusia boleh jadi mengantar seseorang melampaui batas dan berlebihan, sehingga mengalihkan ajaran agama ini dari kemudahan menjadi kesukaran. Padahal Allah SWT menghendaki agar agama-Nya tidak berkurang dan tidak berlebih. Kelebihan dan pelampauan batas serupa dengan pengabaian dan pengurangan, keduanya mengantar agama ini menyimpang dari cirinya yang dikendaki oleh Allah SWT. (Quthb, 1992, h. 630). y y g Dalam tafsir Al-Biqâ’i (w. 885 H) memahami ayat ini (QS. Hud [11]: 112) sebagai bentuk dari makna moderasi, tetapi kenapa tidak dibahas secara langsung dalam ayat tentang pengurangan sesuatu. Karena pengurangan hampir tidak dapat luput darinya kecuali seseorang yang sangat jarang wujudnya, dan pengurangan itu pun biasanya melahirkan kerendahan hati dan rasa takut kepada Allah SWT. Sementara itu, sikap berlebihan melahirkan rasa bangga diri bahkan boleh jadi mengantar seseorang menetapkan ajaran baru yang dapat berakibat dia keluar dari agama (yang benar). (Biqa’i, 2006, h. 451). C. Resepsi Masyarakat Terhadap Konsep Moderasi Beragama i. Al-istiqâmah Karena itulah ayat ini tidak secara langsung menyebut larangan mengurangi dan langsung melarang melebih-lebihkan yakni melampaui batas. Selain itu bagaimana kita menerapkan moderasi (وَسَطِيَّة) itu? Kita wajib mempunyai beberapa pengetahuan mengenai: م 1) Fiqh al-maqâsid (فقهُُاملقاصد) pengetahuan akan sebab atau latar belakang 1) Fiqh al-maqâsid (فقهُُاملقاصد) pengetahuan akan sebab atau latar belakang (‘illat) suatu hukum yang sudah ditetapkan. ام q qمp g g (‘illat) suatu hukum yang sudah ditetapkan. ام 2) Fiqh al-Muwâzanât (فقهُُاملوازانت) yakni dapat membandingkan antara kadar dalam kebaikan atau kemaslahatan agar dapat memilih mana pilihan yang terbaik. 3) Fiqh al-Awlawiyât (فقهُُاألولوايت) yaitu dapat memilah apa yang paling penting dari yang penting sampai yang tidak penting. ام 4) Fiqh al-Ma’âlât (فقهُ ُاملآالت) yakni pengetahuan akan mampu dapat melihat apa hasil dari pilihan yang telah dibuat. (Shihab, 2019, h. 179). Adanya wasaṭiyyah, maka ada keseimbangan antara ruh dan jasad, akhirat dan dunia, iman dan ilmu, akal dan teks keagamaan, taklid dan tajdid, ide dan kenyataan, agama dan negara, individu dan masyarakat, maupun dalam diri manusia seperti cinta dan benci, harap dan cemas, dan lain sebagainya. Dalam menerapkan wasaṭiyyah, diperlukan mujâhadah (mengendalikan diri), ijtihâd (mengolah pikiran), dan jihâd (mencurahkan tenaga), karena wasaṭiyyah bukanlah konsep yang instan, tetapi perlu usaha yang baik dan kerjasama dalam semua kalangan pihak. ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 270 Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an Sebelum membahas mengenai resepsi masyarakat tentang konsep moderasi beragama ini, peneliti terlebih dahulu akan menjelaskan sedikit tentang hukum adat Larvul Ngabal dan filosofi Ain ni Ain. Sebelum membahas mengenai resepsi masyarakat tentang konsep moderasi beragama ini, peneliti terlebih dahulu akan menjelaskan sedikit tentang hukum adat Larvul Ngabal dan filosofi Ain ni Ain. Sejarah terbentuknya hukum adat Larvul Ngabal dan filsafat Ain ni Ain belum ada bukti fisik sama sekali, ini karena masyarakat Kei/leluhur dahulu hanya mengandalkan tuturan lisan (hol tom) dan cenderung subyektif. Jadi tidak ada cerita yang pasti tentang asal-usul sejarah hukum adat ini. Ini juga merupakan kelemahan dari masyarakat ataupun tokoh adat yang belum membukukan secara tepat sehingga tidak ada bukti yang valid ketika ada yang mengkaji lebih dalam tentang sejarah hukum adat Kei. (Wawancara Raja Fer, Mei 2021). Bukti fisik yang ada dan masih dipercaya oleh masyarakat Kei hanya makam Dit Sakmas yang berada di ohoi Semawi kabupaten Maluku Tenggara. i. Al-istiqâmah Tidak ada tanggal yang pasti kapan meninggalnya, karena dari penuturan dari tetua adat, keluarga Kasdew dan Jingra ketika berada di tanah Kei sekitar awal tahun 1500-an sebelum bangsa Eropa menginjakkan kakinya di tanah Maluku. Sedangkan tugu kerbau siw yang menjadi tonggak terbentuknya hukum adat Larvul baru dibangun sekitar tahun 2015. Bukti lainnya seperti tempat ditancapkannya tombak/ngabal belum diketahui secara pasti tempatnya (sumber lain menyebutkan di Ler Ohoilim), dikarenakan semua sejarah ini hanya berdasarkan lisan tanpa adanya sumber yang otentik (base on data). Hukum adat Larvul Ngabal ini menjadi hukum dasar adat yang digunakan oleh masyarakat ke tanah Maluku Tenggara dan juga digunakan sebagai landasan dalam merekonstruksi berbagai fenomena sosial yang terjadi di dalam kehidupan bermasyarakat. Ada beberapa ketentuan-ketentuan dalam hukum diantaranya yakni tujuh pasal utama dan dua puluh empat pasal lanjutan. Pasal lanjutan dibagi lagi menjadi tiga kelompok hukuman dan pelanggaran, yakni hukum pidana (nevnev), hukum etiket (hanilit), dan hukum perdata (hawear balwirin). Pasal utama terdiri dari 7 pasal yaitu: Pasal utama terdiri dari 7 pasal yaitu: 1. Uud su intaluk na etvunad, maksudnya kepala kita bertumpu pada tengkuk kita. 2. Lelad ain fo mahiling, leher kita diluhurkan atau dihormati. 3. Uil hit enwil rumud, dari tanah yang membungkus badan 4. Lar nakmod na rumud, maksudnya darah kita yang tertutup di dalam tubuh. 5. Rek fo kilmutun, maksudnya pernikahan hendaklah ditempatkan pada tempatnya agar tetap suci dan murni. 6. Moryain fo mahiling, tempat untuk perempuan harus diluhurkan dan dihormati. 7. Hira ni fo ini, it did fo it did, maksudnya milik seseorang adalah tetap milik mereka dan milik kita sendiri tetap menjadi milik kita. Dalam pasal yang pertama intinya adalah yang dimaksud dengan kepala yang dekat dengan tempat kita melambangkan pimpinan, perencana, pengatur, penguasa, penyayang, pengayom, dan penyantun. Unsur kepala dalam konteks pasal ini yakni, Tuhan, para leluhur, tokoh adat, pemerintah, dan orang tua. Dalam pasal yang kedua maksudnya yaitu dalam kehidupan manusia itu harus dijaga, diperbaiki, dimuliakan, dan diagungkan. Karena hukum kemanusiaan itu sangat penting dan harus diakui sebagai bentuk upaya menegakkan hukum dan kebenaran dalam menjalin harmonisasi dalam bermasyarakat. Maksud dari pasal ketiga, makna konotasinya ialah bahwa kulit itu menutupi aib seseorang dari kesalahan yang dilakukan agar tidak tercemar nama baik seseorang. Maka harus dijaga dan tidak boleh dinodai dengan fitnah dan untuk 271 Volume 2, nomor 2, September 2022 Rahmah Muharromah Yasin menebus kesalahan orang harus saling memberi hadiah atau barang berharga lainnya kepada sesama. i. Al-istiqâmah Dari pasal keempat yaitu darah yang mengalir dalam tubuh atau darah yang membeku didalam perut. Maksudnya itu manusia tidak boleh disakiti atau dilukai baik secara sengaja maupun tidak sengaja. Oleh karenanya perilaku sewenang- wenang terhadap sesama itu sangat fatal dan mendapat pelanggaran yang sangat keras. Dalam pasal kelima ini artinya sebuah kamar yaitu dalam kamar tidur pasangan suami-istri tidak boleh semua orang memasukinya. Semua ada batas-batasnya. Seperti yang digambarkan di dalam sebuah rumah yang mempunyai bagian-bagian tertentu. Batas-batas tersebut tidak boleh dikunjungi oleh orang lain. Contohnya batasan-batasan pergaulan antara laki-laki dan wanita yang bukan muhrimnya, sehingga tempat wanita itu harus dijaga. Kemudian makna dari pasal keenam yaitu perbedaan antara tempat tidur seseorang yang sudah nikah ataupun yang belum nikah. Dalam perbedaan itu sangat dibedakan. Contohnya seorang wanita yang masih gadis atau yang belum nikah. Tidak boleh dimasuki oleh laki-laki yang bukan muhrim. Sesuai dengan hukum filosofi yang ada, bahwa pernikahan itu tidak boleh dinodai, pernikahan yang suci merupakan bukti suatu ikatan lahir batin yang harus dijaga dan dipelihara. Dalam hal ini masyarakat Kei sangat menjunjung tinggi martabat dan keluhuran kaum wanita. Dan terakhir maksud dari pasal ketujuh yaitu seseorang adalah milik dia sendiri dan diri kita sendiri tetap menjadi milik kita. Pesan ini lebih mengakui dan mengatur terhadap hak-hak seseorang agar tidak terjadi suatu kecurangan ataupun keadilan terhadap orang lain. Dan juga mengatur hubungan sosial tentang hak kepemilikan yang pada akhirnya menimbulkan rasa nyaman, aman, dan damai. Selanjutnya hukum Nevnev adalah hukum yang mengontrol pelanggaran terhadap masalah kriminalitas dan tindakan eksploitasi atau disebut juga hukum pidana. Hukum ini terdiri dari 7 larangan. Hukum ini biasa dikenal dengan sebutan Sasa sorfik hukum nevnev yaitu: a. Mu’ur nar-hebang haung, larangan membahas kelebihan dan kekurangan orang lain di hadapannya serta menyusun tindak kriminalitas terhadap orang lain b. Skut fngahir-suban med, larangan mencicik, mengutuk, dan mencurigai orang lain c. Rasung smu-rudang dad, larangan untuk meracuni sesama dan membunuh sesamanya dengan melalui ilmu tenung d. Kev bangil, larangan memukul d. Kev bangil, larangan memukul e. Tev ahai, fan-sung, tavat, larangan dalam menusuk menembak menikam memanah atau melempar sesama f. Fedan na-tetat vanga, larangan dalam memenggal dan membunuh sesama. g. Tivak, luduk fo vavain, larangan menguburkan seseorang atau menenggelamkan dengan cara hidup hidup. (Difinubun, 2012, h. 27-29). Hukum Hanilit yaitu hukum yang mengelola budi pekerti perbauran antara pria dan wanita. b. Kifuk mat ko, larangan berselingkuh dengan wanita i. Al-istiqâmah Hukum ini berisi tentang etika dan moralitas yang terdiri dari 7 larangan atau yang dikenal dengan sebutan sasa sorfik hukum hanilit yakni: Hukum Hanilit yaitu hukum yang mengelola budi pekerti perbauran antara pria dan wanita. Hukum ini berisi tentang etika dan moralitas yang terdiri dari 7 larangan atau yang dikenal dengan sebutan sasa sorfik hukum hanilit yakni: a. Sis, af, larangan mendesis bersiul dan memikat wanita b. Kifuk mat ko, larangan berselingkuh dengan wanita ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 272 Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an c. Kis kafir, temar u, larangan mengorek, mencubit, dan menghilir anak-anak bila berjalan dengan wanita c. Kis kafir, temar u, larangan mengorek, mencubit, dan menghilir anak-anak bila berjalan dengan wanita d. A lebak, humak voan, larangan mencium dan memeluk wanita e. Tod es, larangan menarik dan memperkosa wanita f. Marvuan fa ivun, larangan membuntingkan wanita di luar nikah g. Manu’u marai, larangan merampas istri orang dan melaksanakan kawin lari. (Difinubun, 2012, h. 29). Dan terakhir hukum Hawear Balwirin adalah hukum yang mengelola hak seseorang atau kelompok atau disebut juga hukum perdata. Hukum ini terdiri dari 7 larangan yang disebut dengan sa sorfit hukum hawear balwirin yaitu: g y g g f y a. Varyatad sa, larangan memimpikan barang orang lain b. Tafbor, dilarang mencuri b. Tafbor, dilarang mencuri b. Tafbor, dilarang mencuri c. It kulik afa borbor, larangan menutupi barang jarahan d. It ba maren, it dad afa waid, larangan hadir dalam kegiatan tanpa ikut berpartisipasi e. It leik hira ni afa, tef en tana il, larangan menjumpai barang tanpa mengembalikannya f. It lavur hira ni afa, larangan merusak hak orang lain g. Taha kuuk umat lian rir welmat, larangan menunda utang sesame. (Difinubun, 2012, h. 29). Pasal-pasal tentang larangan-larangan (Sasa Sorfit) di atas dapat di ringkas njadi: a) Sasa Sorfit hukum Nev-Nev, tujuh tingkat larangan tentang kehidupan pasal 1, 2, 3, dan 4 yang mematuhi pimpinan, jangan melukai orang, jangan membunuh orang, dan menjaga kerahasiaan. b) Sasa Sorfit hukum Hanilit, tujuh tingkat larangan tentang melanggar kehormatan kaum wanita, pasal 5 dan 6 yakni menjaga batas-batas atau sekat dan tidak boleh masuk sembarangan di tempat perempuan yang sudah berkeluarga maupun wanita bujangan. i. Al-istiqâmah c) Sasa Sorfit hukum Hawear Balwirin, tujuh tingkat larangan tentang hak dan kewajiban, pasal 7 yakni tidak melakukan kewajiban/tidak membantu orang ketika diminta (Maren/gotong royong), mengambil hak orang lain yang bukan miliknya (seperti mencuri, korupsi), dan tidak membayar hutang jika berhutang. Selain hukum adat Larvul Ngabal, ada juga falsafah Ain ni Ain sebagai falsafah masyarakat Kei yang sudah terun-temurun dilaksanakan dari para leluhur. Secara historis belum ada naskah yang jelas tentang siapa yang mencetuskan falsafah ini. Sebelum masuknya agama Islam dan Kristen di tanah Kei, falsafah ini sudah ada dan tersebar luas. (Wawancara Raja Fer, Mei 2021). Ain dalam bahasa Kei artinya ‘satu’. Makna satu dalam konteks ini yakni jamak, bukan tunggal. Karena di dalam bahasa Kei, satu tunggal adalah sa, seperti ain-sa, ain-ru, ain-tel, ain-fak, ain-lim (satu-satu (1), satu-dua (2), satu-tiga (3), satu-empat (4), satu-lima (5), dan seterusnya). Dan Ni artinya ‘memiliki, mempunyai’. Jadi ain ni ain secara harfiah artinya satu memiliki satu. Seseorang atau sekelompok orang dalam memandang orang lain (liyan) seperti saudanya sendiri. Atau dalam artian lain, ain yang asli menerima ain yang lain dianggap sebagai saudaranya. (Perda Kota Tual, 2020). Ain ni Ain memiliki padanan yang sama dengan falsafah lar in baba (darah mengalir) wer in soso (tarik dari atas) yang artinya mempunyai hubungan darah secara horizontal dan vertikal. Kedua falsafah ini saling berkaitan satu sama lain. Ain 273 Volume 2, nomor 2, September 2022 Rahmah Muharromah Yasin ni ain menjadi suatu falsafah hidup masyarakat Kei dalam kelangsungan hidup bersama. Karena melalui falsafah ini masyarakat akan sadar bahwa dirinya semuanya berasal dari satu darah/hubungan. Hubungan ini terwujud dalam pepatah masyarakat Kei vu’ut ain mehe ngifun, manut ain mehe tilur. Yang memiliki makna banyaknya telur dari seekor ikan dan seekor ayam (burung) yang sama, sama halnya dengan ibarat orang Kei yang memiliki hubungan dari satu telur ikan atau satu telur burung. Hubungan persaudaraan selalu tetap dijaga baik-baik dan tidak boleh dicerai-beraikan. Kultur dan aktualisasi dari falsafah Ain ni Ain terdiri dari dua tradisi yang selalu dilaksanakan oleh masyarakat Kei agar tetap terpelihara, yakni tradisi Maren dan Yelim. Kedua kultur ini dituangkan dalam berbagai kegiatan sosial budaya yakni dalam pernikahan adat, perayaan keagamaan, pembukaan lahan perkebunan, situasi duka ataupun pembangunan. Kedua adat ini ini dilaksanakan secara bersamaan atau secara bergilir karena saling berkaitan. Moderasi beragama merupakan jalan/usaha dalam menjunjung tinggi ajaran agama, landasan dalam menciptakan kesejahteraan hidup dan dijadikan sebagai karakter dalam bersosialisasi. ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 274 i. Al-istiqâmah Di dalam surah ini disebutkan bagaimana cara terbaik kita dalam hidup berdampingan untuk menciptakan suasana aman dan harmonis. Sesuai dengan resepsi fungsional ini, masyarakat memakai konsep moderasi yang sejalan dengan tradisi masyarakat melalui hukum adat Larvul Ngabal dan filosofi Ain ni ain. Apabila di kaitkan dengan hukum-hukum syariat Islam sangat tepat. Di dalam pasal-pasal hukum Larvul Ngabal tersebut jelas bahwa kerukunan dan moderasi beragama dalam masyarakat Kei telah terbentuk dan terpola sejak lama. Dapat pula dilihat bahwa hukum Larvul Ngabal dan filosofi Ain ni ain bersesuaian dengan kaidah-kaidah agama apapun, dimana setiap agama mengatur tentang hak hidup manusia, hak penghormatan terhadap pemimpin, orang tua, hak penghormatan terhadap kaum perempuan, larangan membunuh, dan juga hak tentang kepemilikan yang tidak boleh diganggu. Kita dapat melihat dari dalam QS. an-Nisa [4] ayat 92 dalam larangan membunuh (hukum Nev-Nev), QS. an-Nisa [4] ayat 34, QS. an-Nahl [16] ayat 72 tentang hak penghormatan kaum perempuan (hukum Hanilit), QS. Az- Zukhruf [43] ayat 32 tentang hak kepemilikan (hukum Hawear Balwirin). (Wawancara Plt. Kandepag Tual, April 2021). p g p Karakteristik kearifan lokal hukum adat Larvul Ngabal di kepulauan Kei sangat efektif dalam menyelesaikan masalah sosial yang menjadi peran penting dalam melihat bagaimana adat kepulauan Kei berperan dalam membentuk kerukunan antar umat beragama. Pasal-pasal dalam hukum adat Larvul Ngabal yang diterapkan dalam penyelesaian konflik agama yang pernah terjadi di Kepulauan Kei di atas adalah pasal 1 sampai dengan pasal 4 yang berkaitan dengan kehidupan manusia. Pasal-pasal ini dengan tegas menggarisbawahi “rasa hormat” yang harus dijaga dalam hidup bersama. Di dalam pasal 1 ditekankan bagaimana pentingnya menghormati orang yang lebih tua. Dalam hal ini penghormatan kepada orang tua diberikan tanpa melihat latar belakang suku, ras maupun agama dari orang yang dihormati. Rasa hormat terhadap orang lain terutama orang yang lebih tua, menjadi nilai yang selalu ditanamkan oleh orang tua kepada anaknya sejak kecil dan ini menjadi salah satu nilai adat yang masih dipertahankan sampai saat ini. Kaitannya dalam syariat Islam ada dalilnya dengan pasal ini seperti dalam QS. Luqman [31] ayat 14-15. Di dalam ayat tersebut kita diharuskan untuk berbuat baik dengan orang yang lebih tua sekalipun dia non muslim, kemudian dalam QS. An-Nisa [4] ayat 36 kita dianjurkan untuk berbuat baik kepada kerabat, teman sejawat, ataupun kepada tetangga kita semua. Selanjutnya pasal 2 hukum adat Larvul Ngabal mendeskripsikan bagaimana masyarakat Kei menempatkan rasa hormat terhadap eksistensi kehidupan manusia terutama terhadap diri sendiri. Ini juga terdapat dalam QS. i. Al-istiqâmah Ada tiga alasan diperlukannya pemikiran moderat dalam beragama, yakni pertama untuk menyeimbangkan ajaran agama dalam aspek kehidupan khususnya dalam hubungan sosial. Kedua mengatasi konflik/perdebatan berlatar agama. Dan ketiga merawat keragaman Indonesia yang memiliki banyak ragama budaya dan agama. g y g Ada tiga hal yang penting untuk menjadi sebuah prinsip dasar moderasi beragama yaitu adil, berimbang dan toleransi. Sebagaimana yang sudah dijelaskan di atas, bahwa makna ayat-ayat wasathiyah terdiri dari nilai keadilan (‘adalah), keseimbangan (tawazun), dan toleransi (tasamuh). Ketiga nilai-nilai ini semua terkandung di dalam hukum adat Larvul Ngabal dan filosofi Ain ni Ain. Seperti hukum Hawear Balwirin atau hukum keadilan dalam bersosialisai, hukum Hanilit atau hukum perilaku dan penghargaan terhadap wanita, hukum Nev-Nev yang memuat hukum pidana/nilai kemanusian. Serta nilai tasamuh yang terdapat dalam filosofi Ain ni Ain. Dalam pelaksanaan hukum adat ini, tak luput juga terdapat maqashid syari’ahnya, contohnya dalam melindungi harta (hukum Hawear Balwirin), melindungi keturunan (hukum Hanilit) dan melindungi jiwa (hukum Nev-Nev). g g j Moderasi beragama yang hadir di kota Tual khususnya di Dullah Utara untuk kalangan awam belum dikenal secara meluas, istilah ini hanya diketahui oleh kalangan akademisi maupun di dalam lingkup Kementerian Agama. Tetapi kalau dijelaskan maknanya, semua masyarakat akan paham maksud dari moderasi beragama ini. Yang mereka ketahui bahwa makna moderasi beragam itu sebuah toleransi antar umat beragama. Dan semua maknanya itu terkandung di dalam hukum adat masyarakat Kei (Larvul Ngabal). Bahwa disini kita diperintahkan dalam menghargai pemeluk agama lain, bukan mengikuti keyakinannya. p g g y y Istilah moderasi ini masih terdengar awam di kalangan para masyarakat umum, hanya beberapa kalangan seperti di akademisi dan lingkungan kemenag. Selain dari hasil wawancara, data yang dipakai oleh peneliti juga menggunakan metode angket. Dari hasil analisis angket dinyatakan bahwa hanya 77,1% yang tahu istilah moderasi beragama, sisanya 22,9% yang baru mengenal/tahu dengan istilah moderasi beragama. Hal ini dikarenakan istilah moderasi muncul beberapa tahun belakangan ini. Yang menjadi salah satu program RPJMN 2020-2024. Selain di dalam QS. al-Baqarah [2] ayat 143 tentang moderasi beragama, terdapat juga di dalam QS. al-Kafirun [109] ayat 6 yang menjelaskan bagaimana menciptakan hubungan harmonis dalam kehidupan masyarakat plural tanpa pencampuran ajaran agama lainnya. Adapun intisari kandungan dari surah ini yaitu ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 274 Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an menanggapi usulan tokoh-tokoh kaum musyrik untuk berkompromi dalam segi aqidah. i. Al-istiqâmah Ibrahim [14] ayat 7, kita dianjurkan bersyukur akan nikmat kehidupan yang diberikan sama Allah. Penghargaan terhadap kehidupan dimaknai dengan sikap menjaga diri agar tidak melakukan aktivitas yang sia-sia dan merugi. Pasal 3 dan 4 hukum adat Larvul Ngabal menghendaki adanya hubungan yang harmonis antar sesama manusia. Pasal ketiga menjelaskan cara masyarakat Kei bersikap dan bertingkah laku yang baik antar sesama manusia, dan pasal empat lebih menitikberatkan tentang adanya tindakan kekerasan yang memicu konflik. Dari penjelasan pasal-pasal hukum adat di atas, bahwa sudah terlihat akan kesesuaian antara nilai-nilai adat dengan hukum syariat Islam yang berlaku di dalam Al-Qur’an, yang pada akhirnya sudah sejalan dengan ajaran agama yang baik yakni bersikap moderat. (Wawancara ketua MUI, April 2021). Pelaksanaan moderasi beragama di Dullah Utara ini tak luput dari pengaruh dasar-dasar agama yakni Al-Qur’an, selain dari hukum adat. Karena agama merupakan peletak/dasar hukum atau aturan yang dibuat oleh manusia. Dan tidak 275 Volume 2, nomor 2, September 2022 Rahmah Muharromah Yasin boleh melenceng dari syariat agama. Dari konteks fenomenologinya yaitu ketika adanya diadakan kegiatan maren, maka anggotanya setiap selesai kegiatan diadakan doa bersama salah satunya dengan pembacaan surah al-Fatihah yang bertujuan agar pelaksanaan kegiatan maren berjalan dengan lancar dan mencapai tujuan yang diinginkan. Disini dapat dilihat bahwa moderasi beragama dalam resepsi masyarakat Dullah Utara yakni cara beragama yang moderat, atau cara ber-Islam yang inklusif atau sikap beragama yang terbuka, tidak bertukar-menukar keyakinan, saling menghargai dalam toleransi, seimbang dan adil. Selain dari hasil wawancara, peneliti memperoleh hasil presentase dari para responden dalam bentuk angket tentang bentuk-bentuk moderasi beragama yang terjadi di lingkungan sekitar. a. Bebas menjalankan ibadah tanpa adanya pengaruh dari agama lain. Responden menyatakan baik dengan presentase 91,7% dan responden menyatakan cukup dengan presentase 8,3%. b. Bakti sosial atau gotong royong (maren). Responden menyatakan baik dengan presentase 89,6% dan responden menyatakan cukup dengan presentase 10,4%. c. Menghormati dan menghargai pemeluk agama lain. Responden menyatakan baik dengan presentase 91,7% dan responden menyatakan cukup dengan presentase 8,3%. d. Tidak ada paksaan dalam hal keyakinan. Responden menyatakan baik dengan presentase 91,7% dan responden menyatakan cukup dengan presentase 8,3%. e. Tidak ada larangan dalam membangun rumah ibadah. Responden menyatakan baik dengan presentase 91,7% dan responden menyatakan cukup dengan presentase 8,3%. f. Tradisi agama dapat dilaksanakan dengan baik. Responden menyatakan baik dengan presentase 89,6% dan responden menyatakan cukup dengan presentase 10,4%. g. Pejabat setempat dan tokoh adat sigap dengan masalah masyarakat. i. Al-istiqâmah Responden menyatakan baik dengan presentase 85,4% dan responden menyatakan cukup dengan presentase 14,6%. Dari hasil angket di atas, dapat disimpulkan bahwa pelaksanaan atau implikasi moderasi beragama sudah sangat baik karena adanya kerjasama antara pemerintah dan semua pihak dalam loyalitasnya dengan hukum adat setempat yakni Larvul ngabal dan filosofi Ain ni ain. Peneliti juga mengambil sampel dari beberapa angket yang telah dikumpulkan mengenai tingkat pengetahuan dan pemahaman responden terhadap ayat-ayat tentang moderasi dalam Al-Qur’an. Adapun hasilnya masih minim, hanya diketahui oleh beberapa responden dari kalangan akademisi saja. Diketahui jumlah responden yang memiliki pengetahuan mengenai ayat-ayat moderasi berjumlah 20,8% (10 orang) dan sisanya berjumlah 79,2% (38 orang) yang tidak mengetahui tentang ayat-ayat wasaṭiyyah. Ini bisa di simpulkan bahwa pengetahuan akan penafsiran Al-Qur’an di masyarakat umum masih kurang/minim. Kesimpulan Moderasi beragama dalam resepsi masyarakat Dullah Utara yakni cara beragama yang moderat, atau cara ber-Islam yang inklusif atau sikap beragama yang terbuka, tidak bertukar-menukar keyakinan, saling menghargai dalam toleransi, perdamaian dan keadilan. Dari hasil wawancara, peneliti dapat menyimpulkan bahwa ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 276 Resepsi Masyarakat Dullah Utara terhadap Konsep Moderasi Beragama Perspektif Al-Qur’an di dalam pasal-pasal hukum Larvul Ngabal dan filosofi Ain ni ain tersebut jelas bahwa kerukunan dan moderasi beragama dalam masyarakat Kei telah terbentuk dan terpola sejak lama. Dapat pula dilihat bahwa hukum adat dan filosofi tersebut bersesuaian dengan hukum syariat Islam khususnya ayat-ayat mengenai moderasi, seperti menghargai sesama muslim maupun non muslim, saling gotong-royong (maren), toleransi (tasamuh), tidak memihak siapapun asal dia masih dalam kebenaran (tawazun). Serta nilai-nilai etika, nilai kejujuran, keadilan (‘adalah), persatuan, musyawarah dan kerjasama. Pola dan bentuk kerukunan ini telah dipegang oleh masyarakat secara turun temurun sejak leluhur, yaitu suatu pola yang menggambarkan peran dan hubungan yang kuat antara agama dan adat. Pola ini sendiri dapat terlihat jelas dalam beberapa aktifitas masyarakat Kei baik dalam upacara adat maupun keagaman. Di dalam nilai adat tersebut peneliti menilai bahwa konflik yang terjadi pun sangat cepat mereda, karena adanya hubungan kerjasama yang sangat baik dan juga kepercayaan yang tinggi terhadap adat istiadat yang dipegang oleh masyarakat di kecamatan Dullah Utara. Pelaksanaan moderasi beragama di kecamatan Dullah Utara sudah terjalin dengan sangat baik, ini karena didukung oleh peran pemerintah dan masyarakat kecamatan Dullah Utara itu sendiri melalui hukum adat yang berlaku di masyarakat Kei, yakni hukum adat Larvul ngabal dan filosofi Ain ni ain. Walaupun pemeluk agama di kecamatan Dullah Utara didominasi oleh muslim, tetapi ini tidak ada pengaruhnya terhadap sikap tasamuhnya umat muslim dengan pemeluk agama lain (non muslim). Interaksi masyarakat dalam beragama sangat kuat karena adanya loyalitas tinggi terhadap hukum adat dan filosofi tersebut. Learner’s Dict Oxford Learner’s Dictionaries. https://www.oxfordleanersdictionaries.com/definition/english/extreme- _1?q=Extreme taken date 11 Januari 2021. Daftar Pustaka tahqiq: Abdul Salam Harun, Kitab al-Waw Bab Waw wa Siin Juz 5 Iran: Daar Al Kutub Al Ilmiyah _________. Mu’jam Maqayis al-Lughah. tahqiq: Abdul Salam Harun, Kitab al-Waw Bab Waw wa Siin, Juz 5, Iran: Daar Al-Kutub Al-Ilmiyah. Gunawan, Wawan (2015). Fikih Kebhinekaan, Pandangan Islam Indonesia tentang Umat, Kewargaan, dan Kepemimpinan Non Muslim. Jakarta: Ma’arif Institut dan Mizan. Al-Jauzi, Muhammad. Zâd Al-Masir fî ‘Ulum At-Tafsir. Jilid 1. t.t.p: Al-Maktab Al- Islami. Kamali, Mohammad Hasyim (2015). The Middle Path of Moderation in Islam, the Qur’anic Principle of Wasaṭiyyah. Oxford: Oxford University Press. Katsir, Ibnu, Tafsir Al-Qur’an Al-‘Azim. tahqiq: Abdul Aziz Ghanim Hamd Ahmad Asyur. Muhammad Ibrahim Al-Bana. Jilid 3. Kairo: Mathba’ah Asy-Sya’ab. __________, Tafsir Al-Qur’an Al-‘Azim. tahqiq: Abdul Aziz Ghanim Hamd Ahmad Asyur. Muhammad Ibrahim Al-Bana. Jilid 4. Kairo: Mathba’ah Asy-Sya’ab. KBBI Online. https://kbbi.kemdikbud.go.id/entri/Toleransi taken date 15 Januar 2021. Daftar Pustaka Amin, Muhammad dan M. Arfah Nurhayat (2020). Resepsi Masyarakat Terhadap Al- Qur’an (Pengantar Menuju Metode Living Qur’an). Jurnal Ilmu Agama. Vol. 21 No. 2. ‘Asyur, Muhammad Thahir Ibn (1984). At-Tahrir wa Tanwîr. Juz 3. Tunisia: Dâr Al Kutub Asy-Syarqiyah. y y q y Al-Asfahani (2009). Mufrodat li al-Fâzil al-Qur’an. Damaskus: Darul Qalam. y y q y Al-Asfahani (2009). Mufrodat li al-Fâzil al-Qur’an. Damaskus: Darul Qalam. Al-Asqalani, Ibnu Hajar (1990). Fathul Bâri bi Syarh Shahih Al-Bukhari. tashih: Abdul Aziz bin Baz. Vol. 12. Beirut: Dar Al-Fikr. Al-Asqalani, Ibnu Hajar (1990). Fathul Bâri bi Syarh Shahih Al-Bukhari. tashih Abdul Aziz bin Baz. Vol. 12. Beirut: Dar Al-Fikr. Aṭ- Ṭabari (1994). Tafsir Aṭ- Ṭabari. Juz 1. Beirut: Muassasah Ar-Risalah. ______________. Tafsir Aṭ- Ṭabari. Juz 2. Beirut: Muassasah Ar-Risalah. ______________. Tafsir Aṭ- Ṭabari. Juz 10. Beirut: Muassasah Ar-Risalah. Aṭ- Ṭabari (1994). Tafsir Aṭ- Ṭabari. Juz 1. Beirut: Muassasah Ar-Risalah. ______________. Tafsir Aṭ- Ṭabari. Juz 2. Beirut: Muassasah Ar-Risalah. ______________. Tafsir Aṭ- Ṭabari. Juz 10. Beirut: Muassasah Ar-Risalah. Bagir, Haidar, Islam Tuhan Islam Manusia Agama dan Spritualitas di Zaman Kacau, Bandung: PT Mizan Pustaka, 2019. g Bhayangkara, Pers. https://persbhayangkara.id/2021/06/11/soal-penyelesaian- pertikaian-akhirnya-2-desa-di-maluku-tenggara-sepakat-berdamai-lewat- pendekatan-adat/ taken date 6 September 2022. g Bhayangkara, Pers. https://persbhayangkara.id/2021/06/11/soal-penyelesaian- pertikaian-akhirnya-2-desa-di-maluku-tenggara-sepakat-berdamai-lewat- pendekatan-adat/ taken date 6 September 2022. Al-Biqa’i (2006). Nudzm ad-Durâr fî Tanâsub al-Ayat wa al-Suwâr. Beirut: Dâr Al Kutub al-‘Ilmiyyah. Bisri, Mohammad (2019). Moderasi Beragama untuk Kebersamaan Umat. Majalah Sejahtera Edisi 1. Tahun V. Januari-Maret 2019. Subbag Informasi Kanwil Kemenag Provinsi Jawa Tengah. Bisri, Mohammad (2019). Moderasi Beragama untuk Kebersamaan Umat. Majalah Sejahtera Edisi 1. Tahun V. Januari-Maret 2019. Subbag Informasi Kanwil Kemenag Provinsi Jawa Tengah. BPS Kabupaten Maluku Tenggara (2020). Kecamatan Pulau Dullah Utara Dalam Angka 2020. Langgur: CV. Aman Jaya. BPS Kabupaten Maluku Tenggara (2020). Kecamatan Pulau Dullah Utara Dalam Angka 2020. Langgur: CV. Aman Jaya. Volume 2, nomor 2, September 2022 277 Rahmah Muharromah Yasin Difinubun, Ahmad (2016). Sinopsis Mengenal Masyarakat Adat Kei. Tual: Dewan Adat Rat Loor Kepulauan Kei. Faris, Ibnu. Mu’jam Maqayis al-Lughah. tahqiq: Abdul Salam Harun, Kitab al-Waw Bab Waw wa Siin Juz 3 Iran: Daar Al-Kutub Al-Ilmiyah Faris, Ibnu. Mu’jam Maqayis al-Lughah. tahqiq: Abdul Salam Harun, Kitab al-Waw Bab Waw wa Siin, Juz 3, Iran: Daar Al-Kutub Al-Ilmiyah. _________. Mu’jam Maqayis al-Lughah. tahqiq: Abdul Salam Harun, Kitab al-Waw Bab Waw wa Siin, Juz 5, Iran: Daar Al-Kutub Al-Ilmiyah. _________. Mu’jam Maqayis al-Lughah. . https://kbbi.web.id/moderasi taken date 10 Januari 2021. _. https://kbbi.web.id/moderasi taken date 10 Januari 2021. Al-Khawarizmi, Az-Zamakhsyari. Al-Kasyaf. Juz 1. Riyadh: Maktabah Al-Ma’arif. Mukrim, Jamaluddin Muhammad bin dan Ibnu Munzhur (1985). Lisan Al-‘Arab. Vol 15. Beirut: Daar Al-Ihya Turath Al-‘Arabi. 5. e ut: aa ya u at ab . Nasional, Kompas. https://nasional.kompas.com/read/2021/01/19/20223241/jokowi- teken-perpres-7-2021-pemerintah-bakal-kembangkan-daerah-percontohan taken date 2 Februari 2021. Oxford Learner’s Dictionaries. https://www.oxfordleanersdictionaries.com/definition/english/extreme- Nasional, Kompas. https://nasional.kompas.com/read/2021/01/19/20223241/jokowi- teken-perpres-7-2021-pemerintah-bakal-kembangkan-daerah-percontohan taken date 2 Februari 2021. Learner’s https://www.oxfordleanersdictionaries.com/definition/english/extreme- _1?q=Extreme taken date 11 Januari 2021. Peraturan Daerah Kota Tual. Nomor 4 Tahun 2020 tentang Ratshap, Ohoi, dan/atau Finua. Pasal 1 No. 30. Peraturan Daerah Kota Tual. Nomor 4 Tahun 2020 tentang Ratshap, Ohoi, dan/atau Finua. Pasal 1 No. 30. Al-Qasimi, Muhammad Jamaluddin (1978). Tafsir Al-Qasimi. Cet. II. Jilid 3. Bairut Daâr Al-Fikr. Al-Qasimi, Muhammad Jamaluddin (1978). Tafsir Al-Qasimi. Cet. II. Jilid 3. Bairut: Daâr Al-Fikr. Al-Qurṭubi (1987). Al-Jami’ li Ahkam Al-Qur’an. Cet. III. Jilid 6. Kairo: Dâr Al Kutub Al-‘Arabiy. Al-Qurṭubi (1987). Al-Jami’ li Ahkam Al-Qur’an. Cet. III. Jilid 6. Kairo: Dâr Al- Kutub Al-‘Arabiy. Quthb, Sayyid (1992). Fî Zhilalil Al-Qur’an. Cet. XVII. Jilid 1. Beirut: Dâr Al Syuruq. Ridha, Muhammad Rasyid. Tafsir Al-Manar. Cet. Ke-II. Beirut: Dâr Al-Manar. Shihab, M. Quraish (2019). Wasaṭiyyah Wawasan Islam Tentang Moderasi Beragama. Ciputat, Lentera Hati. (2017) Wawasan Al Qur’an: Tafsir Mudhu’i atas Berbagai Shihab, M. Quraish (2019). Wasaṭiyyah Wawasan Islam Tentang Moderasi Beragama. Ciputat, Lentera Hati. __________________ (2017). Wawasan Al-Qur’an: Tafsir Mudhu’i atas Berbagai Persoalan Ummat. Bandung: Mizan. Beragama. Ciputat, Lentera Hati. __________________ (2017). Wawasan Al-Qur’an: Tafsir Mudhu’i atas Berbagai Persoalan Ummat. Bandung: Mizan. Tim Penyusun Kamus Bahasa (2002). Kamus Besar Bahasa Indonesia. Edisi 3. Jakarta: Balai Pustaka. Tirto. https://www.google.com/amp/s/amp.tirto.id/daftar-skor-indeks-kerukunan- beragama-versi-kemenag-2019-engH taken date 14 Desember 2020. ULUMUL QUR’AN: JURNAL ILMU AL-QUR’AN DAN TAFSIR 278
https://openalex.org/W3139373992	https://publishing.logos-science.com/index.php/books/article/download/192/191	Ukrainian	null	КОМУНІКАТИВНИЙ ПІДХІД У НАВЧАННІ ГРАМАТИКИ СТУДЕНТІВ ТЕХНІЧНИХ СПЕЦІАЛЬНОСТЕЙ	null	2,021	cc-by-sa	3,229	Опубліковано 16 березня 2021 року Опубліковано 16 березня 2021 року СЕМИДА Оксана Володимирівна СЕМИДА Оксана Володимирівна канд. філол. наук, доцент кафедри англійської мови технічного спрямування № 1 Національний технічний університет України «Київський політехнічний інститут імені Ігоря Сікорського» КРАВЧЕНКО Тетяна Василівна викладач кафедри англійської мови технічного спрямування №1 Національний технічний університет України «Київський політехнічний інститут імені Ігоря Сікорського» УКРАЇНА канд. філол. наук, доцент кафедри англійської мови технічного спрямування № 1 Національний технічний університет України «Київський політехнічний інститут імені Ігоря Сікорського» канд. філол. наук, доцент кафедри англійської мови технічного спрямування № 1 Національний технічний університет України «Київський політехнічний інститут імені Ігоря Сікорського» КРАВЧЕНКО Тетяна Василівна викладач кафедри англійської мови технічного спрямування №1 Національний технічний університет України «Київський політехнічний інститут імені Ігоря Сікорського» УКРАЇНА Анотація: Стаття присвячена особливостям реалізації комунікативного підходу у навчанні граматики студентів технічних спеціальностей. У статті розкрито поняття граматичної компетентності та визначено її компоненти. Розглянуто різні підходи до навчання граматики, їхні переваги та недоліки. Охарактеризовано особливості комунікативного підходу у вивченні іноземних мов та принципи його реалізації при навчанні граматики студентів технічних спеціальностей, наведено етапи ефективної презентації граматичних конструкцій. Розглянуто види вправ та завдань при комунікативному навчанні граматики з урахуванням принципу професійно- комунікативної спрямованості. Визначено роль та функції викладача при організації комунікативного навчання, а також охарактеризовано комунікативну форму контролю засвоєння граматичного матеріалу. Наведено приклади завдань для засвоєння граматичних структур з урахуванням принципу комунікативності. © Семида О.В., Кравченко Т.В., 2021 Розділ дев’ятий Розділ дев’ятий 96 DOI 10.36074/rodmmrfssn.ed-1.09 УДК 378 ВСТУП. На сьогодні вивченню іноземних мов, зокрема, англійської, приділяється багато уваги, оскільки це пов'язано з інтеграційними процесами, в яких приймає участь наша країна, з розвитком програм міжнародного співробітництва, у яких студенти можуть приймати участь як під час навчання, так і у подальшій професійній кар'єрі. Тому, на сьогодні першочерговим є завдання якісної іншомовної підготовки © Семида О.В., Кравченко Т.В., 2021 Колективна наукова монографія DOI 10.36074/rodmmrfssn.ed-1.09 \| 97 97 майбутніх технічних фахівців. З цією метою постійно проводяться дослідження, здійснюються пошуки нових методів та підходів у викладання іноземних мов. Серед різних аспектів методики викладання мови, учені приділяють значну увагу дослідженню питання навчання граматики та формуванню граматичної компетентності, розглядаючи опанування граматичною системою мови як невід’ємну складову опанування мовою. Розробка комунікативного підходу у навчанні іноземної мови постій но знаходиться у фокусі методистів-дослідників, дослідження цього питання продовжують бути актуальними на сьогодні, оскільки залишаються малодосліджені аспекти цього питання. Зокрема, залишається актуальним дослідження питання формування англомовної граматичної компетентності у студентів немовних спеціальностей. Мета наших наукових розвідок полягала в узагальненні та систематизації особливостей навчання граматики при реалізації комунікативного підходу у немовних ЗВО, та у визначенні умов успішного формування професійно-орієнтованої іншомовної граматичної компетенції. З цією метою було розглянуто різні підходи до навчання граматики; розглянуто поняття комунікативної та граматичної компетентностей; проаналізовано особливості навчання граматики при комунікативному підході до навчання; виокремлено основні форми взаємодії студентів на занятті метою підвищення ефективності засвоєння граматичного матеріалу. © Семида О.В., Кравченко Т.В., 2021 ОСНОВНА ЧАСТИНА. Під граматичною компетентністю розуміється “здатність людини до коректного граматичного оформлення своїх усних і писемних висловлювань та розуміння граматичного оформлення мовлення інших. Ця компетентність базується на складній і динамічній взаємодії відповідних навичок і знань та граматичної усвідомленості” [1]. До основних компонентів граматичної компетентності вчені відносять граматичні навички, знання та граматичну усвідомленість, а механізмом формування граматичної компетентності є різні види вправ [2]. Окрім цього вчені вивчають різні фактори, що впливають на формування тієї чи іншої компетентності [3]. Так, О. Касаткіна у своїх дослідженнях відзначає важливість урахування соціокультурного фактору (соціокультурних норм та національно-культурних особливостей носіїв іншої мови) при © Семида О.В., Кравченко Т.В., 2021 Розділ дев’ятий DOI 10.36074/rodmmrfssn.ed-1.09 98 формуванні граматичної компетентності [4]. Т. Стеченко оперує поняттям професійно орієнтованої англомовної граматичної компетентності та наголошує на “професіоналізації” занять при формуванні граматичної компетентності, маючи на увазі професійну орієнтованість мовного навчального матеріалу [5]. Перш за все, при вивченні питання комунікативного навчання граматики, вважаємо за необхідне розглянути основні підходи до навчання граматики у методиці викладання мов. Одним із найдавніших підходів до навчання граматики іноземної мови є граматико- перекладний метод. У його назві відображено основні напрямки видів діяльності — засвоєння і подальше закріплення граматичних структур шляхом перекладу. Прихильниками та розробниками такого підходу були І. Мей дингер, Г. Оллендорф. Особливості цього підходу полягали у засвоєнні граматичних правили та виключень, а також у роботі з оригінальними текстами з метою перекладу. Мовний матеріал брався із писемних літературних джерел. Типовий урок, що базувався на цьому методі, складався зі вступної частини (презентація граматичного матеріалу), тренувального етапу та виконання практичних завдань на переклад [6]. До недоліків цього методу вчені відносять відсутність продуктивного спілкування, недостатність створення умов для самостійного засвоєння матеріалу. Іншим популярним методом навчання граматики був аудіо- лінгвальний метод, запропонований Ч. Фрізом та Р. Ладо в США в 40-50-і роки XX ст. Розроблена ними методика роботи з граматичними структурами полягала у заучуванні моделей шляхом імітації, інтенсивному тренуванню моделей та їх подальшому вільному вживанні. До недоліків цього методу вчені відносять відсутність креативності та мотивації при звичайному завчанні. Серед інших методів навчання мови можна назвати когнітивний, ситуативний тощо. Проте, зі зміною парадигми у навчанні іноземної мови, сучасним пріоритетом стало гасло “діючи, навчаємося”, що дало поштовх для розвитку та популяризації комунікативного підходу у навчанні іноземної мови. ОСНОВНА ЧАСТИНА. Метою комунікативного підходу у викладанні іноземної мови є формування комунікативної компетентності, яка включає вміння та знання щодо використання мови у різних ситуаціях, відповідно до обстановки та учасників; розуміння та використання різних типів текстів (розповіді, звіти, бесіди, інтерв’ю), вміння спілкуватися, маючи обмежені © Семида О.В., Кравченко Т.В., 2021 Колективна наукова монографія DOI 10.36074/rodmmrfssn.ed-1.09 \| 99 99 99 знання мови (різні стратегії спілкування). Розробниками комунікативного підходу у навчанні мов були Г. Уідоусан, У. Литлвуд, Г. Піфо, Ю. Пассов. Учені висувають такі принципи реалізації комунікативного підходу: мовленнєва спрямованість навчального процесу, яка полягає не стільки в тому, що ставиться мовленнєва практична мета, скільки в тому, що шляхом до цієї мети є саме практичне користування іноземною мовою; • індивідуалізація, орієнтація на особистість учня, створення мотивації та активності учнів з урахуванням їх життєвого досвіду, контексту діяльності, сфери інтересів, емоційної сфери і статусу конкретної особистості в колективі; • індивідуалізація, орієнтація на особистість учня, створення мотивації та активності учнів з урахуванням їх життєвого досвіду, контексту діяльності, сфери інтересів, емоційної сфери і статусу конкретної особистості в колективі; • функціональність, яка забезпечує відбір іншомовного матеріалу, адекватного процесу комунікації; • функціональність, яка забезпечує відбір іншомовного матеріалу, адекватного процесу комунікації; • ситуативність, що розглядається як засіб мовленнєвої стимуляції і як умова розвитку мовленнєвих навичок; • ситуативність, що розглядається як засіб мовленнєвої стимуляції і як умова розвитку мовленнєвих навичок; • новизна, яка проявляється в постійній зміні предмета розмови, обставин, завдань та ін [7]. • новизна, яка проявляється в постійній зміні предмета розмови, обставин, завдань та ін [7]. Як можемо побачити, велика увага приділяється реалізації індивідуального підходу до кожного студента під час навчання, акцент робиться не на результат, а на процес. Комунікативний підхід розглядається як викладання через комунікацію, а ситуація обирається/створюється в залежності від потреб студентів. Його основою є створення реальних життєвих ситуацій, та формування умінь і навичок застосовувати знання з іноземної мови в них. Слід відзначити, що така “симуляція” реальності в аудиторії сприяє підвищенню мотивації до навчання, усвідомленню важливості цього процесу та можливості практичного застосування набутих знань, оскільки ці знання одразу набуваються на практиці. Розвиток комунікативного підходу в методиці викладання вплинув і на методику навчання граматики іноземної мови. Суттю навчання граматики у межах цього підходу є вміння студентів самостійно дослідити мовний матеріал та вивести правила. Характерним є використання автентичних матеріалів, які містять культурну інформацію про цільову мову, занурення у реальний світ та відповідність професійним потребам студентів. При комунікативному підході вивчення мови здійснюється у процесі комунікації. © Семида О.В., Кравченко Т.В., 2021 ОСНОВНА ЧАСТИНА. Тобто, комунікативний підхід акцентує увагу на розвитку навичок спілкування, © Семида О.В., Кравченко Т.В., 2021 Розділ дев’ятий DOI 10.36074/rodmmrfssn.ed-1.09 100 розумінні граматичних правил та використанні їх у мові. Знання з граматики повинні бути усвідомлені, систематизовані, так щоб студенти могли їх побігло використовувати у мові. Звідси випливають наступні принципи формування граматичної компетентності при застосуванні комунікативного підходу: принцип врахування індивідуально- психологічних особливостей студентів; принцип комунікативної спрямованості навчання; принцип доступності; принцип поетапного формування іншомовних знань, умінь та навичок; принцип міждисциплінарного взаємозв’язку та взаємообумовленості різних компонентів в системі підготовки студентів; принцип особистісно- орієнтованого напрямку навчання; принцип мовленнєво-поведінкових стратегій; принцип врахування лінгвокультурологічних особливостей граматичних явищ. Крім того, О. Касаткіна наголошує, що система вправ, призначена для формування англомовної граматичної компетенції студентів, має містити соціокультурну інформацію, проблемні завдання культурознавчого характеру, які спонукають студентів до здійснення діалогу культур, до спілкування з представниками іншої культури [4]. При комунікативному підході до навчання граматики переважно використовується індуктивний підхід (надаються приклади, з яких виводяться правила вживання). Щодо форм роботи, то поширеними є парна робота, групова робота та дискусіı̈. Студентам можна запропонувати участь у навчальних іграх, розгадування головоломок, рольову гру, вирішення проблемноı̈ ситуаціı̈, участь у дискусіı̈, висловлення власноı̈ точки зору. Все це сприяє взаємодіı̈ студентів, під час якого відбувається опанування певними граматичними правилами [8]. Н. Прокопенко виокремлює наступні етапи з метою ефективної презентації та засвоєння граматичних конструкцій. Перший етап - це надання мовного зразка шляхом аналізу і синтезу граматичного явища. На цьому етапі студенти осмислюють граматичне явище, з’ясовують його зміст, форму та вживання. Ознайомлення з новим навчальним матеріалом для продуктивного засвоєння здійснюється найчастіше в навчально-мовленнєвих ситуаціях. Завданням викладача є створити умови для демонстрації форми і конструкції як динамічної одиниці, що має певну функціонально-комунікативну роль у спілкуванні. На другому етапі студенти тренуються у використанні виділеного граматичного фрагменту, відбувається формування граматичних мовленнєвих навичок. Навчання граматичній стороні мовлення, автоматизація © Семида О.В., Кравченко Т.В., 2021 Колективна наукова монографія DOI 10.36074/rodmmrfssn.ed-1.09 101 граматичної навички ефективно здійснюється через цілу низку тренувальних вправ. Учені зазначають, що навички спонтанної комунікації необхідно формувати за рахунок системи комунікативно- орієнтованих вправ. На третьому етапі відбувається вживання моделі, остаточне оформлення граматичних мовленнєвих умінь і навичок. Вправи на цьому етапі повинні мати, передусім, комунікативну цінність (комунікативні завдання, навчальні і природні мовленнєві ситуації, комунікативні ігри). Такі завдання повинні бути наближеними до реального спілкування, зверненими до самого студента та передбачати використання його власного життєвого досвіду [9]. ОСНОВНА ЧАСТИНА. Методисти-дослідники відзначають, що разом зі зміною підходу до навчання граматики необхідно змінити і форми контролю сформованості навички: якщо при комунікативному підході робиться акцент на спілкуванні і використанні структури у реальній ситуації, то саме це вміння і потрібно контролювати. Контролюється вміння виконувати дії та операції з граматичними структурами, тобто вміння користуватися граматикою задля здійснення мовленнєвої діяльності, уміння вибирати з усього обсягу засвоєних граматичних засобів лише ті, які відповідають комунікативному завданню, а не точність засвоєння граматичних структури. Це означає, що граматичні явища вивчаються та засвоюються не як «форми» і «структури», а як засоби вираження певних думок і комунікативних намірів, а також введення їх у мовленнєвий досвід. Саме тому за умов застосування комунікативного підходу, контроль також має бути комунікативно спрямованим [10]. Учених цікавить питання оцінки сформованості граматичної компетентності під час комунікативної діяльності. Опитування показали, що студенти і викладачі погоджуються з тим, що якщо вони зможуть використовувати граматичні структури в усному мовленні, в різник комунікативних ситуаціях, то й на письмі вони зможуть вірно застосовувати ці навички [11]. Викладачі зазначають, що контроль сформованості граматичних навичок в процесі комунікації є досить ефективним, а до мінусів такої форми контролю відносять те, що комунікативна діяльність займає багато часу. Завдання викладача полягає у створенні умов та ситуації, сприятливих для вивчення певного мовного матеріалу, у стимуляції пізнавальної активності студентів, актуалізації набутих знань, розвитку креативності та пізнання. О. Тарнопольський відзначає важливість © Семида О.В., Кравченко Т.В., 2021 Розділ дев’ятий DOI 10.36074/rodmmrfssn.ed-1.09 102 створення сприятливих умов для навчання, вказуючи, що створення позитивної мотивації у навчанні відноситься до головних функцій викладача. У своїх працях учений говорить, що для отримання кращого результату вправи повинні даватися у реальному комунікативному оточенні, базуватися на досвіді тих, хто вивчає мову, або викликати у них усвідомлення практичноı̈ необхідності того що вони роблять і вчать [12]. Дослідження показують, що саме досвід, необхідність та можливість подальшого застосування набутих знань, усвідомлення цього студентами, є запорукою успіху у навчанні та у формуванні позитивного ставлення. Постулюється принцип зв’язку навчання з життям, практикою, що передбачає залучення студентів до застосування набутих знань, навичок і вмінь на практиці та виконання ними завдань, які моделюють фрагменти май бутньоı̈ професій ноı̈ діяльності; використання життєвого досвіду студентів. У забезпеченні цих умов велику роль відіграє май стерність викладача. Саме викладач обирає матеріал за спеціальністю, тексти для опрацювання граматичного, лексичного матеріалу. Він прий має рішення стосовно прий омів, методів та форм роботи на занятті. ОСНОВНА ЧАСТИНА. Хоча при комунікативному підході до навчання акцент переноситься на студента, проте це не зменшує ні важливості, ні контролюючоı̈ функціı̈ викладача. Саме викладач регулює ступінь своєı̈ залученості/присутності на занятті, базуючись на своєму досвіді, професій ності та знанні психологічного налаштування групи, з якою він працює. Індивідуальний підхід здій снюється не лише до кожного студента, але до цілоı̈ групи студентів, як до єдиного цілого. “Присутність” викладача у роботі групи не можна зменшувати однаково для кожноı̈ групи, оскільки це може призвести до вилучення біль слабких чи скромних студентів із комунікаціı̈ та виконання завдання [13]. Розглянемо на прикладах, які комунікативні завдання можна запропонувати студентам немовних спеціальностей. Приклад 1. Рольова гра Safety at Work. Приклад 1. Рольова гра Safety at Work. © Семида О.В., Кравченко Т.В., 2021 Граматична тема. Модальні дієслова. Граматична тема. Модальні дієслова. Граматична тема. Модальні дієслова. Situation: You are a chief engineer at the open pit mine. Your task is to show around a new colleague and tell him safety rules. You have to explain all the safety signs. © Семида О.В., Кравченко Т.В., 2021 DOI 10.36074/rodmmrfssn.ed-1.09 103 Колективна наукова монографія Викладач використовує рольові картки із завданнями для студентів та зображення попереджувальних знаків, які використовуються на виробництві, у промисловості. Викладач використовує рольові картки із завданнями для студентів та зображення попереджувальних знаків, які використовуються на виробництві, у промисловості. Task. Fill in the gaps using must, mustn't, can, cannot, may Task. Fill in the gaps using must, mustn't, can, cannot, may g p g , , , , y You _______wear a safety helmet and goggles. You ________ smoke near the rig. ________ I use these tools? You _________ use them. You ______park here. Clients ________ request the draft. You _______wear a safety helmet and goggles. You ________ smoke near the rig. ________ I use these tools? You _________ use them. You ______park here. Clients ________ request the draft. На занятті створюються реальна комунікативна ситуація, з якою студенти зіткнуться під час роботи — правила поведінки на робочому місці, безпека, інструктаж. Іншим завданням на засвоєння граматичної конструкції умовні речення може бути завдання описати схему. Студентам надаються різні види схем електричного кола, і вони повинні описати, що відбудеться на кожній схемі. Також викладач може роздати картки, у яких студенти заповнюють пропуски відповідно до схем електричного кола. Граматична тема. Present Perfect. Situation. You do the project on the underground tunnel construction for Kyivbud. You have received the corrected technical drawings and your task is to discus and implement changes. Analyse two drawing and say about the changes using have/has changed, inserted, moved. C. Борг зазначає, що велику роль у відборі навчального матеріалу, у виборі методів та форм роботи на занятті відіграє освіта та досвід власного навчання викладача. Також, він зауважує, що викладачі, які навчалися за традиційною системою, прогресивні і не схильні до інновацій у викладанні, а так і надаватимуть перевагу традицій ним методам і формам роботи [14]. Приклад 2. Describe the figure (electric circuit). Приклад 2. Describe the figure (electric circuit). Граматична тема. Умовні речення. Граматична тема. Умовні речення. Граматична тема. Умовні речення. Task. Complete the description of electrical circuits using Conditionals Type 0, 1. Task. Complete the description of electrical circuits using Conditionals Type 0, 1. p 1. When current ________ from A to B, electrons ________ from B to A. 2. The current _______ through a conductor if there _______ no electromagnetic force. (Flow) 3. Electromagnetism __________ when an electrical current ________ through a simple conductor. (Produce) 4. The magnetic field of the magnet ________ a force to the tiny electron magnets in the wire so that all the free electrons in the wire _________ anti parallel to the magnetic pole orientation of the magnet. (Exert, orient) На засвоєння часової форми Present Perfect можна запропонувати описати зміни у технічному кресленні. Студентам даються два креслення — після внесення коректив у технічному відділі, і вони порівнюють, що змінилося у кресленні, які виправлення внесені, використовуючи для цього конструкції have/has changed, inserted, moved. Подібне завдання (з описом креслення) можна запропонувати на засвєння Passive Voice. На засвоєння часової форми Present Perfect можна запропонувати описати зміни у технічному кресленні. Студентам даються два креслення — після внесення коректив у технічному відділі, і вони порівнюють, що — після внесення коректив у технічному відділі, і вони порівнюють, що змінилося у кресленні, які виправлення внесені, використовуючи для цього конструкції have/has changed, inserted, moved. Подібне завдання (з описом креслення) можна запропонувати на засвєння Passive Voice. © Семида О.В., Кравченко Т.В., 2021 104 Розділ дев’ятий DOI 10.36074/rodmmrfssn.ed-1.09 Приклад 3. Make changes to the drawing. Приклад 3. Make changes to the drawing. Приклад 3. Make changes to the drawing. Граматична тема. Present Perfect. СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ: [1] Скляренко, Н. К. (2010). Методика формування іншомовної граматичної компетенції в учнів загальноосвітніх навчальних закладів. Іноземні мови, (40), 15-25. [2] Тригуб, І. П. (2014). Формування граматичноı̈ компетенціı̈ у студентів немовних спеціальностей ВНЗ у процесі вивчення англій ськоı̈ мови. Науковий вісник Міжнародного гуманітарного університету. Філологія. Вип. 10(2), 74-77. © Семида О.В., Кравченко Т.В., 2021 ВИСНОВКИ З ТЕМИ ДОСЛІДЖЕННЯ. Отже, практичне оволодіння іноземною мовою, вміння застосовувати набуті знання у реальних комунікативних професійних ситуаціях входять до переліку питань методики викладання іноземних мов. Наразі активно досліджується та розробляється комунікативний підхід до вивчення іноземної мови. Його цінність та ефективність полягає в тому, що здійснюється нерозривне навчання усім видам мовленнєвої діяльності у реальних комунікативних ситуаціях. Такий підхід формує позитивне ставлення до процесу вивчення мови та сприяє усвідомленості практичної необхідності цих знань, умінь і навичок. Формування граматичної компетенції здійснюється на основі реальних комунікативних навчальних матеріалів, контроль сформованості навички здійснюється також у ході реальної комунікації. Це дозволяє студентам побачити можливість практичного застосування набутих знань, оскільки вони їх отримують практикуючи, використовуючи мову у реальних ситуаціях. СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ: © Семида О.В., Кравченко Т.В., 2021 Колективна наукова монографія DOI 10.36074/rodmmrfssn.ed-1.09 105 [3] Saienko, N., Semyda, O., Akhmad, I. (2020). Using social networks in teaching ESP to engineering students. Advanced Education, (14), 38-45. Вилучено з https://doi.org/10.20535/2410- 8286.19808 [4] Касаткіна, О.В. (2011). До питання розвитку граматичноı̈ компетентності студентів на заняттях іноземноı̈ мови. Вісник Житомирського державного університету. Педагогічні науки, (59), 52 55. у ( ) [5] Стеченко, Т.О. (2004). Формування професійно орієнтованої англомовної граматичної компетенції майбутніх філологів (дис. ...канд. пед. Наук). К.иїв, Україна. [6] Мирошниченко, О.А. (2018). Ефективні методи навчання у викладанні англійської мови як іноземої (EFL). Молодий учений, № 1(53), 817 -820. [7] Ніколаєва, С.Ю. (2002). Методика викладання іноземних мов у сер.навч.закладах. Київ: Ленвіт. [8] Chung, Siaw-Fong. (2006). A communicative approach to teaching grammar: theory and practice. The English Teacher, (XXXIV), 33-50. [9] Прокопенко, Н. П. (2008). Формування іншомовної комунікативної компетенції у навчанні граматиці англійської мови студентів напряму підготовки. Міжнародна економіка" та "Менеджмент зовнішньоекономічної діяльності. 29–31 січня. 2008. Київ. Україна. р [10] Осідак, В.В. (2004). Комплексний контроль рівня сформованості англомовної граматичної компетенції майбутніх філологів (дис. канд. пед. наук). К.иїв, Україна. [11] Rukminingsih, (2015). Assessing Grammar By Using Communicative Activities To Employ Students’ Skill And Ability To Use English. Trends and Issues on Teacher Training and Education, 5 -6 November, 2015. Indonesia. (Vol 1. pp. 502 -508). ( pp ) [12] Тарнопольський , О.Б. (2019). Методика викладання іноземних мов та їх аспектів у вищій школі: підручник . Дніпро: Університет імені Альфреда Нобеля. [13] Semyda, O. (2019). Teaching foreign languages in multilevel groupsInternational Scientific Conference Scientific. Development of New Eastern Europe: Conference Proceedings, November 15th, 2019. Riga, Latvia: Baltija Publishing. 22- 25. Вилучено з https://doi.org/10.30525/978- 9934-588-13-6 [14] Borg, S. (1998). Teachers’ Pedagogical Systems and Grammar Teaching: A Qualitative Study. ВИСНОВКИ З ТЕМИ ДОСЛІДЖЕННЯ. TESOL Quaterly, 32(1), 9-38. [14] Borg, S. (1998). Teachers’ Pedagogical Systems and Grammar Teaching: A Qualitative Study. TESOL Quaterly, 32(1), 9-38.
https://openalex.org/W3168052860	https://www.frontiersin.org/articles/10.3389/fonc.2022.778545/pdf	English	null	Clinical Benefit With PARP Inhibitor for Pathogenic Germline FANCA-mutated Relapsed Epithelial Ovarian Cancer: a Case Report	Research Square (Research Square)	2,021	cc-by	3,548	CASE REPORT published: 25 February 2022 doi: 10.3389/fonc.2022.778545 CASE REPORT Clinical Beneﬁt With PARP Inhibitor for Pathogenic Germline FANCA- Mutated Relapsed Epithelial Ovarian Cancer: A Case Report Bing Qian 1, Wenshu Leng 1, Zhengqing Yan 2, Jin Lu 1, Shiqing Chen 2, Huan Yi 2 and Zhi Jiang 1* 1 Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, The Afﬁliated Cancer Hospital of Nanjing Medical University, Nanjing, China, 2 Department of Medical Affairs, The Medical Department, 3D Medicines Inc., Shanghai, China Background: PARP inhibitors have been approved as targeted therapy for BRCA- deﬁcient metastatic ovarian cancer (OC). Fanconi anemia complementation group A (FANCA), one of the homologous recombination repair pathway genes, is a susceptibility gene to breast cancer and OC. Therefore, it is interesting to investigate whether germline FANCA-mutated relapsed epithelial OC could achieve clinical beneﬁt from the treatment of PARP inhibitor. Citation: Qian B, Leng W, Yan Z, Lu J, Chen S, Yi H and Jiang Z (2022) Clinical Beneﬁt With PARP Inhibitor for Pathogenic Germline FANCA- Mutated Relapsed Epithelial Ovarian Cancer: A Case Report. Front. Oncol. 12:778545. doi: 10.3389/fonc.2022.778545 Keywords: PARP inhibitor, ovarian cancer, germline, FANCA, clinical beneﬁt Edited by: Fabio Martinelli, National Cancer Institute Foundation (IRCCS), Italy Edited by: Fabio Martinelli, National Cancer Institute Foundation (IRCCS), Italy Reviewed by: Chen Wang, Mayo Clinic, United States Federica Tomao, European Institute of Oncology (IEO), Italy Correspondence: Zhi Jiang margrate71@163.com Case Presentation: A 49-year-old female patient without a family history of cancer was diagnosed with epithelial OC. This patient underwent surgical resection plus platinum- based treatment twice in 2016 and 2018, successively. After the second relapse in July 2019, the patient underwent another radical resection. The next-generation sequencing analysis results revealed a germline FANCA mutation in the tumor tissue. Subsequently, the third-line treatment of liposomal doxorubicin hydrochloride plus lobaplatin was administrated for ﬁve cycles with the patient’s consent. Then, oral niraparib (200 mg daily) was given for maintenance treatment. During the follow-up, no evidence of tumor recurrence was observed. Currently, the survival with no evidence of disease has already exceeded 21 months, and the treatment is still going on. Reviewed by: Chen Wang, Mayo Clinic, United States Federica Tomao, European Institute of Oncology (IEO), Italy Correspondence: Zhi Jiang margrate71@163.com Specialty section: This article was submitted to Gynecological Oncology, a section of the journal Frontiers in Oncology Specialty section: This article was submitted to Gynecological Oncology, a section of the journal Frontiers in Oncology Received: 17 September 2021 Accepted: 18 January 2022 Published: 25 February 2022 Conclusions: This case highlighted that OC patients harboring pathogenic gene alterations in the homologous recombination pathway might achieve clinical beneﬁt from PARP inhibitors, which should be conﬁrmed in further studies. Received: 17 September 2021 Accepted: 18 January 2022 Published: 25 February 2022 INTRODUCTION Ovarian cancer (OC) is the most common cause of death from gynecological cancer among women worldwide (1). Approximately 80% of patients with newly diagnosed OC have a response to platinum- based chemotherapy. However, most patients would relapse and achieve limited beneﬁts from subsequent therapies. The median progression-free survival (PFS) after the ﬁrst, second, third, fourth, February 2022 \| Volume 12 \| Article 778545 Frontiers in Oncology \| www.frontiersin.org FANCA-Mutated Ovarian Cancer Qian et al. and ﬁfth relapse was 10.2 [95% conﬁdence interval (CI) 9.6–10.7], 6.4 (5.9–7.0), 5.6 (4.8–6.2), 4.4 (3.7–4.9), and 4.1 (3.0–5.1) months, respectively (2). and ﬁfth relapse was 10.2 [95% conﬁdence interval (CI) 9.6–10.7], 6.4 (5.9–7.0), 5.6 (4.8–6.2), 4.4 (3.7–4.9), and 4.1 (3.0–5.1) months, respectively (2). and ﬁfth relapse was 10.2 [95% conﬁdence interval (CI) 9.6–10.7], 6.4 (5.9–7.0), 5.6 (4.8–6.2), 4.4 (3.7–4.9), and 4.1 (3.0–5.1) months, respectively (2). operation, the patient underwent four cycles of paclitaxel/ carboplatin. Given that the CA19-9 level did not drop to the normal range after chemotherapy, carboplatin was replaced by lobaplatin since the ﬁfth cycle. The patient underwent one cycle of paclitaxel/lobaplatin. This patient achieved complete response with normal CA19-9 level. After the ﬁrst relapse with a pelvic metastasis in October 2018, debulking surgery was performed (R0 resection). Postoperative pathology revealed high-grade serous OC. The CA125 level was 21.7 U/ml after operation (normal range, 0–35 U/ml). The patient was administrated with a second-line chemotherapy of paclitaxel/lobaplatin from November 2018 to February 2019. However, CT scanning revealed a cystic- appearing solid mass in the pelvic cavity (Figure 1A), and the CA125 level was increased up to 60+ U/ml in July 2019, suggesting thatshedevelopedtumorprogressionafterthepaclitaxel/platinum- free interval of 4 months in the second-line therapy. Subsequently, sheunderwentradicalsurgerywithR0resection.A7-cm-sizedmass was seen in the rectovaginal pouch during the operation, and a postoperative pathological examination conﬁrmed a poorly differentiated ovarian carcinoma. PARP inhibitors, such as olaparib and niraparib, are new treatment strategies for BRCA 1/2 altered OC and other cancers (3). BRCA1/2-deﬁcient cells utilize error-prone DNA repair pathways, causing increased genomic instability, which might be responsible for their sensitivity to DNA-damaging agents. Niraparib is an oral, highly selective PARP1 and PARP2 inhibitor (4). In the NOVA trial, all patients who received niraparib had a signiﬁcantly longer PFS than those who received placebo. Notably, BRCA-mutated patients could achieve more beneﬁts from PARP inhibitors than non-BRCA-mutated patients (5). INTRODUCTION Similar to BRCA1/2, some other “BRCAness” genes (e.g., ATM, PALB2, and FANC) also play key roles in homologous recombination pathway (6, 7). According to previous reports, a subset of patients harboring deleterious gene mutations in the non-BRCA homologous recombination repair pathway (HRRm) might beneﬁt from PARP inhibitor (8). Furthermore, for patients with non-BRCA HRRm, the extent of beneﬁt from PARP inhibitors was different (9). Thus, identifying non-BRCA HRRm is critical for precision treatment and survival management for OC patients. To further explore the genomic features, both the surgical specimen and the matched white blood cell (WBC) sample underwent next-generation sequencing analysis based on a pan- cancer 733-gene panel (3D Medicines, China). Germline mutation was identiﬁed from WBC sequencing results. The mutation proﬁling of this patient is summarized in Figure 1B. The results suggest that this patient harbored germline FANCA p. P615Hfs25 heterozygous mutation and somatic PIK3CA, AKT1, ARID1A, ARID1B, and TP53 mutations, with a high homologous recombination deﬁciency (HRD) score of 35 (cutoff = 30). It was notedthattheHRDscoreisdeﬁnedastheunweightednumericsum of the loss of heterozygosity score, telomeric allelic imbalance score, and large-scale state transition score, according to 3DMed-HRD algorithm as previously described(17).The HRD scorethreshold of 30 is predeﬁned by analyzing the HRD scores in a Chinese training cohort of breast and ovarian cancer patients with known BRCA1/2 status and identifying a cutoff with 95% sensitivity to detect those tumors with BRCA1/2mutation. Previous works suggested that FANCA could increase the sensitivity to DNA-damaging agents, such as platinum (14, 18). Since August 2019, the third-line treatment of liposomal doxorubicin hydrochloride plus lobaplatin (ﬁvecycles) was administratedwithpatient consent.Noevidenceof disease (NED) was observed during the third-line chemotherapy. Subsequently, oral niraparib (200 mg daily) was administered for maintenance treatment. During the follow-up, no evidence of tumor recurrence was observed (Figure 1A), and the CA125 level remained within the normal range (Figure 1C). No adverse events were observed during niraparib treatment. Currently, the survival with NED has already exceeded 21 months, and the period of niraparib treatment has been more than 15 months. The close follow-up is still going on. Fanconi anemia complementation group A (FANCA) is associated with Fanconi anaemia, a rare autosomal recessive disorder characterized by congenital abnormalities, bone marrow failure, and predisposition to malignancy. Recently, FANCA has emerged mainly as a susceptibility gene to breast cancer and OC (10, 11). BACKGROUND A 49-year-old female patient was admitted to the hospital due to a growing left adnexal mass in August 2016. She had no family history of cancer. The results of laboratory tests showed normal levels of carbohydrate antigen (CA) 125, alpha-fetoprotein, carcinoembryonic antigen (CEA), and human epididymis protein 4. However, the CA 19-9 level was up to 62.65 U/ml. The ultrasound results revealed a 10.9 × 8.5-cm cystic mass on the left ovary. Thus, she underwent a series of surgical procedures, including total hysterectomy, bilateral salpingo-oophorectomy, and omentectomy (R0 resection). Intraperitoneal perfusion of carboplatin was given during the operation. The postoperative pathology conﬁrmed a moderately differentiated ovarian endometrioid adenocarcinoma. After the INTRODUCTION The single-strand annealing activity of FANCA plays a direct role in double-strand break (DSB) repair (12, 13). Preclinical studies demonstrated an association between FANCA mutated cells and sensitivity to PARP inhibitors (7). Compared with control isogenic wild-type cells, FANCA- deﬁcient mouse ﬁbroblast cells demonstrated greater sensitivity to PARP inhibitors. Another study reported that FANCA p.S1088F could induce sensitivity to olaparib in vitro in cancer cell lines or in vivo in patient-derived xenografts (14). Besides this, PARP inhibitors demonstrate promising results in FANCA- altered metastatic castration-resistant prostate cancer (mCRPC) (6, 15, 16). Herein we reported the ﬁrst case of a germline FANCA-mutated relapsed epithelial OC who achieved clinical beneﬁt from PARP inhibitors. DISCUSSION In this report, we presented a case of relapsed epithelial OC harboring germline FANCA p. P615Hfs25 heterozygous February 2022 \| Volume 12 \| Article 778545 Frontiers in Oncology \| www.frontiersin.org 2 Qian et al. FANCA-Mutated Ovarian Cancer A B C FIGURE 1 \| (A) Computed tomography scans: (a) before cytoreductive surgery, (b) before niraparib treatment, and (c) after 9 months of niraparib treatment. (B) List of deleterious or likely deleterious somatic variations by next-generation sequencing in ovarian carcinoma. (C) Changes of serum tumor marker levels of carbohydrate antigen 125. A B C FIGURE 1 \| (A) Computed tomography scans: (a) before cytoreductive surgery, (b) before niraparib treatment, and (c) after 9 months of niraparib treatment. (B) List of deleterious or likely deleterious somatic variations by next-generation sequencing in ovarian carcinoma. (C) Changes of serum tumor marker levels of carbohydrate antigen 125. A A C B C FIGURE 1 \| (A) Computed tomography scans: (a) before cytoreductive surgery, (b) before niraparib treatment, and (c) after 9 months of niraparib treatment. (B) List of deleterious or likely deleterious somatic variations by next-generation sequencing in ovarian carcinoma. (C) Changes of serum tumor marker levels of carbohydrate antigen 125. FIGURE 1 \| (A) Computed tomography scans: (a) before cytoreductive surgery, (b) before niraparib treatment, and (c) after 9 months of niraparib treatment. (B) List of deleterious or likely deleterious somatic variations by next-generation sequencing in ovarian carcinoma. (C) Changes of serum tumor marker levels of carbohydrate antigen 125. Its single-strand annealing activity plays a direct role in DSB repair (12, 13). To the best of our knowledge, several clinical trials demonstrated that FANCA-mutated patients respond to PARP inhibitors via a synthetic lethality mechanism. In phase 2 TRITON2 study, one mCRPC patient with FANCA mutation had complete radiographic and prostate-speciﬁc antigen (PSA) responses to the PARP inhibitor rucaparib (6). In phase 2 GALAHAD study evaluating niraparib in mCRPC, tumor radiographic response was observed in two patients with alterations in FANCA (15). The phase 2 TOPARP-B study presented another mCRPC patient harboring a FANCA mutation who had PSA responses after olaparib monotherapy (16). How FANCA mutations affect the genomic instability and the efﬁciency of PARP inhibitors in FANCA-mutated OC patients should be investigated in the future. REFERENCES 12. Jeong E, Lee SG, Kim HS, Yang J, Shin J, Kim Y, et al. Structural Basis of the Fanconi Anemia-Associated Mutations Within the FANCA and FANCG Complex. Nucleic Acids Res (2020) 48(6):3328–42. doi: 10.1093/nar/ gkaa062 1. Lheureux S, Gourley C, Vergote I, Oza AM. Epithelial Ovarian Cancer. Lancet (2019) 393(10177):1240–53. doi: 10.1016/S0140-6736(18)32552-2 2. Hanker LC, Loibl S, Burchardi N, Pﬁsterer J, Meier W, Pujade-Lauraine E, et al. The Impact of Second to Sixth Line Therapy on Survival of Relapsed Ovarian Cancer After Primary Taxane/Platinum-Based Therapy. Ann Oncol (2012) 23(10):2605–12. doi: 10.1093/annonc/mds203 13. Benitez A, Liu W, Palovcak A, Wang G, Moon J, An K, et al. FANCA Promotes DNA Double-Strand Break Repair by Catalyzing Single-Strand Annealing and Strand Exchange. Mol Cell (2018) 71(4):621–8.e4. doi: 10.1016/j.molcel.2018.06.030 3. Kaufman B, Shapira-Frommer R, Schmutzler RK, Audeh MW, Friedlander M, Balmana J, et al. Olaparib Monotherapy in Patients With Advanced Cancer and a Germline BRCA1/2 Mutation. J Clin Oncol (2015) 33(3):244–50. doi: 10.1200/JCO.2014.56.2728 14. Wilkes DC, Sailer V, Xue H, Cheng H, Collins CC, Gleave M, et al. A Germline FANCA Alteration That Is Associated With Increased Sensitivity to DNA Damaging Agents. Cold Spring Harb Mol Case Study (2017) 3(5): a001487. doi: 10.1101/mcs.a001487 4. Moore KN, Secord AA, Geller MA, Miller DS, Cloven N, Fleming GF, et al. Niraparib Monotherapy for Late-Line Treatment of Ovarian Cancer (QUADRA): A Multicentre, Open-Label, Single-Arm, Phase 2 Trial. Lancet Oncol (2019) 20(5):636–48. doi: 10.1016/S1470-2045(19)30029-4 15. Smith MR, Sandhu SK, Kelly WK, M.R. Scher HI, Efstathiou E, Lara PN, et al. Pre-Speciﬁed Interim Analysis of GALAHAD: A Phase II Study of Niraparib in Patients (Pts) With Metastatic Castration-Resistant Prostate Cancer (mCRPC) and Biallelic DNA-Repair Gene Defects (DRD). Ann Oncol (2019) 30:v884–v5. doi: 10.1093/annonc/mdz394.043 Oncol (2019) 20(5):636–48. doi: 10.1016/S1470-2045(19)30029-4 5. Mirza MR, Monk BJ, Herrstedt J, Oza AM, Mahner S, Redondo A, et al. Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer. N Engl J Med (2016) 375(22):2154–64. doi: 10.1056/NEJMoa 1611310 16. Mateo J, Porta N, Bianchini D, McGovern U, Elliott T, Jones R, et al. Olaparib in Patients With Metastatic Castration-Resistant Prostate Cancer With DNA Repair Gene Aberrations (TOPARP-B): A Multicentre, Open-Label, Randomised, Phase 2 Trial. Lancet Oncol (2020) 21(1):162–74. doi: 10.1016/ S1470-2045(19)30684-9 6. Abida W, Campbell D, Patnaik A, Shapiro JD, Sautois B, Vogelzang NJ, et al. REFERENCES Non-BRCA DNA Damage Repair Gene Alterations and Response to the PARP Inhibitor Rucaparib in Metastatic Castration-Resistant Prostate Cancer: Analysis From the Phase II TRITON2 Study. Clin Cancer Res (2020) 26(11):2487–96. doi: 10.1158/1078-0432.CCR-20-0394 17. Chen Y, Cui L, Zhang B, Zhao X, Xu X. Efﬁcacy of Platinum-Based Chemotherapy in Advanced Triple-Negative Breast Cancer in Association With Homologous Recombination Deﬁciency. J Clin Oncol (2021) 39(suppl 15):abstr e13051. doi: 10.1200/JCO.2021.39.15_suppl.e13051 7. McCabe N, Turner NC, Lord CJ, Kluzek K, Bialkowska A, Swift S, et al. Deﬁciency in the Repair of DNA Damage by Homologous Recombination and Sensitivity to Poly(ADP-Ribose) Polymerase Inhibition. Cancer Res (2006) 66(16):8109–15. doi: 10.1158/0008-5472.CAN-06-0140 18. Park W, Chen J, Chou JF, Varghese AM, Yu KH, Wong W, et al. Genomic Methods Identify Homologous Recombination Deﬁciency in Pancreas Adenocarcinoma and Optimize Treatment Selection. Clin Cancer Res (2020) 26(13):3239–47. doi: 10.1158/1078-0432.CCR-20-0418 8. Hodgson DR, Dougherty BA, Lai Z, Fielding A, Grinsted L, Spencer S, et al. Candidate Biomarkers of PARP Inhibitor Sensitivity in Ovarian Cancer Beyond the BRCA Genes. Br J Cancer (2018) 119:1401–9. doi: 10.1038/ s41416-018-0274-8 19. Huang Z, Liu X, Zou B, Shen Q, Liu Y, Zhou T. Ovarian Cancer Transformation From Adenocarcinoma to Undifferentiated Small Cell Carcinoma: A Case Report. Oncol Lett (2015) 9:2230–2. doi: 10.3892/ ol.2015.3031 9. Lindemann K, Škof E, Colombo N, González-Martı́n A, Davidson R, Blakeley C, et al. Olaparib Maintenance Monotherapy for non-Germline BRCA1/2- Mutated (non-gBRCAm) Platinum-Sensitive Relapsed Ovarian Cancer (PSR OC): Exploratory Biomarker Analyses of the Phase IIIb OPINION Study. Ann Oncol (2021) 32(suppl_5):S725–72. doi: 10.1016/annonc/annonc703 20. Guo J, Yu J, Song X, Mi H. Serum CA125, CA19-9 and CEA Combined Detection for Epithelial Ovarian Cancer Diagnosis: A Meta-Analysis. Open Med (Wars) (2017) 12:131–7. doi: 10.1515/med-2017-0020 10. Thompson E, Dragovic RL, Stephenson SA, Eccles DM, Campbell IG, Dobrovic A. A Novel Duplication Polymorphism in the FANCA Promoter and its Association With Breast and Ovarian Cancer. BMC Cancer (2005) 5:43. doi: 10.1186/1471-2407-5-43 21. Tomao F, D'Incalci M, Biagioli E, Peccatori FA, Colombo N. Platinum Sensitivity in Recurrent Ovarian Cancer by Extending the Platinum-Free Interval: Myth or Reality? Cancer (2017) 123:3450–9. doi: 10.1002/cncr.30830 22. Fierheller CT, Guitton-Sert L, Alenezi WM, Revil T, Oros KK, Gao Y, et al. A Functionally Impaired Missense Variant Identiﬁed in French Canadian Families Implicates FANCI as a Candidate Ovarian Cancer-Predisposing Gene. Genome Med (2021) 13:186. doi: 10.1186/s13073-021-00998-5 11. CONCLUDING REMARKS ZJ contributed to planning and organization. BQ collected clinical data and supervised the ﬁndings of this work. JL and WL aided in the data collection and supervision. ZY, SC, and HY analyzed the results and prepared the manuscript. All authors contributed to the article and approved the submitted version In conclusion, we presented the ﬁrst case of one relapsed epithelial OC harboring a germline FANCA mutation who achieved an impressive PFS after niraparib treatment. This case highlighted that OC patients carrying pathogenic HRRm might ETHICS STATEMENT Written informed consent was obtained from the patient before clinical samples were collected. Consent to publication was also obtained from the patient. The patient was informed of the test results. DISCUSSION FANCA-Mutated Ovarian Cancer achieve the best outcome from PARP inhibitors. Such a conclusion should be conﬁrmed in further studies. the patient harboring pathogenic FANCA achieved clinical beneﬁt with PFS of over 21 months, almost equivalent to that of BRCA- mutated patients with PARP inhibitors. Consistent with a previous study (24), such result highlighted that PAPRP inhibitor was efﬁcacious not only in BRCA-mutated patients but also in patients with unknown alterations. Given the nature of case reports, larger cohort studies should be investigated to further conﬁrm such conclusions. DISCUSSION Though no secondary hits were found on FANCA gene, this patient had a high HRD score of 35 (cutoff = 30), which might be able to explain the signiﬁcant clinical beneﬁt from niraparib. mutation who responded well to the niraparib treatment with PFS of over 21 months. In this case, different histologic subtypes are conﬁrmed after operation, which reﬂect the tumor heterogeneity. Pathological transformation was also observed in a previous OC case, whose pathology transformed to undifferentiated small cell carcinoma from adenocarcinoma (19). In this case, the CA19-9 level was kept at a high level at diagnosis, while the CA125 level was increased to a high level during the second recurrence. Although the CA19-9 level was usually elevated in gastrointestinal cancer, it could also be detected in the blood of some OC patients (20). This case supported that the combination detection of serum CA125, CA19-9, and CEA might have higher sensitivity and speciﬁcity compared to a single serum marker. The paclitaxel/platinum-free interval is less than 6 months during the second-line treatment, suggesting that paclitaxel or platinum resistance was developed during the second recurrence. Notably, current clinical evidence could neither support nor deny the beneﬁt of extending platinum-free interval in patients with recurrent ovarian cancer (21). Thus, lobaplatin-based strategy is still administrated as the third-line treatment in this case. Based on their great clinical beneﬁts to OC patients, PARP inhibitors such as olaparib, rucaparib, and niraparib had been approved for OC maintenance treatment after platinum-based chemotherapy. A previous study demonstrated that the median PFS of third-line chemotherapy in relapsed patients was only 5.6 (4.8–6.2) months (2). When PARP inhibitors are used for maintenance therapy in relapsed OC patients, the median PFS of germline BRCA-mutated patients is up to 21 months, according to the NOVA trial results. In contrast, the PFS is just 9.3 months in these relapsed OC patients with wild-type BRCA (5). In this case, Besides BRCA1/2, pathogenic gene variants involving Fanconi anemia have been reported as candidate ovarian cancer-predisposing genes (10, 22). Though no family cancer history is observed in this case, the relationship between pathogenic germline gene variants and OC should be further studied in the future. About 0.23% of patients harbor germline or somatic FANCA mutations in the TCGA OC cohort (23). February 2022 \| Volume 12 \| Article 778545 Frontiers in Oncology \| www.frontiersin.org 3 Qian et al. 23. Kanchi KL, Johnson KJ, Lu C, McLellan MD, Leiserson MDM, Wendl MC, et al. Integrated Analysis of Germline and Somatic Variants in Ovarian Cancer. Nat Commun (2014) 5:3156. doi: 10.1038/ncomms4156 24. Caruso D, Papa A, Tomao S, Vici P, Panici PB, Tomao F. Niraparib in Ovarian Cancer: Results to Date and Clinical Potential. Ther Adv Med Oncol (2017) 9:579–88. doi: 10.1177/1758834017718775 Conﬂict of Interest: ZY, SC, and YH were employed by the company 3D Medicines Inc. The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. February 2022 \| Volume 12 \| Article 778545 REFERENCES Yang X, Zhang X, Jiao J, Zhang F, Pan Y, Wang Q, et al. Rare Variants in FANCA Induce Premature Ovarian Insufﬁciency. Hum Genet (2019) 138(11- 2):1227–36. doi: 10.1007/s00439-019-02059-9 February 2022 \| Volume 12 \| Article 778545 4 Frontiers in Oncology \| www.frontiersin.org FANCA-Mutated Ovarian Cancer Qian et al. Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Copyright © 2022 Qian, Leng, Yan, Lu, Chen, Yi and Jiang. 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. Conﬂict of Interest: ZY, SC, and YH were employed by the company 3D Medicines Inc. The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. February 2022 \| Volume 12 \| Article 778545 Frontiers in Oncology \| www.frontiersin.org 5
https://openalex.org/W4224268573	https://www.sciendo.com/pdf/10.2478/asn-2022-0003	English	null	An ontological model to support citizen science in the field of invasive species research	Acta Scientifica Naturalis	2,022	cc-by	4,124	ASN, Vol. 9, No 1, Pages 23–32, 2022 Acta Scientifica Naturalis Former Annual of Konstantin Preslavsky University of Shumen: Chemistry, Physics, Biology, Geography Journal homepage: https://content.sciendo.com/view/journals/asn/asn-overview.xml 1Department of Computer Systems and Informatics, Faculty of Business Administration, University of Forestry, 1797 Sofia, Bulgaria 1Department of Computer Systems and Informatics, Faculty of Business Administration, University of Forestry, 1797 Sofia, Bulgaria 1Department of Computer Systems and Informatics, Faculty of Business Administration, University of Forestry, 1797 Sofia, Bulgaria 2Department of Plant Protection, Faculty of Agronomy, University of Forestry, 1797 Sofia, Bulgaria Abstract: Advances in information technology developments have led to improved ways and means of sharing information and good practices in various areas of social development. Providing the necessary tools enables Citizen Sciences (CS) to play an important role in raising awareness and engaging various stakeholders in the prevention of invasive alien species (IAS). In Bulgaria, up until this point, it is poorly developed, and this is largely due to the lack of information to the general public regarding the categorization of species, pathways of introduction and their negative impact. The article examines the possibilities for introduction and use of an advanced ontological model in the area of invasive alien species research, which will aid the process of involving a wide range of stakeholders in various initiatives that will contribute to preventing the introduction and spread of IAS. The researched approach using the advantages of modern information and communication technologies includes acquaintance with the basic concepts in the area of IAS, the processes related to their introduction and spread, as well as taking into account the existing interrelationships, which would provide opportunities for early detection and the rapid eradication of IAS. The developed model will also be applied to measures and policies put in place to change the attitudes of the general public to the problem of IAS. Keywords: Citizen Sciences, Invasive species, Ontologies, information technology 23 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg DOI: 10.2478/asn-2022-0003 Introduction Citizen science is a prerequisite for the successful implementation of initiatives related to conservation of biodiversity and prevention and control of invasive alien species (IAS), in addition its generated data are becoming an increasingly key factor for organizations involved in these issues. The technological development of information and communication technologies (ICT), the facilitated ways of sharing information, the wide choice of use of applications for the purposes of citizen sciences - commercial or open source allow more and more participants to become involved in the process. In Bulgaria, so far, the IAS citizen science is not at a high enough level, despite the provision of necessary tools. In order to increase awareness and engagement, it is necessary to inform the general public about the categorization of species, pathways of introduction and their negative impact. Becoming familiar with the basic concepts and the existing relationships between them, their generalization and summary will help individual participants to gain the necessary confidence, in terms of their proper judgment. Ontologies are an appropriate tool in this regard. The possible introduction and application of a model based on their use in the field of IAS research will support the processes of involving a wide range of stakeholders in individual initiatives. Accurate, up-to-date, well-documented, standardized and openly available information on alien species is at the heart of science-based strategies for dealing with biological invasions [1]. In order to achieve effective collection of information on alien species, with possibilities for its combination and use in individual initiatives, biodiversity-related data need to be interoperable. At this stage, this is hindered due to their dispersion. The benefits of using an ontological model in terms of aggregating and summarizing data related to alien species can be sought in the direction of their documentation, through metadata, as well as their formatting based on existing standards. On the other hand, such an approach will allow for their easier maintenance and updating, in order to ensure their valorization. In many cases, it is necessary to include the spatial component of the data needed in order to achieve the objectives of individual activities. In order to model reliable intelligent applications, as well as to avoid misinterpretation when designing spatial data infrastructures, an ontology-based approach can be used [2]. ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 Corresponding author: gmilchev@ltu.bg DOI: 10.2478/asn-2022-0003 ing author: gmilchev@ltu.bg 78/asn-2022-0003 ©2022 Radoslav Milchev et al., publishe This work is licensed under the Creative Commons Attribution 3.0 Public License ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 from their use in the control and reporting of circumstances related to biological invasions, are caused due to a lack of knowledge or inaccurate detection, or as a result of contextual dependence and ambiguity related to language misunderstanding [4]. The contribution of the use of ontologies in these cases can be sought in the direction of improving the accuracy of information in the lists of IAS, and despite the fact that uncertainty will never be completely eliminated, these approaches will significantly improve the transparency, repeatability and comparability of IAS lists. Having an overall understanding and presentation of citizen science projects, the participants and their results are key to ensuring a seamless exchange of knowledge and data [5]. In this sense, the application of ontologies will be useful in the implementation of a conceptual model related to the presence of an overall understanding and presentation of initiatives pertaining to citizen sciences, participants and the information they have generated. Built on the basis of fundamental citizen science concepts describing the activities and data in a standardized way, and with a focus on the participants and what they have done, it will contribute to a good foundation in ensuring their smooth exchange. In recent years, citizen sciences have been the subject of numerous projects and technological developments aimed at their functional and effective use. Based on the practical experience gained by the members of the research team in the field of developing ontological models for different subject areas and the established practices in the field of determining the characteristics of invasive alien and native species, the aim of the present paper is to develop an ontological model to support the development of Citizen Sciences in the area of IAS in Bulgaria. The aim of the present study is to facilitate the design and development of an ontological model, which on the one hand to facilitate the learning or self-learning processes of a wide range of potential representatives of Citizen Sciences in the area of invasive alien species, and on the other hand to support the development of appropriate desktop and mobile applications to take advantage of the opportunities to formalize the knowledge provided by ontologies. Introduction The benefits of such a blurred approach and level-based approximation will be related to: presenting and managing the uncertainty of volunteers in describing their observations in order to avoid inaccuracies and imperfections concerning said observations; obtaining verified and reliable information to compensate for their poorly defined knowledge in the relevant field. The development of citizen sciences, as an area related to large-scale information gathering, mobilization and social action of participants and organizations developing relevant initiatives, is associated with the careful choice of terminology [3]. For this purpose, the use of ontological models will be of great benefit, and in addition to refining the terms, a detailed explanation of their use is needed after conducting a systematic study of terminological trends in citizen sciences. Most errors with serious consequences, arising 24 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License 24 Corresponding author: gmilchev@ltu.bg DOI: 10.2478/asn-2022-0003 ASN, Vol. 9, No 1, Pages 23–32, 2022 Methods The ontological model is built in the Protégé 5.5.0 application environment [6]. The method for building an ontological model, through the use of information technology tools, allows the integration of mechanisms and tools that facilitate the processes of formalization of knowledge. The ontological model describing IAS with the means of information technology allows the analysis and detection of relationships that are difficult to implement with standard analysis tools, while allowing the model to be integrated for other purposes, such as software application development. The main statistical parameters of the ontology are shown in Figure 1, as well as parameters such as version, project description, authors and other characteristics including 112 classes, 253 axioms, 18 object properties and etc. 25 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License 25 Corresponding author: gmilchev@ltu.bg DOI: 10.2478/asn-2022-0003 ASN, Vol. 9, No 1, Pages 23–32, 2022 Figure 1. Basic statistical parameters of the ontology ASN, Vol. 9, No 1, Pages 23–32, 2022 Figure 1. Basic statistical parameters of the ontology Figure 1. Basic statistical parameters of the ontology Results The model successfully shows the applicability of the ontologies in the given subject area and allows subsequent addition and expansion, which in turn will affect the main statistical parameters of the project, which can be seen in the figure with the ontology description. The hierarchy of classes in the developed ontological model is shown in Figure 2. In this case the class with key terms for determining species is illustrated, which contains three subclasses with annotations in Bulgarian and English and a source of the used definitions content. The model has 12 main classes. The model has also developed a set of object properties (Figure 3), used to describe the individual species that could be registered and identified by the representatives of citizen sciences. 26 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg DOI: 10.2478/asn-2022-0003 Corresponding author: gmilchev@ltu.bg DOI: 10.2478/asn-2022-0003 Corresponding author: gmilchev@ltu.bg DOI: 10.2478/asn-2022-0003 ing author: gmilchev@ltu.bg 78/asn-2022-0003 ©2022 Radoslav Milchev et al., published This work is licensed under the Creative Commons Attribution 3.0 Public License ASN, Vol. 9, No 1, Pages 23–32, 2022 27 Figure 2. Hierarchy of classes in the ontological model Figure 3. Object properties of the individual species ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 , , , g , Figure 2. Hierarchy of classes in the ontological model Figure 2. Hierarchy of classes in the ontological model g y g Figure 3. Object properties of the individual species Figure 3. Object properties of the individual species Figure 3. Object properties of the individual species 27 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License 27 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License ASN, Vol. 9, No 1, Pages 23–32, 2022 Figure 6. OntoGraf visualization tool Figure 6. OntoGraf visualization tool 1 https://www.un.org/sustainabledevelopment/development-agenda/ ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 The individual species belonging to different kingdoms for which there is information in the scientific community at the moment of the research, are in the process of specification. These species are pre-processed before being imported into the model, the data being as shown in Figure 4. Figure 4. Processing and preparation of data before importing into the model Figure 4. Processing and preparation of data before importing into the model Ontological models offer a number of advantages in the analysis, study and presentation of the studied subject areas. Figure 5. OWLViz visualization tool Figure 5. OWLViz visualization tool Figure 5. OWLViz visualization tool Various visualization tools such as OWLViz or OntoGraf can be used for that purpose. The results from using these tools are shown in Figure 5 and Figure 6. 28 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License from using these tools are shown in Figure 5 and Figure 6. 28 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 Results from previous studies [9] related to the use of ontology-based approaches show a number of possibilities they provide in describing complex dependencies in a format where the stimulation code is automatically generated by the definition of the ontology of the model. This determines the possibility for automatic connection of models and data sources, as well as for automatic detection of similarities and differences and generation of model copies of common principles. Ontologically based methods can find application in citizen sciences initiatives related to alien species by supporting activities aimed at engaging additional systematic knowledge using a multi-evidence approach [10]. In this way, the initiatives will enjoy better legitimacy, based on ethical and reciprocal relations, which will reflect on improving the results aimed at managing and maintaining biodiversity. Despite the development of ICT, many good practices, including those in the field of citizen sciences, remain poorly documented. This has a direct impact on the management of results generated by them and especially on their future integration, sharing and dissemination. To make them visible to the general public, the use of an ontological knowledge management approach will enable stakeholders to share and understand knowledge at the domain and instance level, to reduce possible ambiguities, and to integrate with other sources of knowledge compared to traditional approaches for information management [11]. In the formalized description of knowledge regarding the IAS citizen sciences, through the use of ontologies it is necessary to take into account the possibility of including an explicit evaluation procedure, as without a well-structured one it will be difficult to assess the suitability of developed models in related research and their practical application [12]. Ontologies provide the necessary basis for the proper operation of semantic network-based applications - technologies that can provide instructions and clear rules in the processes of data collection, integration and use [13] in IAS citizen sciences initiatives. By providing semantic interoperability of the descriptive models built for presenting the information, the use of ontologies will allow for the generation and analysis of different scenarios in their implementation [14], with the help of built-in visualization tools showing the connection and interaction between the individual classes, as well as the flow of information and the specific properties of the objects within the classes. Discussion and conclusions The design of related ontological models should take into account the information gained from the many platforms acting as centers for knowledge, aggregation, dissemination and popularization of experience related to citizen sciences [6] and in particular invasive species. The conceptual models developed for the operation of some of them consist of the project metadata model, the metadata model from the data set, and the observation data model, specially developed for citizen sciences initiatives. Studies related to citizen sciences initiatives show that cultivating a passion for species identification is key to both "supporting nature" and broader stakeholder education [7]. On the other hand, citizen sciences are a tool for achieving universal and equal access to scientific data and information [8]. This determines the need for an accurate and detailed description, which can be achieved by using a methodology based on ontological models. In this way, the quality of the data the participants receive from the related initiatives will be optimized and improved, which will in turn bring scientific and public benefits in accordance with existing programs, such as the "Sustainable Development Agenda 20301". 29 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License ing author: gmilchev@ltu.bg 78/asn-2022-0003 ©2022 Radoslav Milchev et al., published This work is licensed under the Creative Commons Attribution 3.0 Public License Conflict of interest statement All authors have approved the manuscript with no conflict of interest. Acknowledgements This work was supported within the project №KP-06-COST-14 “State and perspectives of citizen science for invasive alien species in Bulgaria” funded by the National Scientific Fund of Bulgaria. ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 ontological model allows continuous supplementation and updating in the context of dynamic changes which we observe in the environment we inhabit. The development, use and application of ontological models in various subject areas is a powerful and effective method for the application of modern information and communication technologies in the study of said areas. The created ontological model in the field of IAS can significantly improve the participation rates of citizen sciences representatives in the field of registration and detection of invasive species, both at the territory of the Republic of Bulgaria and at regional level. The model improves the opportunities for interaction between professional researchers and civic researchers, setting a bridge to unify terminology and thus create a prerequisite for better registration of phenomena and events, even by people who do not have the necessary specialized knowledge obtained as part of the educational process they went through. The proposed 30 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License 30 Corresponding author: gmilchev@ltu.bg DOI: 10.2478/asn-2022-0003 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License References [1]. Groom, Q.J.; Adriaens, T.; Desmet, P.; Simpson, A.; De Wever, A.; Bazos, I.; Cardoso, A. C.; Charles, L.; Christopoulou, A.; Gazda, A.; Helmisaari, H.; Hobern, D.; Josefsson, M.; Lucy, F.; Marisavljevic, D.; Oszako, T.; Pergl, J.; Petrovic-Obradovic, O.; Prévot, C.; Ravn H-P.; Richards, G.; Roques, A.; Roy, H. E.; Rozenberg, M-AA.; Scalera, R.; Tricarico, E.; Trichkova, T.; Vercayie, D.; Zenetos, A.;Vanderhoeven, S., Seven Recommendations to Make Your Invasive Alien Species Data More Useful. Front. Appl. Math. Stat., 2017, 3, 13. DOI: 10.3389/fams.2017.00013. [1]. Groom, Q.J.; Adriaens, T.; Desmet, P.; Simpson, A.; De Wever, A.; Bazos, I.; Cardoso, A. C.; Charles, L.; Christopoulou, A.; Gazda, A.; Helmisaari, H.; Hobern, D.; Josefsson, M.; Lucy, F.; Marisavljevic, D.; Oszako, T.; Pergl, J.; Petrovic-Obradovic, O.; Prévot, C.; Ravn H-P.; Richards, G.; Roques, A.; Roy, H. E.; Rozenberg, M-AA.; Scalera, R.; Tricarico, E.; Trichkova, T.; Vercayie, D.; Zenetos, A.;Vanderhoeven, S., Seven Recommendations to Make Your Invasive Alien Species Data More Useful. Front. Appl. Math. Stat., 2017, 3, 13. DOI: 10.3389/fams.2017.00013. [2]. Bordogna, G.; Fugazza, C.; Oggioni, A., VGI Imperfection in Citizen Science Projects and Its Representation and Retrieval Based on Fuzzy Ontologies and Level-Based Approximate Reasoning. 2018, DOI: 10.1007/978-3-319-70878-2_10. [2]. Bordogna, G.; Fugazza, C.; Oggioni, A., VGI Imperfection in Citizen Science Projects and Its Representation and Retrieval Based on Fuzzy Ontologies and Level-Based Approximate Reasoning. 2018, DOI: 10.1007/978-3-319-70878-2_10. [2]. Bordogna, G.; Fugazza, C.; Oggioni, A., VGI Imperfection in Citizen Science Projects and Its Representation and Retrieval Based on Fuzzy Ontologies and Level-Based Approximate Reasoning. 2018, DOI: 10.1007/978-3-319-70878-2_10. [3]. Eitzel, M.; Cappadonna, J.; Lang, C.; Duerr, R.; Virapongse, A.; West, S.; Maximillian-Kyba, C.; Bowser, A.; Cooper, C.; Sforzi, A.; Metcalfe, A.; Harris, E.; Thiel, M.; Haklay, M.; Ponciano, L.; Roche, J.; Ceccaroni, L.; Shilling, F.; Dörler, D.; Heigl, F.; Kiessling, T.; Davis, B.; Jiang. Q. Citizen Science Terminology Matters: Exploring Key Terms. Citizen Science: Theory and Practice, 2017, 2(1), 1–20, DOI: https://doi.org/10.5334/cstp.96. [4]. McGeoch, M.; Spear, D.; Kleynhans, E.; Marais, E., Uncertainty in invasive alien species listing. Ecological applications: a publication of the Ecological Society of America. 2012, 22, 959-971. DOI: 10.2307/23213930. [5]. Lemmens R.; Falquet G.; Tsinaraki C.; Klan F.; Schade S.; Bastin L.; Piera J.; Antoniou V.; Trojan J.; Ostermann F.; Ceccaroni L., A Conceptual Model for Participants and Activities in Citizen Science Projects. In: Vohland, K. et al. (eds). The Science of Citizen Science. Springer, Cham. 2021, DOI: https://doi.org/10.1007/978-3-030-58278-4_9. References 31 31 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License ing author: gmilchev@ltu.bg 78/asn-2022-0003 ©2022 Radoslav Milchev et al., publishe This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License ASN, Vol. 9, No 1, Pages 23–32, 2022 ASN, Vol. 9, No 1, Pages 23–32, 2022 [6]. Musen, M. A., The Protégé project: A look back and a look forward. AI Matters. Association of Computing Machinery Specific Interest Group in Artificial Intelligence, 2015, 1(4), DOI: 10.1145/2557001.25757003. [7]. Wagenknecht, K.; Woods, T,; García Sanz, F.; Gold, M,; Bowser, A.; Rüfenacht, S.; Ceccaroni, L.; Jaume Piera, J., EU-Citizen. Science: A Platform for Mainstreaming Citizen Science and Open Science in Europe. Data Intelligence, 2021, 3(1), 136–149. DOI: https://doi.org/10.1162/dint_a_00085. [8]. Sharma, N.; Greaves, S.; Siddharthan, A.; Anderson, H. B.; Robinson, A.; Colucci-Gray, L.; Wibowo, A. T.; Bostock, H.; Salisbury, A.; Roberts, S.; Slawson, D.; van der Wal, R., From citizen science to citizen action: analysing the potential for a digital platform to cultivate attachments to nature. JCOM, 2019, 18 (01), A07. https://doi.org/10.22323/2.18010207. [9]. de Sherbinin, A.; Bowser, A.; Chuang, T-R.; Cooper, C;. Danielsen, F.; Edmunds, R.; Elias, P.; Faustman, E.; Hultquist, C.; Mondardini, R.; Popescu, I.; Shonowo, A.; Sivakumar, K., The Critical Importance of Citizen Science Data. Front. Clim., 2021, 3, 650760. DOI: 10.3389/fclim.2021.650760. [10]. Beck, H.; Morgan, K.; Jung, Y.; Grunwald, S.; Kwon, H.; Wu, J., Ontology-based simulation in agricultural systems modeling. Agricultural Systems. 2010, 103, 463-477. DOI: 10.1016/j.agsy.2010.04.004. [11]. Tengö, M.; Austin, B. J.; Danielsen, F.; Fernández-Llamazares, A., Creating Synergies between Citizen Science and Indigenous and Local Knowledge, BioScience, 2021, 71(5), 503–518, DOI: https://doi.org/10.1093/biosci/biab023. [12]. Ahsan, M.; Motla, Y. H.; Asim, M., Knowledge modeling fore-agriculture using ontology. International Conference on Open Source Systems & Technologies, 2014, 112-122, DOI: 10.1109/ICOSST.2014. 7029330. [13]. Goldstein, A.; Fink, L.; Ravid, G., A Framework for Evaluating Agricultural Ontologies. Sustainability. 2021, 13(11), 6387. DOI: https://doi.org/10.3390/su13116387. [14]. Rodríguez-García, M. Á.; García-Sánchez, F., CropPestO: An Ontology Model for Identifying and Managing Plant Pests and Diseases. In: Valencia-García, R.; Alcaraz-Marmol, G.; Del Cioppo-Morstadt, J.; Vera-Lucio, N.; Bucaram-Leverone, M. (eds) Technologies and Innovation. CITI 2020, Communications in Computer and Information Science, vol. 1309. Springer, Cham. DOI: https://doi.org/10.1007/978-3-030-62015-8_2. [15]. Bonacin, R.; Fernanda, O.; Pierozzi, I., Ontology models of the impacts of agriculture and climate changes on water resources: Scenarios on interoperability and information recovery, Future Generation Computer Systems. 2016, 54, 423–434, DOI: https://doi.org/10.1016/j.future.2015.04.010. [15]. Bonacin, R.; Fernanda, O.; Pierozzi, I., Ontology models of the impacts of agriculture and climate changes on water resources: Scenarios on interoperability and information recovery, Future Generation Computer Systems. 2016, 54, 423–434, DOI: https://doi.org/10.1016/j.future.2015.04.010. 32 Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License Corresponding author: gmilchev@ltu.bg Full Paper DOI: 10.2478/asn-2022-0003 ©2022 Radoslav Milchev et al., published by Sciendo This work is licensed under the Creative Commons Attribution 3.0 Public License
https://openalex.org/W3024892016	https://www.scielo.br/j/ambiagua/a/5jChwgn7vsDG448HVVQh3QP/?lang=en&format=pdf	English	null	Effect of hydraulic retention time on chemical oxygen demand and total nitrogen removal in intermittently aerated constructed wetlands	Revista Ambiente & Água	2,020	cc-by	5,205	Effect of hydraulic retention time on chemical oxygen demand and total nitrogen removal in intermittently aerated constructed wetlands Effect of hydraulic retention time on chemical oxygen demand and total nitrogen removal in intermittently aerated constructed wetlands 1Instituto de Geociências e Ciências Exatas. Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Avenida 24-A, n° 1515, CEP: 13506-900, Rio Claro, SP, Brazil. E-mail: isa_bela_pires@hotmail.com, gabrielacosta-1997@hotmail.com, marcelo.garcia@unesp.br 2Centro de Estudos Ambientais. Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Avenida 24-A, n° 1515, CEP: 13506-900, Rio Claro, SP, Brazil. E-mail: isa_bela_pires@hotmail.com, gabrielacosta-1997@hotmail.com Corresponding author. E-mail: j.queluz@unesp.br ABSTRACT This study evaluated the effect of hydraulic retention time on chemical oxygen demand (COD) and total nitrogen (TN) removal in an intermittently aerated constructed wetlands. Two horizontal subsurface-flow constructed wetlands were used: one without aeration and the other aerated intermittently (1 hour with aeration/7 hours without aeration). Both systems were evaluated treating domestic wastewater produced synthetically. The flow rate into the two CWs was 8.6 L day-1 having a hydraulic retention time of 3 days. The results show that the intermittently aerated constructed wetland were highly efficient in removing COD (98.25%), TN (83.60%) and total phosphorus (78.10%), while the non-aerated constructed wetland showed lower efficiencies in the removal of COD (93.89%), TN (48.60%) and total phosphorus (58.66). These results indicate, therefore, that intermittent aeration allows the simultaneous occurrence of nitrification and denitrification processes, improving the removal of TN in horizontal subsurface-flow constructed wetlands. In addition, the use of intermittent aeration also improves the performance of constructed wetlands in removing COD and total phosphorus. Keywords: ecotechnology, effluent treatment, nutrients removal. 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. Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Effect of hydraulic retention time on chemical oxygen demand and total nitrogen removal in intermittently aerated constructed wetlands ARTICLES doi:10.4136/ambi-agua.2504 Received: 05 Dec. 2019; Accepted: 30 Mar. 2020 Isabela Pires da Silva1,2 ; Gabriela Barbosa da Costa1,2 ; João Gabriel Thomaz Queluz1,2 ; Marcelo Loureiro Garcia1 1Instituto de Geociências e Ciências Exatas. Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Avenida 24-A, n° 1515, CEP: 13506-900, Rio Claro, SP, Brazil. E-mail: isa_bela_pires@hotmail.com, gabrielacosta-1997@hotmail.com, marcelo.garcia@unesp.br 2Centro de Estudos Ambientais. Universidade Estadual Paulista “Júlio de Mesquita Filho” (UNESP), Avenida 24-A, n° 1515, CEP: 13506-900, Rio Claro, SP, Brazil. E-mail: isa_bela_pires@hotmail.com, gabrielacosta-1997@hotmail.com Corresponding author. E-mail: j.queluz@unesp.br Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com Ambiente & Água - An Interdisciplinary Journal of Applied Science ISSN 1980-993X – doi:10.4136/1980-993X www.ambi-agua.net E-mail: ambi.agua@gmail.com 1. INTRODUCTION Constructed wetland (CW) is a simple technology which not only mimics the functions of a natural wetland but is easy to operate and maintain and cost-effective (Al-Isawi et al., 2015; 2017). In recent decades, it has garnered increasing interest in the treatment of several types of wastewater rich in biodegradable organic materials (Matos et al., 2010a). Its main component is the support medium, which can be composed of soil, sand, gravel, plant species characteristic of wetland environments, and microorganisms associated with these elements. Its design is generally aimed at maximizing the wastewater treatment efficiencies (Fia, 2009). Macrophytes play an important treatment role in CWs, especially in systems with large amounts of organic matter and ammonia (Ciria et al., 2005). The most commonly used plants are cattail (Typha sp.), common reed (Phragmites sp.), and bulrush (Scirpus sp.) (Matos and Lo Monaco, 2003). However, plant characteristics such as local climate adaptability, photosynthetic rate, oxygen transport capacity, pollutant absorption capacity, resistance to pests and diseases, and root system development should be considered. y p According to Suliman et al. (2004), the main variables considered while designing CWs are the hydraulic retention time (HRT), width, height and length, and hydraulic application rate. The hydraulic load and HRT significantly influence the efficiency in CWs (Wu et al., 2015). Low HRT can be associated with inefficient denitrification, with a longer retention time required for nitrogen removal compared to that required for organic matter removal (Lee et al., 2009). The present study therefore evaluated the dynamics of chemical oxygen demand (COD) and total nitrogen (TN) removal in relation to the HRT in intermittently aerated horizontal subsurface-flow constructed wetlands. RESUMO O objetivo do presente trabalho foi avaliar o efeito do tempo de detenção hidráulica na remoção da demanda química de oxigênio (DQO) e nitrogênio total (NT) em alagados construídos aerados intermitente. Foram utilizados dois alagados construídos de fluxo subsuperficial horizontal: um sem aeração e outro aerado intermitentemente (1 hora com aeração/7 hora sem aeração). Ambos os sistemas foram avaliados tratando água residuária doméstica sintética. Cada sistema recebeu uma vazão de 8,6 L dia-1, resultando em tempo de detenção de 3 dias. Os resultados mostram que o alagado construído aerado intermitentemente apresentou elevada eficiência na remoção de DQO (98,25%), NT (83,60%) e fósforo total 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. 2 Isabela Pires da Silva et al. (78,10%), enquanto que o sistema sem aeração apresentou menor eficiência na remoção de DQO (93,89%), NT (48,60%) e fósforo total (58,66%). Esses resultados indicam, portanto, que a aeração intermitente permite a ocorrência simultânea dos processos de nitrificação e desnitrificação, melhorando a remoção de NT em alagados construídos de fluxo subsuperficial horizontal. Além disso, o uso de aeração intermitente também melhora o desempenho de alagados construídos na remoção de DQO e fósforo total. (78,10%), enquanto que o sistema sem aeração apresentou menor eficiência na remoção de DQO (93,89%), NT (48,60%) e fósforo total (58,66%). Esses resultados indicam, portanto, que a aeração intermitente permite a ocorrência simultânea dos processos de nitrificação e desnitrificação, melhorando a remoção de NT em alagados construídos de fluxo subsuperficial horizontal. Além disso, o uso de aeração intermitente também melhora o desempenho de alagados construídos na remoção de DQO e fósforo total. Palavras-chave: ecotecnologia, remoção de nutrientes, tratamento de efluente. Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 2. MATERIALS AND METHODS The experiment was conducted in a green-house environment located at the Center for Environmental Studies (Centro de Estudos Ambientais - CEA) of the Institute of Geosciences and Exact Sciences - UNESP, Rio Claro, São Paulo, Brazil. The climate of the region is defined as Cwa (Koppën): warm temperate climate (mesothermal) with rainy summer and dry winter, with the temperature of the hottest month averaging over 22°C. The average annual precipitation and average annual temperatures are 1366.8 mm and 21.6°C, respectively. Two horizontal subsurface-flow constructed wetlands were evaluated: one without aeration (CW1) and the other aerated intermittently (CW2). Each treatment system was composed of a rectangular polypropylene water tank with a capacity of ~61 L and dimensions of 310 × 355 × 555 mm. The media used was gravel (2 ≤ Ø ≤ 9 mm) with a porosity of 53%, having an height of 30 cm and saturated by the effluent up to a height of 25 cm with a useful volume of 26 L. Two 32-mm PVC perforated tubes were installed in the longitudinal section of each constructed wetland with 18.5-cm spacing between them so that wastewater samples could be collected in the system. Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 3 Effect of hydraulic retention time on chemical … 3 CW2 was aerated intermittently for 3 h day-1 (1 h aeration/7 h non aeration) at an average aeration rate of 20 L min-1, resulting in an oxygen surface application rate of ~101.5 L m-2 min- 1. The air was applied to the system using an aquarium air compressor and a tubular air diffuser installed inside the constructed wetland support material. pp The aquatic macrophyte species used in the CWs was cattail (Typha latifolia), which was collected from a natural swamp and pruned at half height. The replanting of macrophytes in the CWs was carried out on March 07, 2018, and the planting density was approximately 15 plants per m2, that is, each system was cultivated with three plants. During the period of macrophyte fixation, the constructed wetlands were saturated with water and only on April 4, 2018, after the fixation of the plant species, was the application of wastewater started. p p pp Figure 1 shows the schematic diagram of the CWs. Figure 1 shows the schematic diagram of the CWs. Figure 1. Schematic diagram of the CWs used in the experiment. Figure 1. 2. MATERIALS AND METHODS Schematic diagram of the CWs used in the experiment. The CWs were operated from April 4, 2018 to December 7, 2018, that is, the systems were kept in operation for 250 consecutive days. The experiment was conducted using synthetic wastewater prepared with tap water with the addition of C12H22O11 (sucrose), (NH4)2SO4 (ammonium sulfate), KH2PO4 (monopotassium phosphate), MgSO4 (magnesium sulfate), FeSO4 (iron(II) sulfate), and CaCl2 (calcium chloride), equal to the synthetic effluent used in Wu et al. (2015), Fan et al. (2016), and Wu et al. (2016) works. Table 1 shows the compounds and their respective concentrations used in the synthetic effluent. Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 4 Isabela Pires da Silva et al. Table 1. Composition of the synthetic primary effluent. Chemical compound Concentration (mg L-1) C12H22O11 386 (NH4)2SO4 188 KH2PO4 18 MgSO4 10 FeSO4 10 CaCl2 10 * Chemical compounds were diluted in tap water to prepare the synthetic effluent. Source: Wu et al. (2015), Fan et al. (2016) and Wu et al. (2016). Table 1. Composition of the synthetic primary effluent. Table 1. Composition of the synthetic primary effluent. Chemical compound* Concentration (mg L-1) ab e . Co pos t o o t e sy t et c p a y e ue t. Chemical compound* Concentration (mg L-1) The effluent was applied to the CWs using peristaltic pumps, with a hydraulic loading rate of 0.044 m3 m-2 d-1 (8,6 L d-1). Therefore, considering the saturated volume of the CWs and the hydraulic loading rate, it was estimated that the HRT was approximately 3 days. However, as perforated tubes were installed along the longitudinal section in each water tank, it was possible to evaluate the concentration of pollutants according to the distance from the entry point of the influent and, consequently, according to different HRT: 0, 1, 2, and 3 days. q y g y In the period from November 1, 2018 to December 7, 2018, five effluent samples were collected from each sampling point, and following parameters were analyzed: COD, TN, ammonium (NH4+), nitrite (NO2-), nitrate (NO3-), total phosphorus (TP), potential of hydrogen (pH), and temperature. The analyses were performed at CEA-UNESP, Rio Claro Campus, according to the methodologies described in Standard Methods (APHA et al., 2012). Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 3. RESULTS AND DISCUSSION The characteristics of the influent and effluent throughout the CWs and their respective removal efficiencies are shown in Table 2. Table 2. Physico-chemical characteristics of the influent and effluent along the CWs and their respective removal efficiencies (ε). ble 2. Physico-chemical characteristics of the influent and effluent along the CWs and their pective removal efficiencies (ε). Parameter Effluent HRT (d) CW1 HRT (d) CW2 1 2 3 1 2 3 COD (mg L-1) 388.±19.7 119±29.9 43±10.4 23.8±6.6 42.2±15 9.6±5.6 6.8±2.9 ε (%) - 69.37±8.0 88.98±2.5 93.89±1.6 89.24±3.6 97.51±1.5 98.25±0.8 NH4+ (mg L-1) 39.16±6.2 25.08±5.6 18.86±8 16.48±2.3 6.52±3.3 0.82±1.9 0.58±0.6 ε (%) - 35.64±7.9 51.54±9.7 57.72±4.5 83.23±3.6 97.89±2.1 98.52±0.7 NO2- (mg L-1) 0.0042 0.043±0.04 0.03±0.05 0.019±0.01 0.059±0.04 0.027±0.02 0.048±0.08 NO3- (mg L-1) 1.36±0.3 8.02±1.2 3.96±1.3 4.26±1.6 6.98±2.6 7.9±1.4 5.98±1.1 TN (mg L-1) 40.52±2.1 33.14±3.3 22.85±5.0 20.76±3.1 13.56±3.2 8.75±1.8 6.61±1.16 ε (%) - 17.81±8.8 43.40±9.3 48.60±6.3 66.42±7.2 78.23±4.7 83.60±3.2 TP (mg L-1) 3.88±0.48 - - 1.58±0.56 - - 0.89±0.69 ε (%) - - - 58.66±17.8 - - 78.1±16.36 pH 6.96±0.25 - - 7.09±0.45 - - 6.68±0.49 S (μS cm-1) 562±19.3 - - 397±15.6 - - 578.5±72.8 T (ºC) 23.0±1.5 - - 21.5±1.7 - - 20.3±1.8 All samples were collected and analyzed between November 1, 2018 to December 7, 2018 (Mean ± SD, n = 5). Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 Effect of hydraulic retention time on chemical … 5 3.1. Chemical Oxygen Demand Figure 2 shows the average COD concentrations along the CWs. The removal efficiencies of both systems within 3 days of HRT was high, with similar removal efficiencies of 93.89% for CW1 and 98.25% for CW2. Similar values were reported in the literature, for example, Matos et al. (2010b) obtained, with HRT of ~4.8 days, COD removal efficiencies ranging from 71% to 96% in non-aerated horizontal subsurface-flow constructed wetlands treating hog wastewater; Brasil et al. (2005) obtained, in non-aerated horizontal subsurface-flow constructed wetlands treating domestic wastewater, average COD removal efficiencies of 84% and 93% for HRTs of 1.9 and 3.8 days, respectively; and Wu et al. (2016) obtained, in intermittent-aerated vertical subsurface-flow constructed wetlands treating synthetic wastewater, COD removal efficiencies >95% for HRT of 3 days. Figure 2. Average COD concentrations along both CWs. Figure 2. Average COD concentrations along both CWs. Figure 2. Average COD concentrations along both CWs. However, the removal efficiency of CW2 for HRT of 1 day was 89.24 ± 3.57%, while the value obtained for CW1 was 69.37 ± 8.04% (Table 2). The results of the present work are similar to the data reported by Wu et al. (2016), in which the authors evaluated vertical subsurface-flow constructed wetlands and with a HRT of 12 hours obtained in intermittent aerated systems and in non-aerated systems COD removal efficiencies of >88% and 76-82%, respectively. The higher efficiency of CW2 compared to that of CW1 after 1day HRT can be attributed to the intermittent artificial aeration, which stimulates the increase of biological activity, thereby contributing to a high efficiency of COD removal, nitrification, and denitrification. This is because during aeration periods aerobic heterotrophic microorganisms use organic matter as a source of energy, leading to COD removal. Moreover, during non- aeration periods (anoxic), denitrifying heterotrophic microorganisms remove COD (Wendling, 2017), indicating the occurrence of denitrification. Our results indicate, therefore, that the use of intermittent aeration improves the COD removal performance of horizontal subsurface-flow constructed wetlands, corroborating the results reported by Wu et al. (2015), in which the authors achieved COD removal efficiencies ranging from 96.42 to 98.49% in intermittently aerated CWs, and ranging from 62.68 to 85.49% in non-aerated systems. Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 3.2. Nitrogen removal The dynamics of nitrogen removal along the CWs can be seen in Figure 3. The results obtained in the present study show that the final average concentration in CW1 was Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 6 Isabela Pires da Silva et al. 16.48 mg L-1 and 20.76 mg L-1 for NH4+ and TN, respectively. Thus, the mean final removal efficiency (Table 2) of CW1 is 57.7 ± 4.5% and 48.6 ± 6.3% while in CW2, it is 98.5 ± 0.7% and 83.6 ± 3.2% for NH4+ and TN, respectively. Although the removal efficiencies of CW1 for NH4+ and TN with only 1 day of HRT are 35.64 ± 7.9% and 17.8 ± 8.8%, respectively, CW2 has a removal efficiency of 83.23 ± 3.6% and 66.4 ± 7.17% with the same HRT. That is, the efficiency levels of the aerated system increase by ~47.6% for NH4+ and by 48.6% for TN, thereby surpassing the final removal efficiency rates of CW1 in the first 24 hours of treatment. Figure 3. Dynamics of nitrogen removal along the CWs. The TN and NH4+ removal efficiencies in the CW2 can be considered satisfactory, since the average values obtained are higher than those reported in the literature for non-aerated constructed wetlands. Ramos et al. (2017) reported average TN removal efficiencies of 38-48% in systems planted with Polygonum punctatum and Chrysopogon zizanioides with HRT of 3.2 days. It should be noted that these removal efficiencies were exceeded in only 24 hours in our Figure 3. Dynamics of nitrogen removal along the CWs. Figure 3. Dynamics of nitrogen removal along the CWs. The TN and NH4+ removal efficiencies in the CW2 can be considered satisfactory, since the average values obtained are higher than those reported in the literature for non-aerated constructed wetlands. Ramos et al. (2017) reported average TN removal efficiencies of 38-48% in systems planted with Polygonum punctatum and Chrysopogon zizanioides with HRT of 3.2 days. It should be noted that these removal efficiencies were exceeded in only 24 hours in our Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 7 Effect of hydraulic retention time on chemical … CW2 system. The results obtained by Wang et al. (2016) showed a mean removal efficiency of 46% for NH4+ and 43% for TN in CWs planted with Canna indica L. with 5-day HRT. 3.2. Nitrogen removal The removal efficiencies obtained in CW2 for both parameters in 1 day of HRT were significantly higher than the values obtained by those authors. Therefore, our results indicate that intermittent aeration has enhanced the aerobic environment for nitrifiers and, in addition, the cattails have aerenchyma tissues that transport oxygen from above-ground parts into the below-ground parts, that is, the macrophyte roots released oxygen in the soil media, also increasing aerobic microbial activity. Both these mechanisms improved the NH4+ and TN removal through nitrification and also plant uptake. Due to the predominantly anaerobic conditions non-aerated subsurface-flow CWs have limitations in nitrogen removal, since the low availability of oxygen in the substrate compromises the nitrification process (Wu et al., 2015). The results obtained in the present study confirm these limitations, as can be seen in the NH4+ and TN average removal efficiencies achieved in the CW1 system, which were 57.7% and 48.8%, respectively. Similar results were also reported in the study developed by Hua et al. (2017), in which the authors obtained NH4+ and TN removal efficiencies in the ranges of, respectively, 20.7-66.9% and 38.1-51.6%. g p y Moreover, as stated above, the intermittently aerated system (CW2) showed high efficiency in the removal of NH4+ and TN, with values of, respectively, 98.5 and 83.6%. These results indicate, therefore, that the use of intermittent aeration allows the simultaneous occurrence of nitrification and denitrification, improving nitrogen removal performance of CWs. These results are similar to those obtained by Fan et al. (2016) and Wu et al. (2016). However, it is observed that the remaining fraction of nitrogen in the CW2 effluent is mainly composed of NO3-. This fact indicates that complete denitrification has not occurred, probably due to the lack of organic matter (carbon). The occurrence of partial denitrification of the effluent due to the absence of organic carbon was also observed by Wu et al. (2016). g y Vymazal and Kröpfelová (2008) concluded that, in most cases, the removal of TN in non- aerated horizontal subsurface-flow constructed wetlands is small (40-50%), as these systems do not provide simultaneous aerobic and anaerobic conditions for the occurrence of nitrification and denitrification, respectively. On the other hand, the results of the present study show that the use of intermittent aeration allows nitrification and denitrification to occur simultaneously, increasing CWs performance in the removal of TN (CW2 was 35% more efficient than CW1). 3.3. Total Phosphorus The results of the phosphorus analysis can be observed in Figure 4. Although they were considered satisfactory, since constructed wetlands are efficient in removing biochemical oxygen demand (BOD) and total suspended solids (TSS), phosphorus removal is still considered a challenge (De Rozari et al., 2015). Moreover, the results partly contradict the results of Ayaz et al. (2003) and Vymazal (2007); the former study stated that the phosphorus removal efficiency varies between 20 and 70%, depending on the plant used and the feeding regime, while according to the latter, total phosphorus removal varies between 40 and 60% among all wetland systems. The final phosphorus concentration in the CW1 was 1.58 mg L-1 and in the CW2 was 0.89 mg L-1, representing average removal efficiencies of, respectively, 58.66% and 78.1%. Although these results are much higher than those obtained by Mendonça et al. (2012), who obtained average removal efficiencies of 33.6% and 34.3% in CWs planted with cattails and with 2 days of HRT, they are similar to the results found by Ramos et al. (2017) who obtained, with 3.2 days of HRT, removal efficiencies of 51±24%, 69±22% and 45±19% in CWs without vegetation, planted with P. punctatum, and planted with and C. zizanioides, respectively. Fia et al. (2017) also reported high phosphorus removal efficiencies (73-78%) in the treatment of swine wastewater in CWSs planted with cattails with HRT of 11.8-12 days. Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 8 Isabela Pires da Silva et al. Figure 4. Total phosphorus input and output concentrations for both CWs. Figure 4. Total phosphorus input and output concentrations for both CWs. It is noteworthy that the CW2 system was on average ~20% more efficient in removing TP than the CW1 system. This difference can be attributed to the use of intermittent aeration, as it promotes an increase in biological activity, including polyphosphate-accumulating organisms (PAOs). In addition, intermittent aeration also promotes the mixing of the effluent, a fact that intensifies the contact between phosphorus and the substrate and, consequently, improves the absorption of this nutrient by the support material. Shi et al. (2017) also reported significant improvements (18-46%) in the removal of phosphorus in intermittently aerated vertical subsurface-flow constructed wetlands compared to non-aerated systems, both filled with gravel. 3.3. Total Phosphorus The results obtained by these authors indicate that intermittent aeration increased TP accumulated by support material storage and also by microbial uptake According to Vymazal (2007), phosphorus removal can be mainly associated with physical processes such as adsorption, precipitation, accumulation of organic matter in the substrate, microbial activity, and plant absorption. According to Greenway and Woolley (2001) plants play a significant role in removing phosphorus, being responsible for the absorption of up to 80% of this nutrient in CWs. However, other studies suggest that plants are responsible for only 4.81–22.33% of phosphorus uptake (Wu et al., 2013; Dzakpasu et al., 2015) and therefore have no significant role in TP removal. It is also important to note that the age of CWs has an impact on the phosphorus removal process. According to Fia et al. (2017) and Sousa et al. (2004), newly constructed wetlands have greater capacity to adsorb and precipitate phosphorus-based compounds and, over time, the system ends up saturated with these compounds, reducing their removal efficiency. Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 4. CONCLUSIONS Based on the results obtained, it can be concluded that intermittent aeration increased the efficiency of organic matter removal (represented by COD) and nutrient removal mainly in the nitrification process. The best average values of COD (98.25%), NH4+ (98.52%), TN (83.6%), and TP (78.1%) removal were obtained in the system using intermittent aeration, which demonstrates the importance of aeration in nitrogen removal by creating excellent aerobic and anaerobic conditions alternately; this also contributes to the removal of organic matter that is consumed in the denitrification process. It was observed that although intermittent aeration in a HRT greater than 3 days does not considerably improve the performance of COD removal as opposed to the results observed in the removal of the nitrogen species, it is an alternative that Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 9 Effect of hydraulic retention time on chemical … significantly increases the efficiency in the treatment of organic matter and nutrients in a HRT of less than 3 days. This suggests that it is an appropriate alternative for obtaining high performance in constructed wetland systems. significantly increases the efficiency in the treatment of organic matter and nutrients in a HRT of less than 3 days. This suggests that it is an appropriate alternative for obtaining high performance in constructed wetland systems. 5. ACKNOWLEDGMENTS The authors thank the São Paulo Research Foundation - (Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP) for their financial support (Process nos. 2017/18075-8, 2018/04320-3 and 2019/20793-1). APHA; AWWA; WEF. Standard Methods for the examination of water and wastewater. 22nd ed. Washington, 2012. 1496 p. APHA; AWWA; WEF. Standard Methods for the examination of water and wastewater. 22nd ed. Washington, 2012. 1496 p. AL-ISAWI, R. H. K.; SANAK, R.; SCHOLZ, M. Comparative study of domestic wastewater treatment by ponds and mature vertical-flow constructed wetlands. Ecological Engineering, v. 100, p. 8-18, 2017. https://doi.org/10.1016/j.ecoleng.2016.12.017 AL-ISAWI, R. H. K.; SANI, A.; ALMUKTAR, S.; SCHOLZ, M. Vertical-flow constructed wetlands treating domestic wastewater contaminated by hydrocarbons. Water Science and Technology, v. 71, n. 6, p. 938–946, 2015. https://doi.org/10.2166/wst.2015.054 AYAZ, S. Ç.; AKÇA, L.; TUNÇSIPER, B. Removal of organic, inorganic and microbial pollution from waters discharged to drinking water dams by constructed wetland systems. Project No:5022410. Istanbul: TUBITAK-Marmara Research Center, 2003. BRASIL, M. S.; MATOS, A. T.; SOARES, A. A.; FERREIRA, P. A. Qualidade do efluente de sistemas alagados construídos, utilizados no tratamento de esgoto doméstico. Revista Brasileira de Engenharia Agrícola e Ambiental, v. 9, (Suplemento), p.133-137, 2005. CIRIA, M. P.; SOLANO, M. L.; SORIANO, P. Role of macrophyte Typha latifolia in a constructed wetland for wastewater treatment and assessment of its potential as a biomass fuel. Biosystems Engineering, v. 92, p. 535-544, 2005. https://doi.org/10.1016/j.biosystemseng.2005.08.007 DZAKPASU, M.; SCHOLZ, M.; MCCARTHY, V.; JORDAN, S. Phosphorus retention and mass balance in an ICW system. Water Environ Journal, v. 29, p. 298-306, 2015. https://doi.org/10.1111/wej.12107 DE ROZARI, P.; GREENWAY, M.; EL HANANDEH, A. An investigation into the effectiveness of sand media amended with biochar to remove BOD5, suspended solids and coliforms using wetland mesocosms. Water Science and Technology, v. 71, p. 1536–1544, 2015. FAN, J.; ZHANG, J.; GUO, W.; LIANG, S.; WU, H. Enhanced long-term organics and nitrogen removal and associated microbial community in intermittently aerated subsurface flow constructed wetlands. Bioresource Technology, v. 214, p. 871–875, 2016. https://doi.org/10.1016/j.biortech.2016.05.083 FIA, F. R. L. Modelos de remoção de matéria orgânica e nutrientes de águas residuárias da suinocultura em sistemas alagados construídos. 2009. 146 f. Tese (doutorado) - Programa de pós-graduação em Engenharia Agrícola, Viçosa, 2009. Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 10 Isabela Pires da Silva et al. FIA, F. R. L.; MATOS, A. T. D.; FIA, R., BORGES, A. C.; CECON, P. R. Efeito da vegetação em sistemas alagados construídos para tratar águas residuárias da suinocultura. Revista Brasileira de Eng. Sanitária e Ambiental, v. 22, n. 2, p. 303-311, 2017. https://doi.org/10.1590/S1413-41522016123972 GREENWAY, M.; WOOLLEY, A. Changes in plant biomass and nutrient removal over 3 years in a constructed wetland, Cairns, Australia. APHA; AWWA; WEF. Standard Methods for the examination of water and wastewater. 22nd ed. Washington, 2012. 1496 p. Water Science and Technology, v. 44, p. 303–310, 2001. https://doi.org/10.2166/wst.2001.0844 HUA, Y.; PENG, L.; ZHANG, S.; HEAL, K. V.; ZHAO, J.; ZHU, D. Effects of plants and temperature on nitrogen removal and microbiology in pilot-scale horizontal subsurface flow constructed wetlands treating domestic wastewater. Ecological Engineering, v. 108, p. 70–77, 2017. https://doi:10.1016/j.ecoleng.2017.08.007 LEE, C.; FLETCHER, T. D.; SUN, G. Nitrogen removal in constructed wetland systems. Engineering in Life Sciences, v. 9, p. 11–22, 2009. https://doi.org/10.1002/elsc.200800049 MATOS, A. T.; ABRAHÃO, S. S; BORGES, A. C.; MATOS, M. P. Influência da taxa de carga orgânica no desempenho de sistemas alagados construídos cultivados com forrageiras. Revista Engenharia Sanitária e Ambiental, v. 15, n. 01, p. 83-92, 2010a. https://doi.org/10.1590/S1413-41522010000100010 MATOS, A. T.; FREITAS, W. S.; LO MONACO, P. A. V. Eficiência de sistemas alagados construídos na remoção de poluentes de águas residuárias da suinocultura. Revista Ambiente & Água, v. 5, n. 2, p. 119-132, 2010b. https://doi.org/10.4136/ambi-agua.142 MATOS, A. T.; LO MONACO, P. A. Tratamento e aproveitamento agrícola de resíduos sólidos e líquidos da lavagem e despolpa de frutos do cafeeiro. Viçosa: UFV, 2003. 68p. Boletim técnico. MENDONÇA, H. V.; RIBEIRO, C. B. M.; BORGES, A. C.; BASTOS, R. R. Remoção de nitrogênio e fósforo de águas residuárias de laticínios por sistemas alagados construídos operando em bateladas. Revista Ambiente & Água, v. 7, n. 2, p. 75-87, 2012. https://doi.org/10.4136/ambi-agua.805 RAMOS, N. D. F. S; BORGES, A. C.; GONÇALVES, G. C.; MATOS, A.T. D. Tratamento de águas residuárias de suinocultura em sistemas alagados construídos, com Chrysopogon zizanioides e Polygonum punctatum cultivadas em leito de argila expandida. Engenharia Sanitária e Ambiental, v. 22, n. 01, p. 123-132, 2017. https://doi.org/10.1590/S1413- 4152201687067 SHI, X.; FAN, J.; ZHANG, J.; SHEN, Y. Enhanced phosphorus removal in intermittently aerated constructed wetlands filled with various construction wastes. Environmental Science and Pollution Research, v. 24, p. 22524-22534, 2017. https://doi.org/10.1007/s11356-017-9870-z SOUSA, J. T.; VAN HAANDEL, A. C.; LIMA, E. P. C.; HENRIQUE, I. N. Utilização de wetland construído no pós-tratamento de esgotos domésticos pré-tratados em reator UASB. Revista de Engenharia Sanitária e Ambiental, v. 9, n. 4, p. 285-290, 2004. http://dx.doi.org/10.1590/S1413-41522004000400004 Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 11 Effect of hydraulic retention time on chemical … SULIMAN, F.; FRENCH, H.; HAUGEN, L.; KLØVE B.; JENSSEN, P. The effect of scale on the flow and transport parameters in horizontal subsurface flow constructed wetlands. Rev. Ambient. Água vol. 15 n. 3, e2504 - Taubaté 2020 APHA; AWWA; WEF. Standard Methods for the examination of water and wastewater. 22nd ed. Washington, 2012. 1496 p. In: INTERNATIONAL CONFERENCE ON WETLAND SYSTEMS FOR WATER POLLUTION CONTROL, 9., 26th of Sept. – 1st of October 2004, Avignon, France. Proceedings[…] Nanterre: IWA/Astee, 2004. 1 CD-ROM. VYMAZAL, J. Removal of nutrients in various types of constructed wetlands. Science of the Total Environment, v. 380, n. 1-3, p. 48-65, 2007. https://doi.org/10.1016/j.scitotenv.2006.09.014 VYMAZAL, J.; KRÖPFELOVÁ, L. Wastewater Treatment in Constructed Wetlands with Horizontal Subsurface Flow. [S.l.]: Springer, 2008. V. 14. 566 p. https://doi.org/10.1007/978-1-4020-8580-2 WANG, W.; DING, Y.; WANG, Y.; SONG, X.; AMBROSE, R. F.; ULLMAN, J. L.; GONG, J. Treatment of rich ammonia nitrogen wastewater with polyvinyl alcohol immobilized nitrifier biofortified constructed wetlands. Ecological Engineering, v. 94, p. 7-11, 2016. https://doi.org/10.1016/j.ecoleng.2016.05.078 WENDLING, A. C. Remoção simultânea de matéria orgânica e nitrogênio total em reator de leito estruturado com aeração intermitente tratando efluente composto por esgoto bruto e efluente de ralf. 2017. Dissertação (Mestrado) - Universidade Estadual de Ponta Grossa, Ponta Grossa, 2017. WU, H.; ZHANG, J.; LI, C.; FAN, J.; ZOU, Y. Mass balance study on phosphorus removal in constructed wetland microcosms treating polluted river water. CLEAN – Soil Air Water, v. 41, p. 844–850, 2013. https://doi.org/10.1002/clen.201200408 WU, H.; FAN, J.; ZHANG, J.; NGO, H. H.; GUO, W.; HU, Z.; LIANG, S. Decentralized domestic wastewater treatment using intermittently aerated vertical flow constructed wetlands: impact of influent strengths. Bioresource Technology, v. 176, p. 163–168, 2015. https://doi.org/10.1016/j.biortech.2014.11.041 WU, H.; FAN, J.; ZHANG, J.; NGO, H. H.; GUO, W.; LIANG, S.; LV, J.; LU, S.; WU, W.; WU, S. Intensified organics and nitrogen removal in the intermittent-aerated constructed wetland using a novel sludge-ceramsite as substrate. Bioresource Technology, v. 210, p. 101–107, 2016. https://doi:10.1016/j.biortech.2016.01.051
https://openalex.org/W2044257264	https://europepmc.org/articles/pmc3866278?pdf=render	English	null	Soluble receptor for advanced glycation end products as an indicator of pulmonary vascular injury after cardiac surgery	BMC pulmonary medicine	2,013	cc-by	5,952	* Correspondence: p.r.tuinman@amc.uva.nl 1Department of Intensive Care Medicine and Laboratory of Experimental Intensive Care and Anesthesiology (L.E.I.C.A.), Academic Medical Center, Meibergdreef 9, Amsterdam 1105, AZ, The Netherlands 4Department of Intensive Care Medicine, Academic Medical Center, Room G3-227, Meibergdreef 9, 1105, AZ Amsterdam, the Netherlands Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access © 2013 Tuinman et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Cardiac surgery is frequently complicated by an acute vascular lung injury and this may be mediated, at least in part, by the (soluble) receptor for advanced glycation end products (sRAGE). Methods: In two university hospital intensive care units, circulating sRAGE was measured together with the 68Gallium-transferrin pulmonary leak index (PLI), a measure of pulmonary vascular permeabiliy, in 60 consecutive cardiac surgery patients stratified by the amount of blood transfusion, within 3 hours of admission to the intensive care. Results: Cardiac surgery resulted in elevated plasma sRAGE levels compared to baseline (315 ± 181 vs 110 ± 55 pg/ml, P = 0.001). In 37 patients the PLI was elevated 50% above normal. The PLI correlated with sRAGE (r2 = 0.11, P = 0.018). Plasma sRAGE discriminated well between those with an elevated PLI and those with a normal PLI (area under the operator curve 0.75; P = 0.035; 95% CI 0.55-0.95), with 91% sensitivity but low specificity of 36% at a cutoff value of 200 pg/mL. Blood transfusion did not influence sRAGE levels. Conclusions: sRAGE is elevated in plasma after cardiac surgery and indicates increased pulmonary vascular permeability. The level of sRAGE is not affected by transfusion. Keywords: sRAGE, Cardiac surgery, Transfusion, Critically ill, Acute lung injury, ARDS, Pulmonary leakage index The mechanism for endothelial changes in increased alveolocapillary permeability after cardiac surgery remains unknown. The receptor for advanced glycation end prod- ucts (RAGE) is a multi-ligand cell-surface receptor, that may be involved in alveolocapillary inflammation [5]. Some of its ligands form in the presence of hyperglycaemia or oxidant stress and are termed advanced glycation endproducts (AGE). RAGE-ligand interactions propa- gate inflammatory responses [6]. RAGE is expressed in systemic vascular endothelium and nervous tissues, but the highest basal levels of RAGE are displayed by alveolar epithelial type 1 cells [7]. Accordingly, soluble RAGE (sRAGE) has been postulated as a potential plasma marker of type I cell injury [8-11]. In ALI, sRAGE is released into the alveolar and plasma com- partments [8] and high circulating levels are associated with severe ALI/ ARDS [9,10]. Also, cardiopulmonary bypass is associated with elevated plasma sRAGE levels [12], as is blood transfusion [11]. AGEs may form in Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Soluble receptor for advanced glycation end products as an indicator of pulmonary vascular injury after cardiac surgery Pieter R Tuinman1,4, Alexander D Cornet2, Maria T Kuipers1, Alexander P Vlaar1, Marcus J Schu Albertus Beishuizen2, AB Johan Groeneveld3 and Nicole P Juffermans1 Pieter R Tuinman1,4, Alexander D Cornet2, Maria T Kuipers1, Alexander P Vlaar1, Marcus J Schultz1, Albertus Beishuizen2, AB Johan Groeneveld3 and Nicole P Juffermans1 Patient data collection Potential risk factors for ALI (and therefore an increased PLI) were scored, including diabetes, smoking and alcohol abuse. Some known risk factors for ALI, such as pneumo- nia, trauma and sepsis were not taken into account as the patients included were elective surgery patients. For this reason, presence of such a risk factor was a reason to cancel surgery in these patients. Also, cardiopulmonary function was scored. Data on operation-time, clamp-time and hours of mechanical ventilation on the ICU were recorded in the electronic patient data system. Cardiogenic pulmonary edema was identified when pulmonary arterial occlusion pressure was > 18 mmHg (during the study period all patients received a pulmonary artery catheter pre-operatively). Chest radiographs routinely taken before surgery and on arrival at the ICU were scored for the pres- ence of new onset bilateral interstitial abnormalities by two independent physicians who were blinded to the predictor variables. Left ventricular function was categorized as pre- served (ejection fraction (EF) > 45%) or reduced (EF ≤45%), from pre-operative routine echocardiograms. Methods Setting The study is a secondary analysis of samples from a pro- spective case–control study (Medical Ethical Commission Academic Medical Center, Amsterdam, The Netherlands, MEC07/098#07.17.0539) in the mixed medical-surgical ICUs of two university hospitals in The Netherlands per- formed in 2006–2009 [14]. With approval from the ethical committee, patients 18 years or older were asked written informed consent for participation in the study prior to valvular and/or coronary artery surgery. Exclusion criteria were off-pump surgery, emergency surgery and the use of immunosuppressive drugs. Pulmonary leakage index (PLI) For the measurement of the PLI, as described previously [15], transferrin was labelled in vivo, after i.v. injection of 67Gallium (Ga)-citrate, 4 to 5 MBq (physical half-life 78 h; Mallinckrodt Diagnostica, Petten, The Netherlands). Patients were in the supine position and two scintillation detection probes (Eurorad C.T.T., Strasburg, France) were positioned over the right and left lung apices. Starting at the time of the i.v. injection of 67Ga, radioactivity was detected during 30 min. The 67Ga counts are cor- rected for background radioactivity, physical half-life, spillover of 67Ga, obtained by in vitro measurement of 67Ga, and expressed as cpm per lung field. At 0, 5, 8, 12, 16, 20, 25 and 30 min after 67Ga injection, blood samples (2 ml aliquots) were taken. Each blood sample was weighed and radioactivity was determined with a single-well well-counter (LKB Wallac 1480 WIZARD, Perkin Elmer, Life Science, Zaventem, Belgium), taking background, spillover of 67Ga and decay into account. Results are expressed as cpm g–1. For each blood sam- ple, a time-matched cpm over each lung was taken. The radioactivity ratio was calculated as (67Galung)/ (67Gablood) and plotted against time. The PLI was cal- culated from the slope of increase of the radioactivity ratio divided by the intercept, to correct for physical Background Cardiac surgery is frequently complicated by acute lung injury (ALI) which, together with postoperative atelectasis, may prolong length of stay in the intensive care unit (ICU) [1]. ALI following cardiac surgery may be associated with trauma, use of cardiopulmonary bypass, blood transfusions, mechanical ventilation and others [1,2]. The pulmonary leak index (PLI) can be used as a measure of alveolocapillary permeability, which plays a pivotal role in the pathogenesis of ALI/acute respiratory distress syndrome (ARDS) in critically ill patients [3], for instance after cardiac surgery [4]. Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Page 2 of 7 stored red blood cells and may ligate RAGE expressed on endothelial cells, resulting in oxidative damage in vitro [13]. To elucidate the mechanistic role of sRAGE in alveo- locapillary injury after cardiac surgery, we hypothesized that plasma sRAGE elevation following cardiac surgery is associated with increased pulmonary vascular leakage. Additionally, we investigated the role of blood transfusion as a determinant of plasma sRAGE. stored red blood cells and may ligate RAGE expressed on endothelial cells, resulting in oxidative damage in vitro [13]. blood cells were transfused to maintain the haemoglobin concentration above 5.0 mmol/L (8.7 g/dL), Platelets and FFP were transfused in the case of (suspected) bleeding. Transfusions administered in the operation room or within the first 3 hours postoperatively were included in the analysis. Design For PLI measurements, a nested case-cohort study was performed. Cardiac surgery patients were included for analysis after they had received no blood product trans- fusion perioperatively (n = 20), a limited transfusion regimen of 1–2 transfusions (n = 20) or multiple trans- fusions (> 2 units of red blood cells, 2 units of fresh frozen plasma (FFP) and 1 unit of platelets pooled from 5 donors (n = 20). Blood samples were taken before surgery and within 3 hours post operatively. In these 60 patients, within 3 hours post operatively, a PLI measure- ment and non-directed bronchoalveolar lavage (NBL) were performed. Assays A BALF and blood samples were centrifuged and stored at −80°C until assays were performed. In plasma and BALF, levels of sRAGE were determined by an ELISA developed in our laboratory [18]. In short, 96-well plates were coated overnight with mouse anti-human RAGE antibody (R&D systems, Minneapolis, Minnesota, USA). Samples diluted as appropriate were added and incu- bated for 2 hours. Next, biotinylated goat anti-human RAGE antibody (R&D Systems) was added and incu- bated for another 2 hours. Streptavidin poly-HRP was added for 30 minutes. Finally, sodium-acetate buffer (pH 5.5) containing 100 microg/ml tetramethylbenzidine and 0.003% H₂0₂ was added and the colour reaction was stopped by MH₂SO₄. All measurements were made in duplicate. For exploration of confounding variables, a logistic regression analysis was performed. Besides sRAGE, operation-time, EuroSCORE and amount of blood products were added to the model. Analysis showed that sRAGE and operation time were risk factors for a strongly increased PLI (Table 2). Of note, an OR of 1.005 for sRAGE applies to each increment of 1 pg/ml. Thereby, a patient with an sRAGE level of 190 pg/mL (which is the median increase in sRAGE in this population) has an OR of 2.59 (95% CI 1.47-3.71) for an increased PLI. Patient characteristics Patients’ baseline and peri-operative characteristics are given in Table 1. Surgery was uneventful in all patients, without the need for rethoracotomy. Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Results factors in radioactivity detection. The PLI represents the transport rate of 67Ga-transferrin from the intravascular to the extravascular space of the lungs and is therefore a measure of pulmonary vascular permeability. The values for both lung fields are averaged. The upper limit normal for the PLI is 14.7x10–3 min–1 and the measurement error is ~10%. PLI and sRAGE Cardiac surgery resulted in an elevated PLI with a mean of 30 ± 18 (normal value < 14.1) x 10-3 min-1. In addition, postoperative plasma sRAGE levels were increased compared to baseline (Figure 1A). Levels of sRAGE did not differ according to stratification by the number of blood transfusions (Figure 1B). Following surgery, levels of sRAGE in BALF were more than 10-fold lower than in plasma (12 (15) vs 287 (192) pg/mL). Patients were ar- bitrarily divided in a group with a strongly elevated PLI (>1.5 x upper limit of normal) and a less elevated PLI (<1.5 x upper limit of normal). The group with high PLI had a lower PaO₂/FiO₂-ratio than the group with lower PLI (Table 1). Patients with a more elevated PLI had a longer duration of surgery, but not of clamp- and pump time. Patients with a strongly elevated PLI (>1.5 x upper limit of normal) had a higher level of sRAGE in plasma compared to those with a less elevated PLI (Figure 2). The levels of sRAGE in BALF did not differ between patients with a strongly elevated or less elevated PLI. There was a small but positive relation between plasma sRAGE and PLI (r 0.32, r2 = 0.11, P = 0.018) (Additional file 1: Figure S3). The area under the curve of the ROC-curve applied to plasma sRAGE and PLI was 0.75 (P = 0.035; 95% CI 0.55-0.95), which represents acceptable discrimination (Additional file 2: Figure S4). For plasma sRAGE, a cut-off value of 200 pg/mL was found to have a good sensitivity of 91% but low specificity of 36% for an elevated PLI. Non-directed bronchoalveolar lavage technique Non-directed bronchoalveolar lavage was performed by instilling 20 ml of sterile 0.9% saline via a standard 50 cm, 14 gauge tracheal suction catheter as described previously [16,17]. In short, the distal end of the catheter was introduced via the endotracheal tube and advanced until significant resistance was encountered. Immediately after instillation over 10–15 seconds, fluid was aspirated before withdrawal of the catheter. Generally, 5–10 ml of fluid was recovered. Cardiothoracic surgery/anesthesia procedures g y Patients were anesthetized according to local protocol, with lorazepam, etomidate, sufentanil, and rocuronium for induction of anesthesia and sevoflurane plus propofol for maintainance of anesthesia. Steroids were given at the discretion of the cardio-anesthesiologist. As part of standard care, a pulmonary artery catheter was inserted for peri-operative monitoring. In all patients, cardiopul- monary bypass was performed under mild to moderate hypothermia (28°C-34°C), using a membrane oxygenator and a non-pulsatile blood flow. During the procedure, lungs were deflated. After the procedure, all patients were transferred to the ICU with mechanical ventilation using a pressure controlled mode with tidal volumes targeted at 6 ml/kg and positive end-expiratory pressure (PEEP). In the ICU adaptive support ventilation was used for automatic weaning in the postoperative phase. Red Page 3 of 7 Page 3 of 7 Page 3 of 7 Statistical analysis Data were checked for distribution by Kolmogorov- Smirnov test. Normally distributed data were analyzed using Students t test. Non-parametric data were analyzed with Mann–Whitney U test. Categorical data were analyzed with the chi-square test. Linear correlation coefficients are reported where appropriate by Spearman correlation. To evaluate independent causal factors for an increase in PLI, a binary logistic regression analysis, using the Enter method, was applied. Statistically and/or clinically relevant factors were added to the model. The model was evaluated using Hosmer-Lemeshow goodness-of-fit test. Patients with bilateral pulmonary changes on chest X-ray at admission to the ICU (n = 5) had a significant lower P/F ratio compared to patients without these changes (n = 54) (188 (60) vs 315 (105), P = 0.025), as expected. In addition, in these patients levels of BALF sRAGE were elevated compared to patients with non-significant pulmonary changes on chest X-ray (97 (168) vs 18 (21) pg/mL, P <0.001). Also, PLI was more elevated in these patients (45 (30) vs 28 (16) x 10-3 min-1, P =0.050). As groups differed in operation time, which may be a Data are reported as mean (± SD) or median (IQR) when appropriate. Receiver-operating-characteristic (ROC) curve was determined, the area under the curve and sensitivity and specificity derived. Statistical significance was defined as P < 0.05 and exact P values are given unless <0.001. Statistical analysis was performed with SPSS 18.0 Tuinman et al. Statistical analysis BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Page 4 of 7 Table 1 Demographic and peri-operative characteristics in cardiac surgery patients according to pulmonary leakage index (PLI) PLI < 1.5 (n = 23) PLI > 1.5 (n = 37) P Age, yearsΦ 66 (±14) 71 (±8.5) 0.43 Sex, male† 19 (82) 25 (68) 0.12 EuroSCOREΦ 4.9 (±3.2) 7.3 (±1.5) 0.45 ASA-scoreΦ 2.7 (±0.6 2.3 (±1.2) 0.67 Diabetes mellitus† 4 (17) 6 (16) 0.82 COPD† 2 (9) 3 (8) 0.87 Left ventricular function† 0.65 Reduced 8 (35) 10(27) Preserved 15 (65) 23 (62) FEV1Φ 80 (±34) 103 (±20) 0.16 Surgery data CABG† 15 (65) 15 (40) 0.06 Valve replacement† 1 (4) 9 (24) 0.09 Other type of surgery† 7 (30) 13 (35) 0.27 Clamp time, minΦ 77 (±35) 87 (±29) 0.20 Pump time, minΦ 114 (±48) 114 (±33) 0.26 Operation-time, minΦ 292 (±81) 333 (±27) 0.02 Perioperative data Red blood cells, unitsΦ 1.5 (±3.4) 2.7 (±3.2) 0.36 Fresh frozen plasma, unitsΦ 1.2 (± 2.4) 1.2 (±2.1) 0.93 Platelets, unitsΦ 0.4 (±0.6) 0.6 (±0.8) 0.13 Postoperative data CVP, mmHgΦ 11 (±5) 12 (±6) 0.35 Cardiac output, liters/min# 5.1 (1.1) 4.3 (0.3) 0.23 Mechanical ventilation, hoursΦ 11.7 (±6.2) 16.9 (±25.8) 0.33 PaO₂/FiO₂ ratio# 336 (162) 280 (112) 0.02 PLI was dichotomized into strongly elevated and modestly elevated, at a cut-off of 1.5 x elevated above upper limit of normal. EuroSCORE: European System for Cardiac Operative Risk Evaluation; ASA-score: physical status classification system according to the American Society of Anesthesiologists; FEV1: forced expiratory volume in 1 second, given in% of predicted; CVP: central venous pressure, measured at time of PLI measurement; Cardiac output and PaO2/FiO2 ratio idem; Data are presented in †counts (percents), Φmean (±SD) or in #median (IQR) when appropriate. Table 1 Demographic and peri-operative characteristics in cardiac surgery patients according to pulmonary leakage index (PLI) Pre OR Post OR 0 100 200 300 400 500 * plasma sRAGE (pg/ml) 0 20 40 200 300 400 500 No (n=20) Limited (n=20) Multiple (n=20) Plasma BALF Transfusion: sRAGE (pg/ml) A B Figure 1 A: Levels of sRAGE in plasma before and after cardiac surgery. B: Levels of sRAGE in plasma BALF after surgery according to amount of transfused products. Non: non-transfused patients; Limited: 1–2 units of blood transfused; Multiple: ≥5 units of blood transfused. * P < 0.001. Data are presented as median + interquartile range. Statistical analysis Pre OR Post OR 0 100 200 300 400 500 * plasma sRAGE (pg/ml) A A 0 20 40 200 300 400 500 No (n=20) Limited (n=20) Multiple (n=20) Plasma BALF Transfusion: sRAGE (pg/ml) B B Figure 1 A: Levels of sRAGE in plasma before and after cardiac surgery. B: Levels of sRAGE in plasma BALF after surgery according to amount of transfused products. Non: non-transfused patients; Limited: 1–2 units of blood transfused; Multiple: ≥5 units of blood transfused. * P < 0.001. Data are presented as median + interquartile range. vascular injury, independent of blood product transfu- sion. Although sRAGE levels are increased after car- diac surgery, a mediating role of endogenous AGES in alveocapillary inflammation cannot be concluded from our results altogether. Plasma levels of sRAGE were enhanced after lung trans- plantation and in ALI/ARDS, and directly associated with a poor clinical outcome [8,10]. In addition, an increase in plasma sRAGE was found in cardiac surgery patients after cardiopulmonary bypass [12]. We now demonstrate that sRAGE is linked to increased pulmonary capillary perme- ability and a decrease in oxygenation, suggesting a possible mediator role in lung injury after cardiac surgery. The higher plasma sRAGE levels were associated with a more elevated PLI, which is an established early marker of increased capillary permeability in the lung [15], so that the elevated plasma sRAGE and PLI may both reflect endothelial injury in the lungs occurring during cardiac surgery, even before the clinical criteria of ALI are met confounder in the increase in sRAGE, patients were divided according to median operation time and re- analyzed. No difference was found in the median level of plasma sRAGE stratified for operation time (267 (205) vs 298 (201) pg/ml. resp., P = 0.191). Discussion This study suggests that sRAGE levels, a biomarker of pulmonary tissue damage, are elevated in plasma after car- diac surgery and may serve as an indicator of pulmonary Page 5 of 7 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 0 10 20 30 40 200 250 300 350 400 PLI 1.5 PLI > 1.5 Plasma BALF * sRAGE (pg/ml) Figure 2 sRAGE in plasma and BALF after cardiac surgery in patients with a pulmonary leakage index (PLI) of ≤1.5 or > 1.5 times the upper limit of normal (< 14.1 x 10-3 min-1); P =0.038. Data are presented as median + interquartile range. and shedding of the receptor in pulmonary endothe- lium was caused by endogenously released AGE. Con- versely, the use of cardiopulmonary bypass elevating sRAGE may have been instrumental, as suggested be- fore [11,12,22]. Alternatively, the rise in plasma sRAGE may be a reflection of alveolar epithelial cell injury, possibly caused in part by deflation of the lungs during surgery. Since in our patients plasma levels were 10-fold higher then in BALF, a systemic production of sRAGE seems more likely then alveolar sRAGE production in this study, unless mainly involving pulmonary endothelium and intravascular shedding. Of note, one must take into account that the BALF samples are diluted to some account. This study may have implications for future efforts to decrease lung injury following cardiac surgery. Previous experimental data suggest that sRAGE may neutralize the ligand-mediated damage by acting as a decoy, thus protecting sensitive cells from the deleterious effects of ligand-RAGE hyperactivity [23,24]. In line, blocking of RAGE-ligand interaction by recombinant sRAGE was protective in murine models of lung injury [25] and ische- mic heart injury [26]. Furthermore, determining sRAGE early in the course of lung injury may be of therapeutic significance, because patients with higher sRAGE levels have been shown to benefit the most from lung protective mechanical ventilation [9]. Our results contribute to the rationale of exploring the therapeutic potential of RAGE as an interventional target in patients at risk of developing ALI/ARDS. Plasma BALF Plasma Figure 2 sRAGE in plasma and BALF after cardiac surgery in patients with a pulmonary leakage index (PLI) of ≤1.5 or > 1.5 times the upper limit of normal (< 14.1 x 10-3 min-1); P =0.038. Data are presented as median + interquartile range. [19]. Discussion The possible diagnostic value of sRAGE in lung injury found in our study may be specific for cardiac surgery pa- tients, since sRAGE was recently found not to be predictive of lung injury in other critically ill patients [20]. However, our results are in line with a previous study in children which found plasma sRAGE to enable prediction of acute lung injury after cardiac surgery [21]. We hypothesize that during progression of lung injury, sRAGE production by al- veolar cells may become more prominent and higher levels of sRAGE in BALF may be found as suggested before [8], since we found higher levels of sRAGE in BALF in patients with marked pulmonary findings on chest X-ray. In line with reflecting lung vascular injury, patients with a more el- evated PLI had a lower PaO₂/FiO₂ ratio and longer duration of mechanical ventilation. This study has several potential limitations. Although we corrected for possible confounding by performing a logistic regression analysis and by dividing patients accord- ing to operation time, we can not exclude confounders unaccounted for. In addition, we performed an in house ELISA and levels were about 4 times lower than those measured in studies using commercially available kits [12]. However, levels were comparable to a previous study in our centre [18]. Furthermore, a comparison of sRAGE to other biomarkers of lung injury (for example surfactant protein B, which leaks in the bloodstream because of alveolar- capillary damage) [27], would have strengthened our data. In contrast to the association between the volume of blood transfused and the plasma levels of sRAGE [11], we did not observe a relation between blood product transfusion and sRAGE, suggesting that upregulation Table 2 Logistic regression analysis of risk factors in cardiac surgery patients for an increased pulmonary leakage index > 1.5 times the upper limit of normal Risk factors OR (95%) p Value sRAGE, pg/mL 1.005 (1.00-1.01) 0.039 Operation time, min 1.010 (1.00-1.02) 0.030 EuroSCORE 1.090 (0.88-1.36) 0.441 Total amount of transfusions, units 0.968 (0.86-1.09) 0.579 PLI was dichotomized into strongly elevated and modestly elevated, at a cut-off of 1.5 x elevated above upper limit of normal. (14.1 x 10-3 min-1). sRAGE: soluble receptor for advanced glycation end products; EuroSCORE: European System for Cardiac Operative Risk Evaluation. Table 2 Logistic regression analysis of risk factors in cardiac surgery patients for an increased pulmonary leakage index > 1.5 times the upper limit of normal Conclusions sRAGE is elevated in plasma after cardiac surgery and indicates increased pulmonary vascular permeability. The level of sRAGE is not affected by transfusion. Authors’ contributions PRT: was instrumental in developing the study hypothesis and was intimately involved in interpretation of the results as well as manuscript preparing. He was also involved in data extraction as well as statistics. He has full access to the data, read the final version of the manuscript and agrees with all reported findings and interpretations. ADC: was instrumental in the performance of coordination of the study, data gathering and analysis. He has revised the manuscript critically for important intellectual content, read the final version of the manuscript and agrees with all reported findings and interpretations. MTK: was instrumental in the performance of laboratory analysis. She was also intimately involved with interpretations of the results and manuscript. She has revised the manuscript critically for important intellectual content, read the final version of the manuscript and agrees with all reported findings and interpretations. APV: was instrumental in the performance of data gathering. He was also intimately involved with interpretations of the results and manuscript. He has read the final version of the manuscript and agrees with all reported findings and interpretations. Furthermore, he is the archival author. MJS: was instrumental in study hypothesis and design. He has revised the manuscript critically for important intellectual content, read the final version of the manuscript and agrees with all reported findings and interpretations. AB: was instrumental in study hypothesis and design. He has revised the manuscript critically for important intellectual content, read the final version of the manuscript and agrees with all reported findings and interpretations. JBG: was instrumental in study hypothesis and design. He was intimately involved in manuscript preparing. He has read the final version of the manuscript and agrees with all reported findings and interpretations. NPJ: was instrumental in developing study hypothesis and was intimately involved in interpretation of the results as well as manuscript preparing and data statistics. She has read the final version of the manuscript and agrees with all reported findings and interpretations. All authors read and approved the final manuscript. 7. Shirasawa M, Fujiwara N, Hirabayashi S, Ohno H, Iida J, Makita K, Hata Y: Receptor for advanced glycation end-products is a marker of type I lung alveolar cells. Genes Cells 2004, 9:165–174. 7. Shirasawa M, Fujiwara N, Hirabayashi S, Ohno H, Iida J, Makita K, Hata Y: Receptor for advanced glycation end-products is a marker of type I lung alveolar cells. Genes Cells 2004, 9:165–174. 8. 1Department of Intensive Care Medicine and Laboratory of Experimental Intensive Care and Anesthesiology (L.E.I.C.A.), Academic Medical Center, Meibergdreef 9, Amsterdam 1105, AZ, The Netherlands. 2Department of Intensive Care Medicine, VU University Medical Center, De Boeleaan 1117, Amsterdam 1081, HZ, The Netherlands. 3Department of Intensive Care Medicine, Erasmus MC, ‘s-Gravendijkwal 230, Rotterdam 3015, CE, The Netherlands. 4Department of Intensive Care Medicine, Academic Medical Center, Room G3-227, Meibergdreef 9, 1105, AZ Amsterdam, the Netherlands. Authors’ contributions Uchida T, Shirasawa M, Ware LB, Kojima K, Hata Y, Makita K, Mednick G, Matthay ZA, Matthay MA: Receptor for advanced glycation end-products is a marker of type I cell injury in acute lung injury. Am J Respir Crit Care Med 2006, 173:1008–1015. 9. Calfee CS, Ware LB, Eisner MD, Parsons PE, Thompson BT, Wickersham N, Matthay MA: Plasma receptor for advanced glycation end products and clinical outcomes in acute lung injury. Thorax 2008, 63:1083–1089. 10. Mauri T, Masson S, Pradella A, Bellani G, Coppadoro A, Bombino M, Valentino S, Patroniti N, Mantovani A, Pesenti A, et al: Elevated plasma and alveolar levels of soluble receptor for advanced glycation endproducts are associated with severity of lung dysfunction in ARDS patients. Tohoku J Exp Med 2010, 222:105–112. 11. Christie JD, Shah CV, Kawut SM, Mangalmurti N, Lederer DJ, Sonett JR, Ahya VN, Palmer SM, Wille K, Lama V, et al: Plasma levels of receptor for advanced glycation end products, blood transfusion, and risk of primary graft dysfunction. Am J Respir Crit Care Med 2009, 180:1010–1015. 12. Agostoni P, Banfi C, Brioschi M, Magri D, Sciomer S, Berna G, Brambillasca C, Marenzi G, Sisillo E: Surfactant protein B and RAGE increases in the plasma during cardiopulmonary bypass: a pilot study. Eur Respir J 2011, 37:841–847. 13. Mangalmurti NS, Chatterjee S, Cheng G, Andersen E, Mohammed A, Siegel DL, Schmidt AM, Albelda SM, Lee JS: Advanced glycation end products on stored red blood cells increase endothelial reactive oxygen species generation through interaction with receptor for advanced glycation end products. Transfusion 2010, 50:2353–2360. 14. Vlaar AP, Cornet AD, Hofstra JJ, Porcelijn L, Beishuizen A, Kulik W, Vroom MB, Schultz MJ, Groeneveld AB, Juffermans NP: The effect of blood transfusion on pulmonary permeability in cardiac surgery patients: a prospective multicenter cohort study. Transfusion 2011, 52:82–90. Abbreviations AGE: Advanced glycation endproducts; ALI: Acute lung injury; ARDS: Acute respiratory distress syndrome; CI: Confidence interval; FFP: Fresh frozen plasma; ICUs: Intensive care units; IQR: Interquartile range; NBL: Non-directed bronchoalveolar lavage; OR: Odds ratio; PLI: Pulmonary leakage index; SD: Standard deviation; sRAGE: Soluble receptor for advanced glycation end products. 3. Verheij J, Van LA, Raijmakers PG, Spijkstra JJ, Girbes AR, Jansen EK, van den Berg FG, Groeneveld AB: Pulmonary abnormalities after cardiac surgery are better explained by atelectasis than by increased permeability oedema. Acta Anaesthesiol Scand 2005, 49:1302–1310. 4. Raijmakers PG, Groeneveld AB, Schneider AJ, Teule GJ, Van LA, Eijsman L, Thijs LG: Transvascular transport of 67Ga in the lungs after cardiopulmonary bypass surgery. Chest 1993, 104:1825–1832. 4. Raijmakers PG, Groeneveld AB, Schneider AJ, Teule GJ, Van LA, Eijsman L, Thijs LG: Transvascular transport of 67Ga in the lungs after cardiopulmonary bypass surgery. Chest 1993, 104:1825–1832. 5. Ramasamy R, Yan SF, Herold K, Clynes R, Schmidt AM: Receptor for advanced glycation end products: fundamental roles in the inflammatory response: winding the way to the pathogenesis of endothelial dysfunction and atherosclerosis. Ann N Y Acad Sci 2008, 1126:7–13. References Additional file 2: Figure S4. Scatterplot of the relation between pulmonary leakage index (PLI) and plasma levels of soluble receptor of advanced glycation end products (sRAGE). 1. Koch C, Li L, Figueroa P, Mihaljevic T, Svensson L, Blackstone EH: Transfusion and pulmonary morbidity after cardiac surgery. Ann Thorac Surg 2009, 88:1410–1418. 2. Koch CG, Li L, Sessler DI, Figueroa P, Hoeltge GA, Mihaljevic T, Blackstone EH: Duration of red-cell storage and complications after cardiac surgery N Engl J Med 2008, 358:1229–1239. 2. Koch CG, Li L, Sessler DI, Figueroa P, Hoeltge GA, Mihaljevic T, Blackstone EH: Duration of red-cell storage and complications after cardiac surgery. N Engl J Med 2008, 358:1229–1239. Acknowledgements W h k A T We thank Anita Tuip- de Boer for excellent technical assistance. 15. Groeneveld AB, Raijmakers PG: The 67gallium-transferrin pulmonary leak index in patients at risk for the acute respiratory distress syndrome. Crit Care Med 1998, 26:685–691. Competing interests h h d l h The authors declare that they have no competing interests. 6. Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B, Stern DM, Nawroth PP: Understanding RAGE, the receptor for advanced glycation end products. J Mol Med 2005, 83:876–886. 6. Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B, Stern DM, Nawroth PP: Understanding RAGE, the receptor for advanced glycation end products. J Mol Med 2005, 83:876–886. Additional files Additional file 1: Figure S3. The receiver operating curve of the ability of sRAGE plasma levels after cardiac surgery to predict the development of a strongly increased pulmonary leakage index of > 1.5 times the upper limit of normal (< 14.1 x 10-3 min-1). Additional file 1: Figure S3. The receiver operating curve of the ability of sRAGE plasma levels after cardiac surgery to predict the development of a strongly increased pulmonary leakage index of > 1.5 times the upper limit of normal (< 14.1 x 10-3 min-1). Page 6 of 7 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Funding source Departmental. 16. Schultz MJ, Millo J, Levi M, Hack CE, Weverling GJ, Garrard CS, van der Poll T: Local activation of coagulation and inhibition of fibrinolysis in the lung during ventilator associated pneumonia. Thorax 2004, 59:130–135. Received: 29 March 2013 Accepted: 5 December 2013 Published: 16 December 2013 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 acute lung injury in critically Ill patients. Am J Respir Crit Care Med 2013, 187(7):736–742. acute lung injury in critically Ill patients. Am J Respir Crit Care Med 2013, 187(7):736–742. 21. Liu X, Chen Q, Shi S, Shi Z, Lin R, Tan L, Yu J, Shu Q, Fang X: Plasma sRAGE enables prediction of acute lung injury after cardiac surgery in children. Crit Care 2012, 16:R91. 22. Wautier JL, Schmidt AM: Protein glycation: a firm link to endothelial cell dysfunction. Circ Res 2004, 95:233–238. 23. Vazzana N, Santilli F, Cuccurullo C, Davi G: Soluble forms of RAGE in internal medicine. Intern Emerg Med 2009, 4:389–401. 24. Santilli F, Vazzana N, Bucciarelli LG, Davi G: Soluble forms of RAGE in human diseases: clinical and therapeutical implications. Curr Med Chem 2009, 16:940–952. 25. Zhang H, Tasaka S, Shiraishi Y, Fukunaga K, Yamada W, Seki H, Ogawa Y, Miyamoto K, Nakano Y, Hasegawa N, et al: Role of soluble receptor for advanced glycation end products on endotoxin-induced lung injury. Am J Respir Crit Care Med 2008, 178:356–362. 25. Zhang H, Tasaka S, Shiraishi Y, Fukunaga K, Yamada W, Seki H, Ogawa Y, Miyamoto K, Nakano Y, Hasegawa N, et al: Role of soluble receptor for advanced glycation end products on endotoxin-induced lung injury. Am J Respir Crit Care Med 2008, 178:356–362. 26. Bucciarelli LG, Kaneko M, Ananthakrishnan R, Harja E, Lee LK, Hwang YC, Lerner S, Bakr S, Li Q, Lu Y, et al: Receptor for advanced-glycation end products: key modulator of myocardial ischemic injury. Circulation 2006 113:1226–1234. 26. Bucciarelli LG, Kaneko M, Ananthakrishnan R, Harja E, Lee LK, Hwang YC, Lerner S, Bakr S, Li Q, Lu Y, et al: Receptor for advanced-glycation end products: key modulator of myocardial ischemic injury. Circulation 2006, 113:1226–1234. 27. Magri D, Brioschi M, Banfi C, Schmid JP, Palermo P, Contini M, Apostolo A, Bussotti M, Tremoli E, Sciomer S, et al: Circulating plasma surfactant protein type B as biological marker of alveolar-capillary barrier damage in chronic heart failure. Circ Heart Fail 2009, 2:175–180. doi:10.1186/1471-2466-13-76 Cite this article as: Tuinman et al.: Soluble receptor for advanced glycation end products as an indicator of pulmonary vascular injury after cardiac surgery. BMC Pulmonary Medicine 2013 13:76. Author details 1 1Department of Intensive Care Medicine and Laboratory of Experimental Intensive Care and Anesthesiology (L.E.I.C.A.), Academic Medical Center, Meibergdreef 9, Amsterdam 1105, AZ, The Netherlands. 2Department of Intensive Care Medicine, VU University Medical Center, De Boeleaan 1117, Amsterdam 1081, HZ, The Netherlands. 3Department of Intensive Care Medicine, Erasmus MC, ‘s-Gravendijkwal 230, Rotterdam 3015, CE, The Netherlands. 4Department of Intensive Care Medicine, Academic Medical Center, Room G3-227, Meibergdreef 9, 1105, AZ Amsterdam, the Netherlands. 17. A’Court CH, Garrard CS, Crook D, Bowler I, Conlon C, Peto T, Anderson E: Microbiological lung surveillance in mechanically ventilated patients, using non-directed bronchial lavage and quantitative culture. Q J Med 1993, 86:635–648. 18. Determann RM, Wolthuis EK, Choi G, Bresser P, Bernard A, Lutter R, Schultz MJ: Lung epithelial injury markers are not influenced by use of lower tidal volumes during elective surgery in patients without preexisting lung injury. Am J Physiol Lung Cell Mol Physiol 2008, 294:L344–L350. 19. Clark SC: Lung injury after cardiopulmonary bypass. Perfusion 2006, 21:225–228. 20. Agrawal A, Matthay MA, Kangelaris KN, Stein J, Chu JC, Imp BM, Cortez A, Abbott J, Liu KD, Calfee CS: Plasma angiopoietin-2 predicts the onset of Page 7 of 7 Page 7 of 7 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 Tuinman et al. BMC Pulmonary Medicine 2013, 13:76 http://www.biomedcentral.com/1471-2466/13/76 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/W3213527394	https://www.frontiersin.org/articles/10.3389/fcell.2021.765992/pdf	English	null	Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Specific Brain Malformations	Frontiers in cell and developmental biology	2,021	cc-by	13,834	Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Speciﬁc Brain Malformations Kristen Park 1, Katelyn J. Hoff 2, Linnea Wethekam 2, Nicholas Stence 3, Margarita Saenz 4 and Jeffrey K. Moore 2* 1Department of Pediatrics and Neurology, Children’s Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO, United States, 2Department of Cell and Developmental Biology, University of Colorado Anschutz Medical Campus, Aurora, CO, United States, 3Section of Pediatric Radiology, Department of Radiology, Children’s Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO, United States, 4Section of Genetics, Department of Pediatrics, Children’s Hospital Colorado, University of Colorado Anschutz Medical Campus, Aurora, CO, United States Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different β-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in β-tubulins and located in the exchangeable GTP-binding pocket of β-tubulin. To determine the impact of T178M on β-tubulin function we created an analogous mutation in the β-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with puriﬁed mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of β-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our ﬁndings demonstrate the importance of tubulin’s GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development. Reviewed by: Reviewed by: Lorraine Santy, The Pennsylvania State University (PSU), United States Curtis Okamoto, University of Southern California, United States Shelley Halpain, University of California, San Diego, United States Reviewed by: Lorraine Santy, The Pennsylvania State University (PSU), United States Curtis Okamoto, University of Southern California, United States Shelley Halpain, University of California, San Diego, United States Correspondence: Jeffrey K. Moore jeffrey.moore@cuanschutz.edu Correspondence: Jeffrey K. Moore jeffrey.moore@cuanschutz.edu Specialty section: This article was submitted to Membrane Trafﬁc, a section of the journal Frontiers in Cell and Developmental Biology Specialty section: This article was submitted to Membrane Trafﬁc, a section of the journal Frontiers in Cell and Developmental Biology Received: 28 August 2021 Accepted: 29 October 2021 Published: 18 November 2021 Received: 28 August 2021 Accepted: 29 October 2021 Published: 18 November 2021 Keywords: cytoskeleton, microtubule, tubulin, tubulinopathy, brain development ORIGINAL RESEARCH published: 18 November 2021 doi: 10.3389/fcell.2021.765992 Edited by: Oscar Marcelo Lazo, University College London, United Kingdom 2.1 Clinical History of TUBB2A and TUBB3 Patients 2.1.1 Patient 1 The index patient was born via vaginal delivery at term to a 31- year-old G3P1 mother after an uncomplicated pregnancy. Birth weight was 8l bs 4 oz (50–75%), length was 22.5 inches (>97%), occipitofrontal circumference (OFC): 35 cm (25–50%) with normal Apgar scores. Developmental delays were noted early on when he did not roll or sit on time. Initial neurologic evaluation was performed due to concern for ventriculomegaly on head ultrasound at 9 months of age. Subsequent MRI revealed signiﬁcant abnormalities, as described below (Figures 1A–E). He was noted on exam to have poor visual ﬁxation and tracking such that Ophthalmology evaluation was recommended. This revealed cortical visual impairment, mild optic nerve hypoplasia, right exophoria, and strabismic amblyopia. He was referred to Neurology at 11 months of age due to emerging microcephaly (head circumference 44 cm, 7%, Z −1.5) in addition to the above concerns. Myoclonic seizures began at approximately 16 months of age and have not resolved with trials of two appropriate anticonvulsant medications. Most recent electroencephalogram (EEG) performed at 2 years 11 months showed diffuse slowing and attenuation along with an absence of a posterior dominant rhythm, generalized spikes, as well as multifocal independent spike discharges. He underwent a number of non-diagnostic genetic and metabolic evaluations with several providers culminating in trio whole exome sequencing at 4 years of age. This identiﬁed a heterozygous de novo c.533C>T (p.T178M) substitution in the TUBB2A gene. His most recent examination at 11 years of age noted dysmorphic facial features including widely spaced teeth, a pronounced maxilla, and bilateral epicanthal folds. From a developmental Over the past two decades, mutations impacting the genes encoding tubulins have emerged as major genetic causes of brain development disorders. These include ‘tubulinopathies’ which are characterized as cortical malformations linked to de novo, heterozygous missense mutations in tubulin isotypes (Keays et al., 2007; Poirier et al., 2010; Bahi-Buisson et al., 2014; Cushion et al., 2014; Fukumura et al., 2016; Mutch et al., 2016; Aiken et al., 2017; Di Donato et al., 2017; Ejaz et al., 2017; Rodan et al., 2017; Severino et al., 2020; Brock et al., 2021). Citation: These differences in assembly kinetics drive dynamic instability, the ability of microtubules to stochastically transition between phases of growth and shortening, that permit rapid generation and remodeling of the microtubule cytoskeleton. p g g y The tubulin heterodimer consists of α- and β-tubulin subunits, which are each encoded by families of genes known as tubulin isotypes. The human genome encodes 9 α-tubulin isotypes and 8–10 β-tubulin isotypes (Findeisen et al., 2014). In general, tubulin isotypes exhibit unique expression programs that differ across cell types and across development (Luduena and Banerjee, 2008). Because of these unique expression programs, tubulin isotypes are hypothesized to play distinct roles throughout human development, particularly during the complicated process of neurodevelopment. In support of this hypothesis, mouse models harboring null mutations in the α-tubulin isotype Tuba1a exhibit severe defects in brain development, while null mutations in the β-tubulin isotypes Tubb2a and Tubb2b exhibit milder cortical malformations (Bittermann et al., 2019). In contrast, a separate study examined mice with null mutations in the β-tubulin isotype, Tubb3, which is expressed in neurons during brain development (Burgoyne et al., 1988; Chen et al., 2003; Katsetos et al., 2003), and reported no clear brain development phenotypes (Latremoliere et al., 2018). This suggests that loss of function mutations in some isotypes may be compensated for by upregulation of alternative isotypes. The roles of isotypes in metazoan development remains a frontier in the microtubule ﬁeld. Citation: Tubulin is the protein building block of the microtubule cytoskeleton and accounts for 25% of total protein in the mammalian brain, by far the most abundant GTPase in the brain (Hiller and Weber, 1978). GTPase activity is essential for tubulin function, particularly during brain development when dynamic microtubules support billions of cell divisions and the subsequent migration and process extension events that generate the complex architecture of the human adult brain. Each tubulin heterodimer binds to two molecules of GTP. One GTP binds to a site buried within tubulin and Park K, Hoff KJ, Wethekam L, Stence N, Saenz M and Moore JK (2021) Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype- Speciﬁc Brain Malformations. Front. Cell Dev. Biol. 9:765992. doi: 10.3389/fcell.2021.765992 November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org Disease Mutations Stabilize Beta Tubulins Park et al. This study seeks to address both questions. We describe two patients with analogous T178M mutations in either the TUBB2A or TUBB3 β-tubulin isotypes exhibiting cortical malformations consistent with tubulinopathies. Our analysis of RNA expression is consistent with roles for TUBB2A and TUBB3 during brain development, particularly in neurons. In contrast, a previous report identiﬁed a T178M mutation in the TUBB4A isotype of a patient exhibiting H-ABC (Tonduti et al., 2016). Our analysis indicates that TUBB4A expression is primarily limited to oligodendrocytes and increases during postnatal brain development. To investigate the mechanistic impact of T178M on tubulin protein activity, we modeled the mutation in yeast β-tubulin. Our results show that T178M weakens the assembly activity of puriﬁed tubulin protein, but when expressed as a heterozygous mutation in cells promotes the formation of stable microtubules with diminished dynamic instability. We conclude that T178M disrupts the control of dynamic instability by tubulin’s GTPase to create kinetically stabilized microtubules. When present in different β-tubulin isotypes, this mutation dominantly alters microtubule activity in cells that express the affected isotype. hydrolyzes very slowly (Weisenberg et al., 1968; Spiegelman et al., 1977). The second GTP binds to the so-called exchangeable site on β-tubulin, is hydrolyzed during assembly into microtubules and exchanges when tubulin is in solution (Kobayashi, 1975; Nogales et al., 1998). GTPase activity at the exchangeable site controls a switch in tubulin conformation that creates a six-fold greater microtubule association for the GTP-bound state (Carlier and Pantaloni, 1978). 2.1 Clinical History of TUBB2A and TUBB3 Patients 2.1.1 Patient 1 (J) shows more extensive and intense abnormal T2 hyperintensity in the juxtacortical white matter of the bilateral occipital lobes (arrowheads), with preservation of overlying cortex. FIGURE 1 \| MR images of TUBB2A-T178M and TUBB3-T178M patients. Selected axial T1 (A–D) and T2 (E) MR images of patient #1 (with TUBB2A mutation) obtained at age 4 demonstrate common features of a moderate tubulinopathy MR phenotype, including (A) mildly small pons and vermis, thin corpus callosum; (B) small, asymmetric-appearing brainstem, mild superior vermian disorganization; (C) abnormal, asymmetric basal ganglia with enlarged caudate and thin anterior limb internal capsule separating the caudate and putamen; (C,D) diffuse pattern of tubulinopathy-associated dysgyria consisting of normal-thickness but closely spaced and disorganized-appearing sulci with orientation directed radially to a central point; (D) ventriculomegaly with low white matter volume. (E) also demonstrates asymmetric T2 hyperintensity in the juxtacortical white matter of the left occipital lobe (arrow), with preservation of overlying cortex. Selected axial T1 (F–I and T2 (J) MR images of patient #2 (with TUBB3 mutation) obtained at age 3 demonstrate similar features as patient #1, but of a more severe MRI phenotype, including (F) moderately to markedly small pons and vermis with posterior vermian rotation, mildly thickened tectum relative to brainstem size, thin corpus callosum; (G) small, asymmetric-appearing brainstem, moderate/severe superior vermian and cerebellar foliar disorganization; (H) abnormal, asymmetric basal ganglia with no discernable separation of the caudate head and putamen; (H,I) tubulinopathy-associated dysgyria with shallow, closely spaced and disorganized-appearing sulcation also directed radially to a central point; (I) ventriculomegaly with low white matter volume. (J) shows more extensive and intense abnormal T2 hyperintensity in the juxtacortical white matter of the bilateral occipital lobes (arrowheads), with preservation of overlying cortex. and the ketogenic diet (initiated at age 3 years). Chromosomal microarray (180K with 5-cell screen) was performed prior to discharge from the NICU and did not identify any copy number variations. She had a mildly elevated Creatine Kinase (CK) on serum from DOL1 (247, normal range 29–168 IU/L) such that a muscular dystrophy next generation sequencing (NGS) panel (sequencing of 33 genes and del/dup of 4; see Materials and Methods) was performed without signiﬁcant ﬁndings (Mendell et al., 2012). This was followed by testing for congenital disorders of glycosylation which was also negative. 2.1 Clinical History of TUBB2A and TUBB3 Patients 2.1.1 Patient 1 In addition, cases of the postnatal brain development disorder hypomyelination with atrophy of the basal ganglia and cerebellum (H-ABC) are linked to heterozygous missense mutations in the β-tubulin isotype TUBB4A (Hersheson et al., 2013; Lohmann et al., 2013; Simons et al., 2013; Hamilton et al., 2014; Miyatake et al., 2014; Purnell et al., 2014; Lu et al., 2017). As the spectrum of phenotypes and associated mutations continues to increase, two major questions remain unresolved: 1) how do the different ⍺- and β-tubulin isotypes contribute to normal brain development such that mutations in different genes might lead to unique developmental consequences; and 2) what are the mechanisms through which patient-associated missense mutations alter tubulin protein function to lead to aberrant development? November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 2 Park et al. Disease Mutations Stabilize Beta Tubulins FIGURE 1 \| MR images of TUBB2A-T178M and TUBB3-T178M patients. Selected axial T1 (A–D) and T2 (E) MR images of patient #1 (with TUBB2A mutation) obtained at age 4 demonstrate common features of a moderate tubulinopathy MR phenotype, including (A) mildly small pons and vermis, thin corpus callosum; (B) small, asymmetric-appearing brainstem, mild superior vermian disorganization; (C) abnormal, asymmetric basal ganglia with enlarged caudate and thin anterior limb internal capsule separating the caudate and putamen; (C,D) diffuse pattern of tubulinopathy-associated dysgyria consisting of normal-thickness but closely spaced and disorganized-appearing sulci with orientation directed radially to a central point; (D) ventriculomegaly with low white matter volume. (E) also demonstrates asymmetric T2 hyperintensity in the juxtacortical white matter of the left occipital lobe (arrow), with preservation of overlying cortex. Selected axial T1 (F–I and T2 (J) MR images of patient #2 (with TUBB3 mutation) obtained at age 3 demonstrate similar features as patient #1, but of a more severe MRI phenotype, including (F) moderately to markedly small pons and vermis with posterior vermian rotation, mildly thickened tectum relative to brainstem size, thin corpus callosum; (G) small, asymmetric-appearing brainstem, moderate/severe superior vermian and cerebellar foliar disorganization; (H) abnormal, asymmetric basal ganglia with no discernable separation of the caudate head and putamen; (H,I) tubulinopathy-associated dysgyria with shallow, closely spaced and disorganized-appearing sulcation also directed radially to a central point; (I) ventriculomegaly with low white matter volume. 2.1 Clinical History of TUBB2A and TUBB3 Patients 2.1.1 Patient 1 She was referred to our neurogenetics clinic at 3 years of age where repeat review of her MRI was suggestive of a tubulinopathy such that targeted testing for cortical brain malformation genes via NGS was performed (56 in panel; see Materials and Methods) and identiﬁed a heterozygous pathogenic c.533C>T (p.T178M) substitution in TUBB3. Parental testing was not completed. At her last examination (6 years) she was non-verbal and non-ambulatory with mixed tone. She spends time in a stander for weight bearing. She is fed by gastrostomy tube only due to a combination of swallowing dysfunction and oral aversion. Both epileptic spasms and focal motor seizures occur on a daily basis despite combination therapy with vigabatrin and clobazam in addition to the ketogenic diet, although family does report improved alertness with the latter. Most recent EEG performed at 4 years of age showed a diffusely standpoint, he acquired head control at 6 months and sat at approximately 3 years of age. Now at age 11, he crawls for locomotion, can walk short distances with a walker, is working on feeding himself, and uses an assistive communication device to indicate his wants and needs as he is largely non-verbal. Family has never reported mirror movements (Nissenkorn et al., 2021) nor has this been indicated on reviewed neurologic examinations. Current seizure frequency is 2–3 myoclonic seizures per day on valproic acid monotherapy, which has been stable. 2.1.2 Patient 2 Patient 2 was born at 38 weeks gestation by vaginal delivery to a 40-year-old G11P4-5 mother after a pregnancy complicated only by the diagnosis of fetal brain malformations on routine ultrasound, felt to be consistent with semilobar holoprosencephaly. Birth weight was 6 lbs 12 oz (10–25%), length was 20 inches (50–75%), and OFC: 30.5 cm (0.1%, Z −4.4) with normal Apgar scores. Neonatal MRI performed on DOL1 showed several abnormalities, as described below (Figure 1F–J). Ophthalmology evaluation revealed intermittent R exotropia and hyperopia, dysconjugate gaze, and strabismus. Seizures began at 1–2 months and were initially myoclonic in nature but subsequently, infantile spasms and focal seizures emerged which have been refractory to multiple medications November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 3 GURE 2 \| β-tubulin isotype composition is speciﬁc during developmental time periods and different cell types. (A–C) Expression of human β-tubulin isotype nscripts, TUBB4A (A), TUBB3 (B), TUBB2A (C), across development. Data obtained from cortical and subcortical samples publicly available RNA-sequencing data ler et al., 2014). Data binned according to developmental time point; fetal (8–37 pcw), postnatal (4 months–11 years), adult (13–40 years). Each data point represents a from an individual patient. Bars are the mean values and error bars represent the 95% conﬁdence interval. p-values were determined by one-way ANOVA. –G) Transcript expression of human β-tubulin isotypes in the adult whole cortex (D), neurons (E), oligodendrocytes (F), and fetal and mature astrocytes (G). Data ained from publicly available RNA-sequencing data (Zhang et al., 2016). Each data point represents data from an individual patient. Bars are the mean values and or bars represent the standard error of the mean. et al. Disease Mutations Stabilize Beta Tubulins Disease Mutations Stabilize Beta Tubulins Park et al. FIGURE 2 \| β-tubulin isotype composition is speciﬁc during developmental time periods and different cell types. (A–C) Expression of human β-tubulin isotype transcripts, TUBB4A (A), TUBB3 (B), TUBB2A (C), across development. Data obtained from cortical and subcortical samples publicly available RNA-sequencing data (Miller et al., 2014). Data binned according to developmental time point; fetal (8–37 pcw), postnatal (4 months–11 years), adult (13–40 years). Each data point represents data from an individual patient. Bars are the mean values and error bars represent the 95% conﬁdence interval. p-values were determined by one-way ANOVA. 2.1.2 Patient 2 (D–G) Transcript expression of human β-tubulin isotypes in the adult whole cortex (D), neurons (E), oligodendrocytes (F), and fetal and mature astrocytes (G). Data obtained from publicly available RNA-sequencing data (Zhang et al., 2016). Each data point represents data from an individual patient. Bars are the mean values and error bars represent the standard error of the mean. slow background with poor organization, slow spike and wave patterns, and multifocal epileptiform discharges. 2021), although the phenotype for patient #2 was generally more severe than that of patient #1. These ﬁndings included thin corpus callosum, dysgyria (sometimes referred to as a tubulinopathy- associated dysgyria) (Di Donato et al., 2017), small and disorganized cerebellar vermis, small and asymmetric- appearing brainstem, abnormal basal ganglia, and ventriculomegaly with low white matter volume (Figures 2.4 T178M Decreases Tubulin Assembly Activity A previous study reported an analogous T178M mutation in the TUBB4A β-tubulin isotype, in a patient exhibiting hypomyelinating leukodystrophy (Tonduti et al., 2016). The different clinical features of that patient, compared to the patients described here motivated us to ask whether differences could be attributed to the expression of the TUBB4A, TUBB3, and TUBB2A β-tubulin isotypes throughout neurodevelopment. We next examined how the T178M substitution impacts tubulin function. Threonine 178 is positioned within β-tubulin’s T5 loop, a highly conserved region that forms part of the exchangeable nucleotide-binding pocket between tubulin heterodimers (Figures 3A,B). Because the cycle of GTP binding, hydrolysis and GDP release at the exchangeable site plays a central role in regulating tubulin’s assembly activity, we tested how T178M affects tubulin assembly. We generated the analogous T178M mutation in the budding yeast β-tubulin gene TUB2 and used an inducible expression system to purify yeast ⍺β-tubulin heterodimers containing tub2-T178M (Johnson et al., 2011). We measured assembly activity by incubating a range of concentrations of tub2-T178M tubulin heterodimers or wild- type control tubulin heterodimers in seeded assembly assays, and imaged microtubule dynamics by DIC microscopy (see Materials and Methods). We ﬁnd that tub2-T178M tubulin assembles into microtubules; but only at concentrations ≥1 µM. In contrast, wild-type tubulin assembles at a ﬁve-fold lower concentration (Figure 3C). We determined the concentration dependent rate of microtubule assembly for tub2-T178M tubulin to be 14.7 µm/h/ µM, compared to 28.7 µm/h/µM for wild-type tubulin (Figure 3C). Together, these data indicate that the T178M substitution in β-tubulin weakens the assembly activity of tubulin heterodimers. The demand for tubulin varies throughout the course of neurodevelopment, and the expression of different tubulin isotypes changes to meet this demand. To gain insight into how TUBB4A, TUBB3, and TUBB2A expression temporally changes during development, we analyzed publicly available RNA-sequencing data from whole brain cortex samples (Miller et al., 2014). TUBB4A transcript levels increase signiﬁcantly between the fetal and postnatal samples, followed by a smaller increase between postnatal and adult brains (Figure 2A). This is consistent with a role for TUBB4A in later stages of development, such as the myelination that occurs in postnatal brains (Miyatake et al., 2014; Tonduti et al., 2016; Joyal et al., 2019). In contrast, TUBB3 transcript levels decrease approximately 30% between the fetal and postnatal brain samples (Figure 2B). TUBB2A transcript levels decrease steadily from fetal to postnatal to adult brains (Figure 2C). 2.2 Magnetic Resonance Imaging Both patients demonstrated most of the typical imaging features previously described in tubulin gene mutations (Mutch et al., 2016; Di Donato et al., 2017; Severino et al., 2020; Brock et al., November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org Frontiers in Cell and Developmental Biology \| www.frontiersin.org 4 Disease Mutations Stabilize Beta Tubulins Park et al. 1A–D, F–I). While both patient’s white matter volumes were low, they were for the most part normally myelinated. However, small areas of white matter in the occipital lobes of both patients demonstrated abnormal T2 hyperintensity with preserved overlying cortex (Figures 1E,J; see arrows). in oligodendrocytes, and middling in fetal and mature astrocytes (Figures 2D–G). Together, this RNA-sequencing data supports the hypothesis that the T178M mutation in the TUBB3 and TUBB2A isotypes disrupt tubulin function during early neurodevelopment, and particularly in neurons, leading to changes in cortical architecture and brain size consistent with the clinical observations made in these patients. 2.4 T178M Decreases Tubulin Assembly Activity These data suggest an enhanced role for TUBB3 and TUBB2A during early brain development. Tubulin isotype expression varies not only across the timing of neurodevelopment, but also across different cell types. Therefore, we used publicly available RNA-sequencing data to compare transcript levels of nine β-tubulin isotypes in whole cortex samples and samples of isolated neurons, oligodendrocytes and astrocytes (Zhang et al., 2016). These data provide insight into both the blend of β isotypes in different cell types in the brain, as well as how the relative abundance of each isotype changes between cell types. TUBB4A transcript is abundant in the whole cortex sample and accounts for the largest portion of β isotypes in oligodendrocytes, but is diminished in neurons and astrocytes (Figures 2D–G). Patients with the T178M mutation in TUBB4A suffer from hypomyelination, which is in accordance with this isotype being dominantly expressed and important in oligodendrocytes. In contrast, TUBB3 makes up a very small portion of the β-tubulin transcripts in the whole cortex and cell- type speciﬁc samples in this dataset (Figures 2D–G). However, these data (aside from the astrocytes) are obtained from adult tissue samples and would not reﬂect TUBB3 transcript levels during fetal and postnatal neurodevelopment. TUBB2A transcript is expressed at moderate levels in samples from the whole adult cortex, but also shows cell-type speciﬁc enrichment. It dominates the β-tubulin pool in neurons, is scarcely expressed While conducting these experiments, we found that microtubules assembled from tub2-T178M mutant tubulin exhibit fewer catastrophes—transitions from assembly or pause states to disassembly—than microtubules assembled from wild- type control tubulin. Whereas microtubules assembled from wild-type yeast tubulin exhibit an average of one catastrophe per 20 min of assembly time (Geyer et al., 2015), we observed no catastrophe events for all T178M mutant microtubules during 402 min of total observed assembly time. 2.5 T178M Dominantly Inhibits Microtubule Dynamics Having established that the T178M weakens tubulin’s assembly activity, we next examined microtubule dynamics in cells, where tubulin activity is controlled by a wide variety of extrinsic regulatory proteins. We generated the T178M mutation at the chromosomal TUB2 locus, the sole β-tubulin isotype in budding yeast. Heterozygous diploids expressing one copy of tub2-T178M and one copy of wild-type TUB2 are viable; however, we were unable to recover haploid cells expressing tub2-T178M as the only source of β-tubulin. This result indicates that tub2-T178M November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 5 Disease Mutations Stabilize Beta Tubulins Park et al. FIGURE 3 \| T178M impairs the assembly activity of β-tubulin. (A) Model of the GTP-bound state of the exchangeable site in tubulin, showing the position of T178 in the β-tubulin T5 loop and N101 in the T3 loop. The structure shown here is from porcine brain tubulin assembled in GTP and saturated in BeF3 −to mimic the GTP-bound state (pdb 6gze; Estevez-Gallego et al., 2020). (B) Alignment of the T5 loop from 10 human β-tubulin isotypes and the budding yeast β-tubulin Tub2. (C) Microtubule growth rates for indicated concentrations of wild-type and tub2-T178M mutant tubulin. Error bars represent SD. FIGURE 3 \| T178M impairs the assembly activity of β-tubulin. (A) Model of the GTP-bound state of the exchangeable site in tubulin, showing the position of T178 in the β-tubulin T5 loop and N101 in the T3 loop. The structure shown here is from porcine brain tubulin assembled in GTP and saturated in BeF3 −to mimic the GTP-bound state (pdb 6gze; Estevez-Gallego et al., 2020). (B) Alignment of the T5 loop from 10 human β-tubulin isotypes and the budding yeast β-tubulin Tub2. (C) Microtubule growth rates for indicated concentrations of wild-type and tub2-T178M mutant tubulin. Error bars represent SD. are signiﬁcantly faster than T178M and dynamicity is signiﬁcantly greater (Figures 4D,E; Table 1). Furthermore, microtubules in tub2Δ heterozygous null cells exhibit a level of pause time that is similar to wild-type controls, and signiﬁcantly less than that observed in tub2-T178M heterozygotes (Table 1). We conclude that the changes in microtubule dynamics seen in tub2-T178M heterozygous cells represents a dominant phenotype. alone is not sufﬁcient to support β-tubulin function in yeast. Our characterization of T178M in yeast is therefore conducted in heterozygous diploids expressing one copy of tub2-T178M and one copy of wild-type TUB2. 2.5 T178M Dominantly Inhibits Microtubule Dynamics We measured microtubule dynamics by fusing three copies of GFP to the microtubule plus-end tracking protein Bik1/CLIP-170 at its native chromosomal locus (Figure 4A). We also attempted an alternative approach of ectopically expressing a fusion of GFP to ⍺-tubulin (GFP-Tub1); however, expressing this fusion severely impaired the viability of tub2-T178M heterozygotes (data not shown). Figure 4B shows representative “lifeplots” of individual astral microtubule lengths over time in a wild- type diploid cell and a tub2-T178M heterozygous cell expressing Bik1-3GFP. The complete set of microtubule dynamics parameters measured in this experiment is reported in Table 1. Our analysis reveals key differences for microtubule dynamics in tub2-T178M heterozygotes compared to wild-type controls: 1) astral microtubules are longer (Figure 4C), 2) exhibit slower polymerization rates (Figure 4D), and 3) exhibit an overall lower level of microtubule length change over time, known as “dynamicity” (Figure 4E). In general, microtubules in the tub2- T178M heterozygotes tend to persist in a “paused” state where they do not undergo sustained polymerization or depolymerization (Table 1). p yp As a second test, we compared the sensitivity of heterozygous tub2 mutants to drugs that bind to soluble tubulin and disrupt microtubule dynamics—benomyl and nocodazole (Kilmartin, 1981; Vasquez et al., 1997). We ﬁnd that tub2-T178M heterozygotes are inhibited by low levels of benomyl (5 µg/ml) where tub2Δ heterozygous null mutants grow similarly to wild- type controls (Figure 4F). This is consistent with tub2-T178M acting as a dominant negative. We also compared the benomyl sensitivity of cells heterozygous for tub2-C354S, a mutation substituting cysteine 354 for serine. Cysteine 354 is located far from the exchangeable GTP binding site, but the tub2-C354S mutation blocks the conformational changes in β-tubulin that normally accompany GTP hydrolysis (Geyer et al., 2015). The tub2-C354S mutation was previously shown to stabilize microtubules and exhibit long and persistently paused astral microtubules, reminiscent of tub2-T178M heterozygotes (Gupta et al., 2002). Despite the similar phenotypes for microtubule dynamics in cells, we ﬁnd opposite phenotypes in the benomyl sensitivity assay—whereas tub2-T178M heterozygotes are highly sensitive to benomyl, tub2-C354S heterozygotes are strongly resistant (Figure 4F). We next tested how cell proliferation was affected by nocodazole. While tub2-T178M heterozygotes do not exhibit a proliferation phenotype in the absence of drug, upon the addition of low doses of nocodazole (5 µM) tub2-T178M mutants exhibit a signiﬁcantly slower doubling time compared to wild-type controls and tub2Δ heterozygous null mutants (p < 0.01; Figure 4G). Frontiers in Cell and Developmental Biology \| www.frontiersin.org 2.5 T178M Dominantly Inhibits Microtubule Dynamics Interestingly, tub2-C354S mutants exhibit increased doubling time in the presence of 10 µM nocodazole compared to wild-type controls in 10 µM nocodazole Our ﬁnding that microtubules in tub2-T178M heterozygotes are longer and relatively stable compared to wild-type controls is surprising since our in vitro experiments indicate that puriﬁed tub2-T178M heterodimers exhibit loss of assembly activity (Figure 3). To determine whether the tub2-T178M acts as a recessive or dominant allele in cells, we compared tub2-T178M heterozygous cells to tub2Δ heterozygous null cells that possess one copy of wild-type TUB2 and the second copy is excised and replaced with a selectable marker (referred to as “tub2Δ”; see Materials and Methods). If T178M acts as a recessive, loss of function mutant then it would be predicted to behave similarly to tub2Δ heterozygous null. We ﬁnd that tub2Δ heterozygous null cells exhibit slower polymerization rates that are similar to T178M; but depolymerization rates in heterozygous null cells November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 6 Disease Mutations Stabilize Beta Tubulins Park et al. FIGURE 4 \| The β-tubulin T178M mutation dominantly decreases microtubule dynamics in vivo. (A) Representative image of microtubules in a cell labelled with Bik1-3GFP. Scale bar 1 µm. (B) Representative life plots of astral microtubules in wild type, TUB2/tub2Δ, and TUB2/tub2-T178M cells. Astral microtubule length was measured over time by plotting the distance between plus-end-associated Bik1-3GFP and the proximal spindle pole. Red and blue lines on wild type plot show a single polymerization and depolymerization rate respectively. (C) Histogram of astral microtubule length measurements in pre-anaphase cells. +/+, n 27 cells; Δ/+, n 30 cells; T178M/+, n 28 cells; T178V/+, n 18 cells. (D) Polymerization rates of astral microtubules in pre-anaphase cells. Dots are individual polymerization events. Lines indicate the mean. Asterisks indicate statistical signiﬁcance (alpha 0.05) determined by Tukey-Kramer post hoc test. (E) Dynamicity of astral microtubules in pre- anaphase cells. Dots are values from individual cells, each imaged for 8 min. Lines indicate the mean. Asterisks indicate statistical signiﬁcance (alpha 0.05) determined by Tukey-Kramer post hoc test. (F) 10-fold dilution series of indicated strains were spotted to rich media (YPD; ‘‘no drug’’) or rich media supplemented with benomyl (5 or 15 µg/ml) and grown at 30°C. 2.5 T178M Dominantly Inhibits Microtubule Dynamics (G) Doubling time in minutes at 30°C of diploid yeast cells with indicated genotypes were calculated from change in absorbance (OD600) over time in the presence of indicated concentration nocodazole or DMSO-only control. Dots represent doubling time measurements for each technical replicate from three separate experiments, and the mean value of the technical replicates within each experiment is represented as a triangle. Bars indicate mean with 95% CI. (H,I) Western blots of dilution series of puriﬁed yeast α- and β-tubulin proteins and protein lysates from cells with indicated genotypes. (J) Estimated molecules/cell for wild type, TUB2/tub2Δ, and TUB2/tub2-T178M cells. Each pair of connected dots represents estimates for α- and β-tubulin from a technical replicate. Park et al. Disease Mutations Stabilize Beta Tubulins FIGURE 4 \| The β-tubulin T178M mutation dominantly decreases microtubule dynamics in vivo. (A) Representative image of microtubules in a cell labelled with Bik1-3GFP. Scale bar 1 µm. (B) Representative life plots of astral microtubules in wild type, TUB2/tub2Δ, and TUB2/tub2-T178M cells. Astral microtubule length was measured over time by plotting the distance between plus-end-associated Bik1-3GFP and the proximal spindle pole. Red and blue lines on wild type plot show a single polymerization and depolymerization rate respectively. (C) Histogram of astral microtubule length measurements in pre-anaphase cells. +/+, n 27 cells; Δ/+, n 30 cells; T178M/+, n 28 cells; T178V/+, n 18 cells. (D) Polymerization rates of astral microtubules in pre-anaphase cells. Dots are individual polymerization events. Lines indicate the mean. Asterisks indicate statistical signiﬁcance (alpha 0.05) determined by Tukey-Kramer post hoc test. (E) Dynamicity of astral microtubules in pre- anaphase cells. Dots are values from individual cells, each imaged for 8 min. Lines indicate the mean. Asterisks indicate statistical signiﬁcance (alpha 0.05) determined by Tukey-Kramer post hoc test. (F) 10-fold dilution series of indicated strains were spotted to rich media (YPD; ‘‘no drug’’) or rich media supplemented with benomyl (5 or 15 µg/ml) and grown at 30°C. (G) Doubling time in minutes at 30°C of diploid yeast cells with indicated genotypes were calculated from change in absorbance (OD600) over time in the presence of indicated concentration nocodazole or DMSO-only control. Dots represent doubling time measurements for each technical replicate from three separate experiments, and the mean value of the technical replicates within each experiment is represented as a triangle. Bars indicate mean with 95% CI. 2.5 T178M Dominantly Inhibits Microtubule Dynamics (H,I) Western blots of dilution series of puriﬁed yeast α- and β-tubulin proteins and protein lysates from cells with indicated genotypes. (J) Estimated molecules/cell for wild type, TUB2/tub2Δ, and TUB2/tub2-T178M cells. Each pair of connected dots represents estimates for α- and β-tubulin from a technical replicate. FIGURE 4 \| The β-tubulin T178M mutation dominantly decreases microtubule dynamics in vivo. (A) Representative image of microtubules in a cell labelled with Bik1-3GFP. Scale bar 1 µm. (B) Representative life plots of astral microtubules in wild type, TUB2/tub2Δ, and TUB2/tub2-T178M cells. Astral microtubule length was measured over time by plotting the distance between plus-end-associated Bik1-3GFP and the proximal spindle pole. Red and blue lines on wild type plot show a single polymerization and depolymerization rate respectively. (C) Histogram of astral microtubule length measurements in pre-anaphase cells. +/+, n 27 cells; Δ/+, n 30 cells; T178M/+, n 28 cells; T178V/+, n 18 cells. (D) Polymerization rates of astral microtubules in pre-anaphase cells. Dots are individual polymerization events. Lines indicate the mean. Asterisks indicate statistical signiﬁcance (alpha 0.05) determined by Tukey-Kramer post hoc test. (E) Dynamicity of astral microtubules in pre- anaphase cells. Dots are values from individual cells, each imaged for 8 min. Lines indicate the mean. Asterisks indicate statistical signiﬁcance (alpha 0.05) determined by Tukey-Kramer post hoc test. (F) 10-fold dilution series of indicated strains were spotted to rich media (YPD; ‘‘no drug’’) or rich media supplemented with benomyl (5 or 15 µg/ml) and grown at 30°C. (G) Doubling time in minutes at 30°C of diploid yeast cells with indicated genotypes were calculated from change in absorbance (OD600) over time in the presence of indicated concentration nocodazole or DMSO-only control. Dots represent doubling time measurements for each technical replicate from three separate experiments, and the mean value of the technical replicates within each experiment is represented as a triangle. Bars indicate mean with 95% CI. (H,I) Western blots of dilution series of puriﬁed yeast α- and β-tubulin proteins and protein lysates from cells with indicated genotypes. (J) Estimated molecules/cell for wild type, TUB2/tub2Δ, and TUB2/tub2-T178M cells. Each pair of connected dots represents estimates for α- and β-tubulin from a technical replicate. November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 7 Disease Mutations Stabilize Beta Tubulins Park et al. 2.5 T178M Dominantly Inhibits Microtubule Dynamics TABLE 1 \| Dynamics of astral microtubules measured in diploid yeast cells during preanaphase. Microtubule dynamics +/+ +/Δ +/T178M +/T178V p-value from single factor ANOVA Polymerization rate (µm/min) 1.51 ± 0.14 1.17 ± 0.11a 0.96 ± 0.13a 1.29 ± 0.24 2.20E-06 Depolymerization rate (µm/min) 2.28 ± 0.21 1.88 ± 0.16a 1.15 ± 0.12a 1.66 ± 0.24a 1.72E-14 Dynamicity (subunits/s) 0.46 ± 0.05 0.39 ± 0.04 0.28 ± 0.03a 0.34 ± 0.05a 1.35E-07 Catastrophe frequency (events/min of polymerization time) 0.92 ± 0.12 0.86 ± 0.11 0.78 ± 0.14 0.69 ± 0.11 0.07 Rescue frequency (events/min of depolymerization time) 1.43 ± 0.23 1.33 ± 0.24 0.99 ± 0.29 0.97 ± 0.28 0.03 % Time paused 20.8 ± 5.0 22.5 ± 5.8 35.6 ± 7.7a 31.4 ± 8.7 0.01 Number of cells analyzed 27 30 28 18 — Image series were collected at 5 s intervals for 8 min and astral microtubule lengths were measured at each timepoint. Values shown are the mean ± 95% conﬁdence intervals of pooled data from at least three separate experiments. aStatistical signiﬁcance (alpha 0.05) for comparison to wild-type controls using Tukey-Kramer post hoc test. TABLE 1 \| Dynamics of astral microtubules measured in diploid yeast cells during preanaphase. therefore explored how the T178M substitution might impact GTP-binding and/or hydrolysis activity to alter the nucleotide- dependence of tubulin assembly. (Figure 4G). Together, these results indicate that T178M exhibits a gain of function phenotype when expressed as a heterozygous allele, creating long and stable microtubules with exquisite sensitivity to the destabilizing drugs. p y We considered two predictions based on structural studies of tubulin. The ﬁrst prediction comes from a cryo-EM study comparing the conformational rearrangement of tubulin in different nucleotide states, which indicates a potentially state- speciﬁc role for threonine 178. When the GTP-mimicking analogue GMPCPP is bound at the exchangeable site, the hydroxyl group in the side chain of threonine 178 appears to hydrogen bond to the ribose of the guanosine nucleotide; but when GDP is bound, the T5 loop is repositioned and the hydrogen bond is lost (Manka and Moores, 2018). We reasoned that replacing threonine’s hydroxyl group with methionine’s S-methyl thioether could either weaken afﬁnity for GTP and/or disrupt the ordering of the T5 loop exchangeable site when GTP is bound. 2.5 T178M Dominantly Inhibits Microtubule Dynamics An alternative prediction comes from crystal structures of tubulin oligomers mimicking the GTP-bound or GDP-bound states, which demonstrate a different role for the T5 loop (Nawrotek et al., 2011; Estevez-Gallego et al., 2020). Analysis of these structures indicates when GDP is bound at the exchangeable site, the T5 loop visits a “ﬂipped in” conformation where threonine 178 hydrogen bonds to asparagine 101 in β-tubulin’s T3 loop. When GTP is bound at the exchangeable site, the T5 loop adopts a ‘ﬂipped out’ conformation that allows asparagine 101 in the T3 loop to interact with α-tubulin across the longitudinal interface, and is thought to promote lateral interactions through an allosteric mechanism (Nawrotek et al., 2011). We reasoned that the T178M substitution could destabilize the “ﬂipped in” conformation of T5 and thereby disrupt nucleotide-dependent regulation of interdimer interactions. Both of these predictions depend on the highly conserved hydroxyl group of the threonine 178 side chain. y g g We considered two hypotheses to explain the hypersensitivity of tub2-T178M heterozygotes to benomyl and nocodazole: either the destabilizing drugs act in an additive fashion with the T178M mutation to suppress microtubule dynamics, or T178M causes underproduction of tubulin protein that leads to a higher proportion of drug-bound tubulin in the cells. To test the second hypothesis, we measured levels of total α- and β-tubulin in tub2-T178M heterozygotes and controls. We ﬁrst used dilutions of puriﬁed yeast tubulin on western blots to create standard curves of signal per nanogram of α- or β-tubulin (Figure 4H). We then prepared lysates from wild-type, tub2- T178M heterozygous and tub2Δ heterozygous null cells under conditions to depolymerize microtubules and shift tubulin into the soluble fraction, and used western blots to estimate the number of α- and β-tubulin molecules per cell (see Materials and Methods; Figure 4I). Whereas tub2Δ heterozygous null cells show little change in the amount of α- or β-tubulin compared to wild-type controls, tub2-T178M heterozygotes exhibit signiﬁcantly higher concentrations of both α- and β-tubulin (Figure 4J). We conclude that T178M does not cause underproduction of tubulin protein. Instead, T178M appears to cause an increase in the supply of α- and β-tubulin. Based on these results and our measurements of microtubule dynamics in tub2-T178M heterozygotes, we favor the hypothesis that the hypersensitivity of these mutants to benomyl and nocodazole reﬂects an additive suppression of microtubule dynamics. 2.6 Investigating the Mechanism of Kinetic Stabilization by T178M To test whether the effects of the T178M mutation can be attributed to the loss of hydrogen bonding, we created an alternative substitution that lacks the hydroxyl group by introducing a valine substitution at position 178 of budding yeast TUB2. We predicted that the tub2-T178V mutant might phenocopy tub2-T178M in our assays of microtubule dynamics and drug sensitivity. However, our results show unique phenotypes for tub2-T178V. Astral microtubules in tub2- Our ﬁndings that T178M mutant tubulin exhibits decreased assembly activity in vitro but creates stable microtubules in heterozygous cells is reminiscent of the effects of microtubule- targeting drugs that suppress tubulin on-off kinetics. For example, the drug vinblastine is thought to kinetically stabilize tubulin by increasing the afﬁnity between heterodimers and eliminating the differences in assembly rate between GTP- and GDP-bound tubulin (Toso et al., 1993; Castle et al., 2017). We November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 8 Disease Mutations Stabilize Beta Tubulins Park et al. T178V heterozygous cells exhibit length distributions and dynamicity that are similar to microtubules in tub2-T178M heterozygotes, but with signiﬁcantly faster rates of polymerization and depolymerization, and less time in pause (Figures 4D,E; Table 1). In addition, tub2-T178V cells exhibit different levels of sensitivity to benomyl and nocodazole. tub2- T178V heterozygotes show greater resistance to high concentrations of benomyl (15 µg/ml) than wild-type controls (Figure 4F) but are slightly more sensitive to high levels of nocodazole (10 µM) than wild-type controls (Figure 4G). Our results for tub2-T178V are reminiscent of the tub2-C354S mutant that constitutively mimics the GTP-bound state tub2 (Figures 4F,G; Gupta et al., 2002; Geyer et al., 2015). We conclude that hydrogen bonding by threonine 178 is important for normal tubulin function and that loss of the threonine hydroxyl group may create a constitutive GTP-like state at the exchangeable site. However, the different phenotypes of T178M vs T178V mutants in our assays indicate that the introduction of the methionine side chain in T178M further disrupts the exchangeable site leading to slower polymerization and depolymerization and increased time in the paused state. linking ocular motility disorders to mutations in TUBB3 (Tischﬁeld et al., 2010). A previous study reported a T178M mutation in TUBB4A in a patient with a milder hypomyelinating phenotype (Tonduti et al., 2016), although no MR images from the patient were published. 3 DISCUSSION We report two new tubulinopathy cases linked to de novo, heterozygous T178M missense mutations in either the TUBB2A or TUBB3 β-tubulin isotypes. Comparing the structural features of brain development in these patients with a previously reported hypomyelinating leukodystrophy linked to an analogous T178M mutation in the TUBB4A isotype suggests speciﬁc developmental requirements for each β-tubulin isotype. In addition, we ﬁnd that the T178M substitution creates a kinetically stabilized population of tubulin heterodimers that dominantly alters microtubule dynamics, representing a novel tubulinopathy mechanism. Together, our results underscore the importance of tubulin as the most abundant GTPase in the brain, and demonstrate how the family of tubulin isotypes may be used to meet shifting demands for tubulin activity at different times of development and in different cell types. The speciﬁc impacts of tubulinopathy mutations on brain development may therefore be related to both the timing and cell-type speciﬁc expression of the affected tubulin isotype and also the degree of molecular-level dysfunction in the mutant tubulin protein. Although the TUBB4A isotype is also abundantly and speciﬁcally expressed in the mammalian brain, expression analysis and patient features indicate a role that is distinct from that of TUBB2A (Lewis et al., 1985). TUBB4A expression increases during postnatal brain development, and is strongly enriched in oligodendrocytes (Figure 2). Furthermore, it is well- established that mutations in TUBB4A are linked to hypomyelinating leukodystrophies, consistent with an important role for TUBB4A in oligodendrocyte function and the myelination of axons during postnatal brain development (Figure 5; Hersheson et al., 2013; Lohmann et al., 2013; Simons et al., 2013; Hamilton et al., 2014; Miyatake et al., 2014; Purnell et al., 2014; Lu et al., 2017). Thus the expression of TUBB4A in oligodendrocytes during postnatal brain development, or TUBB2A in neurons of the developing brain may provide an important mechanism for regulating tubulin activity in those cells; but it also creates speciﬁc vulnerabilities to isotype mutations. The MRI ﬁndings in both patients are consistent with other published cases of tubulin mutations in general and speciﬁcally in TUBB2A and TUBB3 (Figure 1). These include the frequently reported ﬁndings of abnormal basal ganglia, thin corpus callosum, abnormal brainstem and vermis, and disorganized cerebral cortex referred to as dysgyria or tubulinopathy- associated dysgyria. All of these ﬁndings could reasonably be expected to occur due to defects in neuronal migration. 2.6 Investigating the Mechanism of Kinetic Stabilization by T178M In both of our patients, the cerebral white matter was low in volume but normally myelinated as is frequently the case in tubulinopathies. However, both of our patients had areas of abnormal white matter signal in the occipital regions. The appearance is non-speciﬁc, and while it could indicate a prior injury, given the association of tubulin mutations with hypomyelination, areas of focal hypomyelination are possible. In principle, tubulin isotypes provide metazoans with discrete transcriptional modules for meeting demands for tubulin subunits that shift during development and according to cell type. Our ﬁndings support the general conclusion that brain malformations caused by tubulinopathies are related to the timing and cell-type speciﬁc expression of the affected tubulin gene. The βII isotype is the most abundant β-tubulin in the adult mammalian brain (Banerjee et al., 1988). Our analysis shows that TUBB2A, one of the two genes for βII in humans, is expressed during brain development and maintained at high expression levels in neurons of the adult brain (Figure 2). Consistent with a role in brain development, a variety of TUBB2A mutations have been previously linked to cortical malformations (Figure 5; Cushion et al., 2014; Lee et al., 2014; Rodan et al., 2017; Ejaz et al., 2017; Brock et al., 2021). These patients commonly exhibit dysgyria, dysmorphic or hypoplastic corpus callosum and basal ganglia, and microcephaly; reminiscent of patient #1 (Figure 1). These ﬁndings are consistent with TUBB2A providing a major source of β-tubulin in neurons during brain development. Frontiers in Cell and Developmental Biology \| www.frontiersin.org 3 DISCUSSION While both patients had a similar array of malformations, patient #2’s MRI phenotype was overall more severe than patient #1. Patient #2 also presented distinct ocular motility phenotypes that suggest impairment of cranial nerves, consistent with previous reports The TUBB3 isotype presents a more complicated story. TUBB3 is expressed in very few tissues of the human body. In the brain it is expressed in neurons, but not glia, and its expression is most abundant during fetal brain development and declines postnatally (Figure 2; Burgoyne et al., 1988; Chen et al., 2003; Katsetos et al., 2003). Importantly, TUBB3 is expressed at much lower levels in neurons than other β-tubulin isotypes, including TUBB2A (Figure 2). Despite its low level of November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 9 Park et al. Park et al. Disease Mutations Stabilize Beta Tubulins FIGURE 5 \| Diagram of identiﬁed patient mutations in TUBB2A, TUBB3 and TUBB4A. Amino acid sequence alignment of human TUBB2A, TUBB3 and TUBB4A overlayed with a model of β-tubulin domain structure adapted from Lowe et al. (2001). Residues mutated in tubulinopathy cases are highlighted in pink. Residues mutated in CFOEM3 cases are highlighted in blue. Residues mutated in hypomyelinating leukodystrophies are highlighted in yellow. Boxed residues contact nucleotide in the E-site, while dashed boxed residues contact nucleotide in the N-site. FIGURE 5 \| Diagram of identiﬁed patient mutations in TUBB2A, TUBB3 and TUBB4A. Amino acid sequence alignment of human TUBB2A, TUBB3 and TUBB4A overlayed with a model of β-tubulin domain structure adapted from Lowe et al. (2001). Residues mutated in tubulinopathy cases are highlighted in pink. Residues mutated in CFOEM3 cases are highlighted in blue. Residues mutated in hypomyelinating leukodystrophies are highlighted in yellow. Boxed residues contact nucleotide in the E-site, while dashed boxed residues contact nucleotide in the N-site. expression, mutations in TUBB3 are linked to a variety of neuronal disorders, ranging from cortical malformations (Poirier et al., 2010; Oegema et al., 2015; Fukumura et al., 2016) to ocular motility disorders that arise from the failure of motor neurons to innervate eye muscles (Figure 5; Tischﬁeld et al., 2010). Whereas the latter cases do not present obvious cortical malformations, patient #2 described in our study exhibits both cortical malformations and ocular motility disorders. This suggests that the developmental consequences of TUBB3 missense mutations may be highly variable for different mutations. 3 DISCUSSION One possible explanation for this variability is that cortical neurons and motor neurons may require different levels of TUBB3 protein activity, and the impact of a particular TUBB3 mutation may reﬂect the severity of its impact on protein function. Our understanding of the molecular impact of TUBB3 mutations may therefore present a valuable window into unique functions of TUBB3 that are important for neuronal development, despite its relatively low abundance. increased, but the ratio of α- to β-tubulin is not obviously altered. It is therefore unlikely that the phenotypes of T178M mutant yeast cells are attributable to superstoichiometric levels of β-tubulin, which is known to be toxic in yeast cells (Burke et al., 1989; Weinstein and Solomon, 1990). The cause of the increased α- and β-tubulin in T178M heterozygous yeast is unclear, but could be related to the long and stable microtubules observed in mutant cells, which would be expected to deplete the cell’s soluble tubulin pool and might stimulate increased tubulin biogenesis through an autoregulatory mechanism that has been described in mammalian cells (Cleveland et al., 1981; Lin et al., 2020). Whether cells in T178M patients would also be expected to exhibit increased levels of α- and β-tubulin is unclear. Levels of α- and β-tubulin isotype proteins have not been measured in human cells, including the cells of the developing brain. If our mRNA analysis in Figure 2 is representative of protein levels, then TUBB2A protein, for example, could comprise approximately half of the β-tubulin in neurons. Therefore a heterozygous TUBB2A T178M mutant allele could supply approximately one quarter of the β-tubulin in neurons. But this is purely speculation, and it is important to note that mRNA levels are often not representative of protein levels and whether this is the case for tubulin has not been established. Our yeast experiments are designed to assess the impact of the T178M mutant in a system where the mutant allele is under endogenous expression and avoids artifacts generated by ectopic expression. Going forward, it will be important for the tubulinopathy ﬁeld to determine isotype composition at the protein level for the cells of the developing brain, and then create experimental models that faithfully recapitulate this composition to test how shifts in the isotype blend may contribute to disease. Several models have been proposed to explain the dominance of tubulinopathy mutations. 3 DISCUSSION Fundamentally, the mutant tubulin protein produced by heterozygous tubulinopathy mutations represents no more than half of the tubulin pool in the cell; and in some cases, such as TUBB3 tubulinopathies, may represent a much smaller fraction. In multiple reports, tubulinopathy mutations in α- or β-tubulin have been shown to disrupt the formation of stable heterodimers and lead to diminished tubulin protein levels in neurons and other cell expression models (Keays et al., 2007; Tian et al., 2010; Tischﬁeld et al., 2010; Gartz Hanson et al., 2016; Buscaglia et al., 2020). This suggests that the cellular and developmental consequences of tubulinopathies can arise from haploinsufﬁciency of tubulin protein. Our results for the T178M mutation do not support this model. We ﬁnd that the heterozygous expression of T178M β-tubulin in yeast cells leads to increased levels of α- and β-tubulin protein, compared to wild- type controls (Figure 4I). Importantly, the amount of soluble α- and β-tubulin protein measured in these experiments is Our results present a possible alternative model in which the mutant tubulin acts in trans to dominantly suppress microtubule dynamics. Although puriﬁed T178M tubulin exhibits weakened assembly activity, it can co-assemble with wild-type tubulin in yeast cells to form microtubules that assemble and disassemble November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 10 Disease Mutations Stabilize Beta Tubulins Park et al. slowly, and dwell in a paused state with a stable polymer length (Figures 4B–E; Table 1). Microtubule dynamics vary across species and across cell types within a species, so it is important to consider that the magnitude of the effect of T178M on microtubule dynamics and related physiological consequences may be different in the human brain, where microtubules are regulated by a host of extrinsic factors. Going forward it may be informative to conduct further in vitro reconstitution experiments to measure microtubule dynamics when T178M mutant tubulin is blended at different proportions with wild-type tubulin. These experiments would provide direct evidence that T178M can co-assemble with wild- type tubulin, even in the absence of extrinsic microtubule regulators, and demonstrate how much T178M must be present in the lattice to exert effects on microtubule dynamics. heterozygotes (Figure 4D; Table 1), and the cells are hypersensitive to benomyl (Figure 4F). 3 DISCUSSION These effects stand in contrast to the TUBB3 E410K mutation that was identiﬁed in patients with ocular motility disorder and accelerates microtubule polymerization (Tischﬁeld et al., 2010; Ti et al., 2018). To our knowledge, kinetic stabilization of microtubules by T178M represents a novel mechanism for tubulinopathies. Whether this model of kinetic stabilization is speciﬁc to the T178M mutation in β-tubulin or represents a mechanistic theme for other α and β tubulinopathy mutations will be the focus of future investigations. 4 MATERIALS AND METHODS The attenuation of microtubule dynamics by T178M is reminiscent of the effects of microtubule-targeting drugs, and exempliﬁed by vinblastine. Whereas adding vinblastine to soluble tubulin inhibits microtubule assembly, adding substoichiometric concentrations of vinblastine to pre-formed microtubules suppresses plus-end growth and shortening, leading to paused microtubules (Wilson et al., 1982; Toso et al., 1993; Castle et al., 2017). This apparent contradiction of inhibiting tubulin assembly and inhibiting microtubule disassembly is explained by vinblastine stabilizing conformations of tubulin at the microtubule ends that are not compatible with microtubule dynamics. Indeed, vinblastine binds near the longitudinal interface between tubulin heterodimers and stabilizes high- afﬁnity tubulin-tubulin complexes that assemble into oligomeric spirals, quite distinct from the straight microtubule polymer (Na and Timasheff, 1982). Thus, even at low concentrations where only a fraction of tubulin is bound to drug (Toso et al., 1993), vinblastine appears to trap tubulin conformational states that potently suppress microtubule dynamics. 4.1 Patient Sequencing Whole exome sequencing and analysis of patient #1 was conducted by GeneDx (Gaithersberg, MD) using the Cortical Brain Malformations Panel, which analyzed the following genes: ACTB, ACTG1, ADGRG1, AKT3, ARFGEF2, ARX, ASPM, ATP6V0A2, B3GALNT2, B3GNT1, CCND2, CUL4B, DCX, DYNC1H1, ERMARD, FAT4, FKRP, FKTN, FLNA, GMPPB, GPSM2, ISPD, KIF1BP, KIF2A, KIF5C, LAMB1, LAMC3, LARGE, NDE1, OCLN, LIS1, POMGNT1, POMGNT2, POMK, POMT1, POMT2, PQBP1, RAB18, RAB3GAP1, RAB3GAP2, RELN, RTTN, SRD5A3, SRPX2, TBC1D20, TMEM5, TUBA1A, TUBA8, TUBB, TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBG1, VLDLR, WDR62. Data are available in the ClinVar archive under accession SCV000321987. Initial Next Generation Sequencing of patient #2 was conducted by Athena Diagnostics (Marlburough, MA) using a Congenital Muscular Dystrophy Advanced Sequencing Evaluation panel, which analyzed the following genes: B3GALNT2, B3GNT1, CHKB, COL6A1, COL6A2, COL6A3, DNM2, DPM2, FHL1, FKRP, FKTN, ISPD, ITGA7, LAMA2, LARGE, LMNA, POMGNT1, POMGNT2, POMT1, POMT2, SEPN1, TCAP, TMEM5. Subsequently, genome sequencing analysis was conducted by GeneDx using the Cortical Brain Malformations Panel described above. 4.1 Patient Sequencing q g Whole exome sequencing and analysis of patient #1 was conducted by GeneDx (Gaithersberg, MD) using the Cortical Brain Malformations Panel, which analyzed the following genes: ACTB, ACTG1, ADGRG1, AKT3, ARFGEF2, ARX, ASPM, ATP6V0A2, B3GALNT2, B3GNT1, CCND2, CUL4B, DCX, DYNC1H1, ERMARD, FAT4, FKRP, FKTN, FLNA, GMPPB, GPSM2, ISPD, KIF1BP, KIF2A, KIF5C, LAMB1, LAMC3, LARGE, NDE1, OCLN, LIS1, POMGNT1, POMGNT2, POMK, POMT1, POMT2, PQBP1, RAB18, RAB3GAP1, RAB3GAP2, RELN, RTTN, SRD5A3, SRPX2, TBC1D20, TMEM5, TUBA1A, TUBA8, TUBB, TUBB2A, TUBB2B, TUBB3, TUBB4A, TUBG1, VLDLR, WDR62. Data are available in the ClinVar archive under accession SCV000321987. Initial Next Generation Sequencing of patient #2 was conducted by Athena Diagnostics (Marlburough, MA) using a Congenital Muscular Dystrophy Advanced Sequencing Evaluation panel, which analyzed the following genes: B3GALNT2, B3GNT1, CHKB, COL6A1, COL6A2, COL6A3, DNM2, DPM2, FHL1, FKRP, FKTN, ISPD, ITGA7, LAMA2, LARGE, LMNA, POMGNT1, POMGNT2, POMT1, POMT2, SEPN1, TCAP, TMEM5. Subsequently, genome sequencing analysis was conducted by GeneDx using the Cortical Brain Malformations Panel described above. y We propose that the T178M mutant may also create complexes at microtubule plus ends between mutant tubulins or between mutant and wild-type tubulins that suppress microtubule dynamics. Mechanistically, this is likely to involve breaking the network of conformational changes in β-tubulin’s T5 and T3 loops that normally accompany tubulin’s GTPase cycle and facilitate the free energy changes between nucleotide states that underlie dynamic instability (Nawrotek et al., 2011). T3 has been proposed to act as analogously to the Switch II region of canonical GTPases; it binds to the γ-phosphate of GTP and to the α-tubulin of the adjacent heterodimer, and is reordered after GTP hydrolysis to interact with T5 through hydrogen bonding between the T3 asparagine 101 and the T5 threonine 178 (Nogales et al., 1999; Nawrotek et al., 2011; Estevez-Gallego et al., 2020). Our results from heterozygous T178M or T178V mutants indicate that preventing this hydrogen bond in a portion of a cell’s tubulin pool creates hyperstable microtubules with suppressed dynamic instability (Figures 4B–E). Introduction of the methionine side chain in T178M appears to have consequences beyond loss of hydrogen bonding, since microtubules in T178M heterozygotes exhibit slower polymerization and depolymerization than those in T178V 4.5 In Vivo Microtubule Dynamics Assays 4.5 In Vivo Microtubule Dynamics Assays Asynchronous cultures of cell expressing Bik1-3GFP were grown to early log phase in nonﬂuorescent medium and mounted in imaging chambers (Fees et al., 2017). Images were collected on a Nikon Ti-E microscope equipped with a 1.45 NA 100× CFI Plan Apo objective, piezo electric stage (Physik Instrumente, Auburn, MA), spinning disk confocal scanner unit (CSU10; Yokogawa), 488 and 561-nm lasers (Agilent Technologies, Santa Clara, CA), and an EMCCD camera (iXon Ultra 897; Andor Technology, Belfast, United Kingdom) using NIS Elements software (Nikon). During imaging, the temperature of the stage was maintained at 30°C. 4.4 Yeast Strains and Manipulation 4.4 Yeast Strains and Manipulation General yeast manipulation, media and transformation were performed by standard methods. A list of yeast strains is provided in Supplementary Table S1. To construct T178M and T178V mutations, we used site-directed mutagenesis (Agilent Technologies; Santa Clara, CA) of a plasmid containing the TUB2 locus, from 450 base pairs 5’ of the coding sequence through 427 base pairs 3’ of the coding sequence. Mutations were conﬁrmed by sequencing, and then the mutant TUB2 plus a TRP1 selectable marker was ampliﬁed from the plasmid by PCR, and integrated into the native TUB2 genomic locus. The integrated mutants were conﬁrmed by sequencing. The Bik1-3GFP integrating plasmid was a gift from Dr. David Pellman (Harvard University). 4.6 Benomyl Sensitivity y y Cells with were grown to saturation at 30°C in rich media and a 10-fold dilution series of each was spotted to either YPD or YPD supplemented with 5 or 15 µg/ml benomyl. Plates were incubated at 30°C for 2–3 days before imaging. The full experiment was completed at least three times for each genotype. 4.2 RNA Transcript Expression Data Image series of individual microtubules were converted into kymographs, and rates were determined as the change in length divided by the change in time for each polymerization event. 4.2 RNA Transcript Expression Data RNA-sequencing data comparing RNA transcript expression across pre- and post-natal developmental time were obtained from both male and female cortical and subcortical samples published in the developmental transcriptome on brainspan. org (Miller et al., 2014). Data for TUBB4A, TUBB3, and TUBB2A from 8 pcw to 40 years were binned into three categories representative of different phases of development. “Fetal” includes data from 8 to 37 pcw, “postnatal” includes 4 months to 11 years, and “adult” includes 13–40 years. RNA transcript expression data of the nine human β-tubulin isotypes were obtained from brainrnaseq.org (Zhang et al., 2016). Data were collected from adult samples of whole cortex, neurons, oligodendrocytes, and astrocytes. Fetal astrocyte samples were also obtained. November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 11 Disease Mutations Stabilize Beta Tubulins Park et al. 4.3 In Vitro Tubulin Assembly Assays Wild-type or T178M mutant tubulin was puriﬁed using the protocol developed by Johnson and colleagues (Johnson et al., 2011). The T178M mutation was introduced using site-directed mutagenesis (Agilent Technologies; Santa Clara, CA) into a plasmid-bound copy of budding yeast TUB2 under the control of a galactose inducible promoter. Puriﬁed tubulin was incubated with fragments of sea urchin axonemes at 30°C and imaged by Differential Interference Contrast microscopy to measure microtubule dynamics. Image series of individual microtubules were converted into kymographs, and rates were determined as the change in length divided by the change in time for each polymerization event. and pause states. Rescues were deﬁned as transitions from disassembly or pause to assembly. Rescue frequencies were determined for individual astral microtubules by dividing the number of rescue events by the total time disassembly and pause states. Microtubule dynamicity was calculated by the total change in length (growing and shrinking) divided by the change in time and expressed in tubulin subunits changed per second (Toso et al., 1993). 4.3 In Vitro Tubulin Assembly Assays Wild-type or T178M mutant tubulin was puriﬁed using the protocol developed by Johnson and colleagues (Johnson et al., 2011). The T178M mutation was introduced using site-directed mutagenesis (Agilent Technologies; Santa Clara, CA) into a plasmid-bound copy of budding yeast TUB2 under the control of a galactose inducible promoter. Puriﬁed tubulin was incubated with fragments of sea urchin axonemes at 30°C and imaged by Differential Interference Contrast microscopy to measure microtubule dynamics. 4.7 Liquid Growth Assay Cells were grown to saturation at 30°C in rich media and diluted 50-fold into fresh rich media. One hundred and ninety- eight microlitres of each diluted culture was then aliquoted into the wells of a 96-well plate, with three replicates of each condition per experiment. Nocodazole in DMSO was diluted 1: 100 into each well to maintain 1% DMSO. The plate was incubated at 30°C with single orbital shaking in an Epoch 2 plate reader (BioTek; Winooski, VT). The OD600 was measured every 5 min for 24 h. Doubling time was calculated by ﬁtting the growth curves to a nonlinear exponential growth curve as previously published (Fees et al., 2018). Data points plotted as dots represent doubling time measurements for each technical replicate from three separate experiments, and the mean value of the technical replicates within each experiment is represented as a triangle. ANOVA with a Tukey post-hoc test was completed on the means of the technical replicates using Prism 9 (GraphPad Software; San Diego, CA). REFERENCES Burke, D., Gasdaska, P., and Hartwell, L. (1989). Dominant Effects of Tubulin Overexpression in saccharomyces Cerevisiae. Mol. Cell Biol. 9, 1049–1059. doi:10.1128/mcb.9.3.1049-1059.1989 Aiken, J., Buscaglia, G., Bates, E. A., and Moore, J. K. (2017). The α-Tubulin Gene TUBA1A in Brain Development: A Key Ingredient in the Neuronal Isotype Blend. J. Dev. Biol. 5, 8. doi:10.3390/jdb5030008 Buscaglia, G., Northington, K. R., Moore, J. K., and Bates, E. A. (2020). Reduced Tuba1a Tubulin Causes Defects in Trafﬁcking and Impaired Adult Motor Behavior. eNeuro 7. doi:10.1523/ENEURO.0045-20.2020 Carlier, M. F., and Pantaloni, D. (1978). Kinetic Analysis of Cooperativity in Tubulin Polymerization in the Presence of Guanosine di- or Triphosphate Nucleotides. Biochemistry 17, 1908–1915. doi:10.1021/bi00603a017 Bahi-Buisson, N., Poirier, K., Fourniol, F., Saillour, Y., Valence, S., Lebrun, N., et al. (2014). The Wide Spectrum of Tubulinopathies: What are the Key Features for the Diagnosis? Brain 137, 1676–1700. doi:10.1093/brain/awu082 Castle, B. T., McCubbin, S., Prahl, L. S., Bernens, J. N., Sept, D., and Odde, D. J. (2017). Mechanisms of Kinetic Stabilization by the Drugs Paclitaxel and Vinblastine. Mol. Biol. Cell 28, 1238–1257. doi:10.1091/mbc.e16-08-0567 Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Ludueña, R. F. (1988). A Monoclonal Antibody against the Type Ii Isotype of Beta-Tubulin. Preparation of Isotypically Altered Tubulin. J. Biol. Chem. 263, 3029–3034. doi:10.1016/s0021-9258(18)69171-9 Chen, S. S., Revoltella, R. P., Papini, S., Michelini, M., Fitzgerald, W., Zimmerberg, J., et al. (2003). Multilineage Differentiation of Rhesus Monkey Embryonic Stem Cells in Three-Dimensional Culture Systems. Stem Cells 21, 281–295. doi:10.1634/stemcells.21-3-281 Bittermann, E., Abdelhamed, Z., Liegel, R. P., Menke, C., Timms, A., Beier, D. R., et al. (2019). Differential Requirements of Tubulin Genes in Mammalian Forebrain Development. Plos Genet. 15, e1008243. doi:10.1371/ journal.pgen.1008243 Cleveland, D. W., Lopata, M. A., Sherline, P., and Kirschner, M. W. (1981). Unpolymerized Tubulin Modulates the Level of Tubulin Mrnas. Cell 25, 537–546. doi:10.1016/0092-8674(81)90072-6 Brock, S., Vanderhasselt, T., Vermaning, S., Keymolen, K., Régal, L., Romaniello, R., et al. (2021). Deﬁning the Phenotypical Spectrum Associated with Variants in Tubb2a. J. Med. Genet. 58, 33–40. doi:10.1136/jmedgenet-2019-106740 Cushion, T. D., Paciorkowski, A. R., Pilz, D. T., Mullins, J. G. L., Seltzer, L. E., Marion, R. W., et al. (2014). De Novo Mutations in the Beta-Tubulin Gene Tubb2a Cause Simpliﬁed Gyral Patterning and Infantile-Onset Epilepsy. Am. J. Hum. Genet. 94, 634–641. doi:10.1016/j.ajhg.2014.03.009 Burgoyne, R. D., Cambray-Deakin, M. A., Lewis, S. A., Sarkar, S., and Cowan, N. J. (1988). DATA AVAILABILITY STATEMENT We thank the families for their participation. We are grateful to Dr. Sarah Schreiner (CU Boulder) for her contributions to this work; Dr. Luke Rice and members of the Rice lab (UT Southwestern) for generous advice and assistance with the in vitro tubulin assembly experiments; and members of the Moore and Dr. Emily Bates (CU AMC) labs for helpful discussions. The data presented in the study are deposited in the ClinVar repository, accession number SCV000321987. 4.8 QUANTITATIVE WESTERN BLOT Tubulin protein abundance per cell was determined by western blot of whole yeast cell extract (Zhang et al., 2011). Wild-type, heterozygous null, or mutant tubulin cells were grown to log phase at 30°C in 50 ml of rich media, then moved to a nutator at 4°C for 1 h to depolymerize microtubules. Prior to lysate preparation, cell density was determined by hemocytometer counts. For each culture in each experiment, approximately 5 × 107 cells were pelleted at 4°C. Cell pellets were resuspended in 2 M lithium acetate and incubated on ice for 5 min, then pelleted and resuspended in 400 mM NaOH for another 5 min on ice to permeabilize the cell wall. Permeabilized cells were pelleted and resuspended in 50 µl of 2.5× Laemmli buffer, boiled for 5 min for complete cell lysis, and centrifuged before loading onto the gel. Standards of puriﬁed yeast tubulin were prepared by diluting protein to 2.5 ng/µl in 2.5× Laemmli buffer. Dilution series of puriﬁed protein (4, 10, 15, 30, and 40 ng) and lysate samples corresponding to 3.5, 4.5, 6, and 8 × 106 cells and were run on a 10% Bis-Tris PAGE, Microtubule dynamics were analyzed by measuring the distance between Bik1-3GFP foci at astral microtubule plus ends and spindle poles at 5 s intervals for 8 min. This analysis was conducted in pre-anaphase cells, which typically exhibit one or two astral microtubules emanating from each spindle pole. For these experiments, the sample sizes were as follows: +/+, n 27 cells; Δ/+, n 30 cells; T178M/+, n 28 cells; T178V/+, n 18 cells. Assembly and disassembly events were deﬁned as at least three contiguous data points that produced length change greater than 500 nm and a coefﬁcient of variation ≥0.80. Pause events were deﬁned as data points that did not meet the criteria for assembly or disassembly, and did not show signiﬁcant length change. Catastrophes were deﬁned as transitions from assembly or pause to disassembly. Catastrophe frequencies were determined for individual astral microtubules by dividing the number of catastrophe events by the total time spent in assembly November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 12 Disease Mutations Stabilize Beta Tubulins Park et al. FUNDING This work was supported by NIH R01 GM112893, R35 GM 136253 (JKM); KH was supported by T32 GM136444; and LW was supported by T32 GM136444 and the Bolie Scholar Award. ngp 1 Da 1.66p10−15ngp 1 kDa 1000 Dap1 molecule MW kDa p 1 cells loaded molecules cell AUTHOR CONTRIBUTIONS transferred to PVDF and blocked for 1 h at room temperature. Membranes were probed with mouse-anti-α-tubulin (4A1; at 1:100; Piperno and Fuller, 1985), mouse-anti-β-tubulin (E7; at 1:100; Developmental Studies Hybridoma Bank, University of Iowa), rabbi-anti-Zwf1 (Glucose-6-phosphate dehydrogenase; Sigma A9521; at 1:10,000) and followed by goat-anti-mouse- 680 (LI-COR 926-68070, Superior, NE; at 1:15,000) and goat- anti-rabbit-800 (LI-COR 926-32211; at 1:15,000) secondary antibodies and imaged on an Odyssey Imager (LI-COR Biosciences). Band intensities were quantiﬁed using the gel analysis plug-in in FIJI. KP contributed to the conceptualization and design of the study, acquired and analyzed data, and wrote the manuscript. KH acquired and analyzed data, and wrote the manuscript. LW acquired and analyzed data, and wrote the manuscript. NS acquired and analyzed data, and wrote the manuscript. M.S. contributed to the conceptualization and design of the study, acquired and analyzed data. JM contributed to the conceptualization and design of the study, acquired and analyzed data, and wrote the manuscript. To calculate the number of tubulin molecules per cell, a standard curve of signal per nanogram of tubulin was ﬁrst calculated for the puriﬁed proteins. Nanograms was converted to molecules of α- or β-tubulin by: SUPPLEMENTARY MATERIAL The studies involving human participants were reviewed and approved by Colorado Multiple Institutional Review Board (COMIRB). Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021.765992/ full#supplementary-material REFERENCES Differential Distribution of Beta-Tubulin Isotypes in Cerebellum. EMBO J. 7, 2311–2319. doi:10.1002/j.1460-2075.1988.tb03074.x November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 13 Disease Mutations Stabilize Beta Tubulins Park et al. Function but Is Essential for Timely Axon Regeneration. Cell Rep. 24, 1865–1879.e9. doi:10.1016/j.celrep.2018.07.029 Di Donato, N., Chiari, S., Mirzaa, G. M., Aldinger, K., Parrini, E., Olds, C., et al. (2017). Lissencephaly: Expanded Imaging and Clinical Classiﬁcation. Am. J. Med. Genet. 173, 1473–1488. doi:10.1002/ajmg.a.38245 Lee, H., Deignan, J. L., Dorrani, N., Strom, S. P., Kantarci, S., Quintero-Rivera, F., et al. (2014). Clinical Exome Sequencing for Genetic Identiﬁcation of Rare Mendelian Disorders. JAMA 312, 1880–1887. doi:10.1001/jama.2014.14604 Ejaz, R., Lionel, A. C., Blaser, S., Walker, S., Scherer, S. W., Babul-Hirji, R., et al. (2017). De Novo pathogenic Variant in Tubb2a Presenting With Arthrogryposis Multiplex Congenita, Brain Abnormalities, and Severe Developmental Delay. Am. J. Med. Genet. 173, 2725–2730. doi:10.1002/ ajmg.a.38352 Lewis, S. A., Lee, M. G., and Cowan, N. J. (1985). Five Mouse Tubulin Isotypes and Their Regulated Expression during Development J Cell Biol 101, 852–861 Lewis, S. A., Lee, M. G., and Cowan, N. J. (1985). Five Mouse Tubulin Isotypes and Lewis, S. A., Lee, M. G., and Cowan, N. J. (1985). Five Mouse Tubulin Isotypes and Their Regulated Expression during Development. J. Cell Biol. 101, 852–861. doi:10.1083/jcb.101.3.852 Estevez-Gallego, J., Josa-Prado, F., Ku, S., Buey, R. M., Balaguer, F. A., Prota, A. E., et al. (2020). Structural Model for Differential Cap Maturation at Growing Microtubule Ends. Elife 9, e50155. doi:10.7554/eLife.50155 Lin, Z., Gasic, I., Chandrasekaran, V., Peters, N., Shao, S., Mitchison, T. J., et al. (2020). Ttc5 Mediates Autoregulation of Tubulin via Mrna Degradation. Science 367, 100–104. doi:10.1126/science.aaz4352 Fees, C. P., Estrem, C., and Moore, J. K. (2017). High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast. J. Vis. Exp. 122, 55610. doi:10.7554/elife.50155 Lohmann, K., Wilcox, R. A., Winkler, S., Ramirez, A., Rakovic, A., Park, J.-S., et al. (2013). Whispering Dysphonia (DYT4 Dystonia) Is Caused by a Mutation in theTUBB4gene. Ann. Neurol. 73, 537–545. doi:10.1002/ana.23829 Fees, C. P., and Moore, J. K. (2018). Regulation of Microtubule Dynamic Instability by the Carboxy-Terminal Tail of Beta-Tubulin. Life Sci. Alliance 1, e201800054. doi:10.26508/lsa.201800054 Löwe, J., Li, H., Downing, K. H., and Nogales, E. (2001). Reﬁned Structure of αβ-tubulin at 3.5 Å Resolution 1 1Edited by I. A. Wilson. J. Mol. Biol. 313, 1045–1057. REFERENCES doi:10.1093/brain/awu110 Mutch, C. A., Poduri, A., Sahin, M., Barry, B., Walsh, C. A., and Barkovich, A. J. (2016). Disorders of Microtubule Function in Neurons: Imaging Correlates. Am. J. Neuroradiol. 37, 528–535. doi:10.3174/ajnr.a4552 Na, G. C., and Timasheff, S. N. (1982). In Vitro Vinblastine-Induced Tubulin Paracrystals. J. Biol. Chem. 257, 10387–10391. doi:10.1016/s0021-9258(18) 34031-6 Hersheson, J., Mencacci, N. E., Davis, M., MacDonald, N., Trabzuni, D., Ryten, M., et al. (2013). Mutations in the Autoregulatory Domain of β-tubulin 4a Cause Hereditary Dystonia. Ann. Neurol. 73, 546–553. doi:10.1002/ana.23832 Hiller, G., and Weber, K. (1978). Radioimmunoassay for Tubulin: A Quantitative Comparison of the Tubulin Content of Different Established Tissue Culture Cells and Tissues. Cell 14, 795–804. doi:10.1016/0092-8674(78)90335-5 Nawrotek, A., Knossow, M., and Gigant, B. (2011). The Determinants that Govern Microtubule Assembly from the Atomic Structure of Gtp-Tubulin. J. Mol. Biol. 412, 35–42. doi:10.1016/j.jmb.2011.07.029 Johnson, V., Ayaz, P., Huddleston, P., and Rice, L. M. (2011). Design, Overexpression, and Puriﬁcation of Polymerization-Blocked Yeast αβ-Tubulin Mutants. Biochemistry 50, 8636–8644. doi:10.1021/bi2005174 Nissenkorn, A., Yosovich, K., Leibovitz, Z., Hartman, T. G., Zelcer, I., Hugirat, M., et al. (2021). Congenital Mirror Movements Associated with Brain Malformations. J. Child. Neurol. 36, 545–555. doi:10.1177/0883073820984068 Nogales, E., Whittaker, M., Milligan, R. A., and Downing, K. H. (1999). High- resolution Model of the Microtubule. Cell 96, 79–88. doi:10.1016/s0092- 8674(00)80961-7 Joyal, K. M., Michaud, J., van der Knaap, M. S., Bugiani, M., and Venkateswaran, S. (2019). SevereTUBB4A-Related Hypomyelination with Atrophy of the Basal Ganglia and Cerebellum: Novel Neuropathological Findings. J. Neuropathol. Exp. Neurol. 78, 3–9. doi:10.1093/jnen/nly105 Nogales, E., Wolf, S. G., and Downing, K. H. (1998). Structure of the αβ Tubulin Dimer by Electron Crystallography. Nature 391, 199–203. doi:10.1038/34465 Katsetos, C. D., Legido, A., Perentes, E., and Mörk, S. J. (2003). Class III β-Tubulin Isotype: A Key Cytoskeletal Protein at the Crossroads of Developmental Neurobiology and Tumor Neuropathology. J. Child. Neurol. 18, 851–866; discussion 867. doi:10.1177/088307380301801205 Oegema, R., Cushion, T. D., Phelps, I. G., Chung, S.-K., Dempsey, J. C., Collins, S., et al. (2015). Recognizable Cerebellar Dysplasia Associated with Mutations in Multiple Tubulin Genes. Hum. Mol. Genet. 24, 5313–5325. doi:10.1093/hmg/ ddv250 Keays, D. A., Tian, G., Poirier, K., Huang, G.-J., Siebold, C., Cleak, J., et al. (2007). Mutations in α-Tubulin Cause Abnormal Neuronal Migration in Mice and Lissencephaly in Humans. Cell 128, 45–57. doi:10.1016/j.cell.2006.12.017 Piperno, G., and Fuller, M. T. (1985). REFERENCES doi:10.1006/jmbi.2001.5077 Findeisen, P., Muhlhausen, S., Dempewolf, S., Hertzog, J., Zietlow, A., Carlomagno, T., et al. (2014). Six Subgroups and Extensive Recent Duplications Characterize the Evolution of the Eukaryotic Tubulin Protein Family. Genome Biol. Evol. 6, 2274–2288. doi:10.1093/gbe/evu187 Lu, Y., Ondo, Y., Shimojima, K., Osaka, H., and Yamamoto, T. (2017). A Novel Tubb4a Mutation G96r Identiﬁed in a Patient with Hypomyelinating Leukodystrophy Onset beyond Adolescence. Hum. Genome 4, 17035. doi:10.1038/hgv.2017.35 Fukumura, S., Kato, M., Kawamura, K., Tsuzuki, A., and Tsutsumi, H. (2016). A Mutation in the Tubulin-Encoding Tubb3 Gene Causes Complex Cortical Malformations and Unilateral Hypohidrosis. Child. Neurol. Open 3, 2329048X16665758. doi:10.1177/2329048X16665758 Luduena, R. F., and Banerjee, A. (2008). “The Isotypes of Tubulin,” in The Role of Microtubules in Cell Biology, Neurobiology, and Oncology. Editor T. Fojo (Totowa, NJ: Humana Press). 123–175. Manka, S. W., and Moores, C. A. (2018). The Role of Tubulin-Tubulin Lattice Contacts in the Mechanism of Microtubule Dynamic Instability. Nat. Struct. Mol. Biol. 25, 607–615. doi:10.1038/s41594-018-0087-8 Gartz Hanson, M., Aiken, J., Sietsema, D. V., Sept, D., Bates, E. A., Niswander, L., et al. (2016). Novel α-tubulin Mutation Disrupts Neural Development and Tubulin Proteostasis. Develop. Biol. 409, 406–419. doi:10.1016/ j.ydbio.2015.11.022 Mendell, J. R., Shilling, C., Leslie, N. D., Flanigan, K. M., al-Dahhak, R., Gastier- Foster, J., et al. (2012). Evidence-based Path to Newborn Screening for Duchenne Muscular Dystrophy. Ann. Neurol. 71, 304–313. doi:10.1002/ ana.23528 Geyer, E. A., Burns, A., Lalonde, B. A., Ye, X., Piedra, F. A., Huffaker, T. C., et al. (2015). A Mutation Uncouples the Tubulin Conformational and Gtpase Cycles, Revealing Allosteric Control of Microtubule Dynamics. Elife 4, e10113. doi:10.7554/eLife.10113 Miller, J. A., Ding, S.-L., Sunkin, S. M., Smith, K. A., Ng, L., Szafer, A., et al. (2014). Transcriptional Landscape of the Prenatal Human Brain. Nature 508, 199–206. doi:10.1038/nature13185 Gupta, M. L., Bode, C. J., Thrower, D. A., Pearson, C. G., Suprenant, K. A., Bloom, K. S., et al. (2002). β-Tubulin C354 Mutations that Severely Decrease Microtubule Dynamics Do Not Prevent Nuclear Migration in Yeast. Mol. Biol. Cel 13, 2919–2932. doi:10.1091/mbc.e02-01-0003 Miyatake, S., Osaka, H., Shiina, M., Sasaki, M., Takanashi, J.-i., Haginoya, K., et al. (2014). Expanding the Phenotypic Spectrum of Tubb4a-Associated Hypomyelinating Leukoencephalopathies. Neurology 82, 2230–2237. doi:10.1212/wnl.0000000000000535 Hamilton, E. M., Polder, E., Vanderver, A., Naidu, S., Schiffmann, R., Fisher, K., et al. (2014). Hypomyelination with Atrophy of the Basal Ganglia and Cerebellum: Further Delineation of the Phenotype and Genotype-Phenotype Correlation. Brain 137, 1921–1930. REFERENCES Monoclonal Antibodies Speciﬁc for an Acetylated Form of Alpha-Tubulin Recognize the Antigen in Cilia and Flagella from a Variety of Organisms. J. Cel Biol 101, 2085–2094. doi:10.1083/ jcb.101.6.2085 Kilmartin, J. V. (1981). Puriﬁcation of Yeast Tubulin by Self-Assembly In Vitro. Biochemistry 20, 3629–3633. doi:10.1021/bi00515a050 y Kobayashi, T. (1975). Dephosphorylation of Tubulin-Bound Guanosine Triphosphate during Microtubule Assembly. J. Biochem. 77, 1193–1197. Poirier, K., Saillour, Y., Bahi-Buisson, N., Jaglin, X. H., Fallet-Bianco, C., Nabbout, R., et al. (2010). Mutations in the Neuronal β-tubulin Subunit TUBB3 Result in Malformation of Cortical Development and Neuronal Migration Defects. Hum. Mol. Genet. 19, 4462–4473. doi:10.1093/hmg/ddq377 Latremoliere, A., Cheng, L., DeLisle, M., Wu, C., Chew, S., Hutchinson, E. B., et al. (2018). Neuronal-Speciﬁc Tubb3 is not Required for normal Neuronal November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 14 Disease Mutations Stabilize Beta Tubulins Park et al. Purnell, S. M., Bleyl, S. B., and Bonkowsky, J. L. (2014). Clinical Exome Sequencing Identiﬁes a Novel Tubb4a Mutation in a Child with Static Hypomyelinating Leukodystrophy. Pediatr. Neurol. 50, 608–611. doi:10.1016/ j.pediatrneurol.2014.01.051 Vasquez, R. J., Howell, B., Yvon, A. M., Wadsworth, P., and Cassimeris, L. (1997). Nanomolar Concentrations of Nocodazole Alter Microtubule Dynamic Instability In Vivo and In Vitro. MBoC 8, 973–985. doi:10.1091/mbc.8.6.973 Weinstein, B., and Solomon, F. (1990). Phenotypic Consequences of Tubulin Overproduction in Saccharomyces Cerevisiae: Differences between Alpha- Tubulin and Beta-Tubulin. Mol. Cell Biol. 10, 5295–5304. doi:10.1128/ mcb.10.10.5295-5304.1990 Rodan, L. H., El Achkar, C. M., Berry, G. T., Poduri, A., Prabhu, S. P., Yang, E., et al. (2017). De Novo tubb2a Variant Presenting with Anterior Temporal Pachygyria. J. Child. Neurol. 32, 127–131. doi:10.1177/ 0883073816672998 Weisenberg, R. C., Broisy, G. G., and Taylor, E. W. (1968). Colchicine-binding Protein of Mammalian Brain and its Relation to Microtubules. Biochemistry 7, 4466–4479. doi:10.1021/bi00852a043 Severino, M., Geraldo, A. F., Utz, N., Tortora, D., Pogledic, I., Klonowski, W., et al. (2020). Deﬁnitions and Classiﬁcation of Malformations of Cortical Development: Practical Guidelines. Brain 143, 2874–2894. doi:10.1093/ brain/awaa174 Wilson, L., Jordan, M. A., Morse, A., and Margolis, R. L. (1982). Interaction of Vinblastine with Steady-State Microtubules In Vitro. J. Mol. Biol. 159, 125–149. doi:10.1016/0022-2836(82)90035-3 Simons, C., Wolf, N. I., McNeil, N., Caldovic, L., Devaney, J. M., Takanohashi, A., et al. (2013). A De Novo Mutation in the β-Tubulin Gene TUBB4A Results in the Leukoencephalopathy Hypomyelination with Atrophy of the Basal Ganglia and Cerebellum. Am. J. Hum. Genet. REFERENCES 92, 767–773. doi:10.1016/ j.ajhg.2013.03.018 Zhang, T., Lei, J., Yang, H., Xu, K., Wang, R., and Zhang, Z. (2011). An Improved Method for Whole Protein Extraction from Yeast saccharomyces Cerevisiae. Yeast 28, 795–798. doi:10.1002/yea.1905 Zhang, Y., Sloan, S. A., Clarke, L. E., Caneda, C., Plaza, C. A., Blumenthal, P. D., Zhang, Y., Sloan, S. A., Clarke, L. E., Caneda, C., Plaza, C. A., Blumenthal, P. D., et al. (2016). Puriﬁcation and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse. Neuron 89, 37–53. doi:10.1016/j.neuron.2015.11.013 Spiegelman, B. M., Penningroth, S. M., and Kirschner, M. W. (1977). Turnover of Tubulin and the N Site Gtp in Chinese Hamster Ovary Cells. Cell 12, 587–600. doi:10.1016/0092-8674(77)90259-8 Ti, S.-C., Alushin, G. M., and Kapoor, T. M. (2018). Human β-Tubulin Isotypes Can Regulate Microtubule Protoﬁlament Number and Stability. Dev. Cell 47, 175–190.e5. doi:10.1016/j.devcel.2018.08.014 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. 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. Tian, G., Jaglin, X. H., Keays, D. A., Francis, F., Chelly, J., and Cowan, N. J. (2010). Disease-associated Mutations in Tuba1a Result in a Spectrum of Defects in the Tubulin Folding and Heterodimer Assembly Pathway. Hum. Mol. Genet. 19, 3599–3613. doi:10.1093/hmg/ddq276 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Tischﬁeld, M. A., Baris, H. N., Wu, C., Rudolph, G., Van Maldergem, L., He, W., et al. (2010). Human Tubb3 Mutations Perturb Microtubule Dynamics, Kinesin Interactions, and Axon Guidance. Cell 140, 74–87. doi:10.1016/ j.cell.2009.12.011 Copyright © 2021 Park, Hoff, Wethekam, Stence, Saenz and Moore. 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. Copyright © 2021 Park, Hoff, Wethekam, Stence, Saenz and Moore. 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. Frontiers in Cell and Developmental Biology \| www.frontiersin.org November 2021 \| Volume 9 \| Article 765992 REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Tonduti, D., Aiello, C., Renaldo, F., Dorboz, I., Saaman, S., Rodriguez, D., et al. (2016). Tubb4a-related Hypomyelinating Leukodystrophy: New Insights from a Series of 12 Patients. Eur. J. Paediatric Neurol. 20, 323–330. doi:10.1016/ j.ejpn.2015.11.006 Toso, R. J., Jordan, M. A., Farrell, K. W., Matsumoto, B., and Wilson, L. (1993). Kinetic Stabilization of Microtubule Dynamic Instability In Vitro by Vinblastine. Biochemistry 32, 1285–1293. doi:10.1021/bi00056a013 November 2021 \| Volume 9 \| Article 765992 Frontiers in Cell and Developmental Biology \| www.frontiersin.org 15
https://openalex.org/W2759996959	https://scholarlypublications.universiteitleiden.nl/access/item%3A2948811/view	English	null	Intradermal vaccination with hollow microneedles: A comparative study of various protein antigen and adjuvant encapsulated nanoparticles	Journal of controlled release	2,017	cc-by	11,360	A R T I C L E I N F O In this study, we investigated the potential of intradermal delivery of nanoparticulate vaccines to modulate the immune response of protein antigen using hollow microneedles. Four types of nanoparticles covering a broad range of physiochemical parameters, namely poly (lactic-co-glycolic) (PLGA) nanoparticles, liposomes, meso- porous silica nanoparticles (MSNs) and gelatin nanoparticles (GNPs) were compared. The developed nano- particles were loaded with a model antigen (ovalbumin (OVA)) with and without an adjuvant (poly(I:C)), fol- lowed by the characterization of size, zeta potential, morphology, and loading and release of antigen and adjuvant. An in-house developed hollow-microneedle applicator was used to inject nanoparticle suspensions precisely into murine skin at a depth of about 120 μm. OVA/poly(I:C)-loaded nanoparticles and OVA/poly(I:C) solution elicited similarly strong total IgG and IgG1 responses. However, the co-encapsulation of OVA and poly (I:C) in nanoparticles signiﬁcantly increased the IgG2a response compared to OVA/poly(I:C) solution. PLGA nanoparticles and liposomes induced stronger IgG2a responses than MSNs and GNPs, correlating with sustained release of the antigen and adjuvant and a smaller nanoparticle size. When examining cellular responses, the highest CD8+ and CD4+ T cell responses were induced by OVA/poly(I:C)-loaded liposomes. In conclusion, the applicator controlled hollow microneedle delivery is an excellent method for intradermal injection of nano- particle vaccines, allowing selection of optimal nanoparticle formulations for humoral and cellular immune responses. Keywords: Intradermal vaccination Hollow microneedles Nanoparticles Antigen Adjuvant payload in the superﬁcial skin layers potentially without pain, owing to the limited penetration depth of microneedles (typically < 500 μm) [3]. Intradermal vaccination with hollow microneedles: A comparative study of various protein antigen and adjuvant encapsulated nanoparticles M Guangsheng Dua, Rania M. Hathouta,b, Maha Nasra,b, M. Reza Nejadnika, Jing Tuc, Roman I. Koningd, Abraham J. Kosterd, Bram Slüttera,e, Alexander Krosc, Wim Jiskoota, Joke A. Bouwstraa,⁎, Juha Mönkärea a Division of Drug Delivery Technology, Cluster BioTherapeutics, Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands b Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt c Department of Supramolecular & Biomaterials Chemistry, Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands d Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden University, Leiden, The Netherlands e Division of Biopharmaceutics, Cluster BioTherapeutics, Leiden Academic Centre for Drug Research, Leiden University, Leiden, The Netherlands ⁎ Corresponding author. E-mail address: bouwstra@lacdr.leidenuniv.nl (J.A. Bouwstra). Citation Citation Du, G., Mohammed Hafez Mohammed Hathout, R., Nasr Sayed Aly, M., Nejadnik, M. R., Tu, J., Koning, R. I., … Monkare, J. T. (2017). Intradermal vaccination with hollow microneedles: A comparative study of various protein antigen and adjuvant encapsulated nanoparticles. Journal Of Controlled Release, 266, 109-118. doi:10.1016/j.jconrel.2017.09.021 Version: Not Applicable (or Unknown) License: Leiden University Non-exclusive license Downloaded from: https://hdl.handle.net/1887/55805 Note: To cite this publication please use the final published version (if applicable). Version: Not Applicable (or Unknown) License: Leiden University Non-exclusive license Downloaded from: https://hdl.handle.net/1887/55805 Version: Not Applicable (or Unknown) License: Leiden University Non-exclusive license Downloaded from: https://hdl.handle.net/1887/55805 Note: To cite this publication please use the final published version (if applicable). Note: To cite this publication please use the final published version (if applicable). Journal of Controlled Release 266 (2017) 109–118 Contents lists available at ScienceDirect http://dx.doi.org/10.1016/j.jconrel.2017.09.021 Received 6 July 2017; Received in revised form 12 September 2017; Accepted 13 September 2017 ⁎ Corresponding author. E-mail address: bouwstra@lacdr.leidenuniv.nl (J.A. Bouwstra). Available online 21 Septem ber 2017 0168-3659/ © 2017 The Authors. Published by Elsevier B.V. This is an open access article under the CC 1. Introduction Sodium dodecyl sulfate (SDS) was purchased from Merck Millipore (Hohenbrunn, Germany). Vivaspin 2 centrifugal concentrators (PES membrane, MWCO 1000 kDa) were obtained from Sartorius Stedim (Nieuwegein, The Netherlands). Sterile phosphate buﬀered saline (PBS, 163.9 mM Na+, 140.3 mM Cl−, 8.7 mM HPO4 2−, 1.8 mM H2PO4−, pH 7.4) was obtained from Braun (Oss, The Netherlands). Cell culture medium was prepared by mixing Roswell Park Memorial Institute medium (RPMI) with 10% Fetal bovine serum (FBS), 1% L-glutamine and 1% Penicillin-streptamycin. 1 mM phosphate buﬀer (PB, 0.77 mM Na2HPO4, 0.23 mM NaH2PO4, pH 7.4), 10 mM PB (7.7 mM Na2HPO4, 2.3 mM NaH2PO4, pH 7.4), 5 mM 4-(2- hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, pH 7.4) buﬀer, lysis buﬀer (150 mM ammonium chloride, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2), and FACS buﬀer (2% FBS in PBS, pH 7.4) were prepared in the lab. All the other chemicals used are of analytical grade and Milli- Q water (18 MΩ/cm, Millipore Co.) was used for the preparation of all solutions. advantageous when studying optimization of formulations or para- meters for the immunization (e.g. penetration depth or vaccine dose). Furthermore, if required, a higher dose can be injected into the skin compared to dissolving and coated microneedles. p g Subunit antigens are based on puriﬁed antigens and are regarded safer than traditional whole bacterium- or virus-based vaccines [7]. However, these antigens have often lower immunogenicity and there- fore adjuvants, such as toll-like receptor (TLR) ligands or toxoids, are needed to increase the immune response [8]. Recently, nanoparticles have gained growing attention for the delivery of subunit vaccines because of their capability of protecting antigens from degradation, forming a depot at the site of injection, and facilitating antigen uptake by dendritic cells (DCs) [9–11]. Studies have additionally shown that co-formulation of antigen and adjuvant into a nanoparticle might be crucial to improve immune responses against subunit vaccines [12–15]. However, it is not well understood how the physicochemical properties such as size, material, surface charge or release behavior of antigen/ adjuvant inﬂuence the immune response. Previously, it has been pro- posed that positively charged nanoparticles with a size smaller than about 200 nm might be optimal for the interaction with antigen-pre- senting cells [9,16–18]. Moreover, sustained release of antigen and adjuvant from nanoparticles and a depot eﬀect of nanoparticles on the cell surface could allow the co-delivery of antigen and adjuvant to antigen-presenting cells [17,19]. 2.1. Materials 2.1. Materials PLGA (acid terminated, lactide glycolide 50:50, Mw 24–38 kDa), gelatin from porcine skin (bloom 300), OVA for in vitro studies (albumin from chicken egg white, lyophilized), bovine serum albumin (BSA) ≥96%, gluteraldehyde, glycine, cholamine chloride hydrochloride (2- aminoethyl)-trimethylammoniumchloride hydrochloride, 1-ethyl-3-(3- dimethyl-aminopropyl) carbodiimide hydrochloride (EDC), cholesterol (≥99%) and hydroﬂuoric acid ≥48% were purchased from Sigma- 2.2.1. Preparation of PLGA nanoparticles OVA loaded PLGA nanoparticles (PLGA-OVA) were prepared by double emulsion with solvent evaporation method as previously re- ported with modiﬁcations [31]. Brieﬂy, 75 μl OVA (20 mg/ml) in PBS was dispersed in 1 ml ethyl acetate containing 25 mg/ml PLGA by using a Branson soniﬁer 250 (Danbury, CT) for 15 s with a power of 20 W. The obtained water-in-oil emulsion was emulsiﬁed with 2 ml aqueous solution containing 2% (w/v) PVA with the soniﬁer (15 s, 20 W). The water-in-oil-in-water double emulsion was added dropwisely into 25 ml 0.3% (w/v) PVA (40 °C) under stirring. The ethyl acetate was evapo- rated by a rotary evaporator (Buchi rotavapor R210, Flawil, Switzer- land) for 3 h (150 mbar, 40 °C). The nanoparticles were collected by centrifugation (Avanti™J-20XP centrifuge, Beckman Coulter, Brea, CA) at 35000 g for 10 min. Finally, they were washed twice with 1 mM PB to remove the excess OVA and PVA and dried in an ice condenser (Alpha 1–2, Osterode, Germany) in freeze vacuum (−49 °C, 90 mbar) overnight for further use and storage. A model antigen, ovalbumin (OVA), with and without a TLR3 agonist, poly(I:C), was encapsulated into the nanoparticles. First, the physicochemical properties and the in vitro release of antigen and ad- juvant of the diﬀerent nanoparticulate formulations were characterized. Next, mice were immunized with the formulations by using a hollow microneedle device followed by the analysis of humoral and cellular immune responses. The results reveal that the immune response de- pends on encapsulation of antigen/adjuvant and the characteristics of nanoparticles. Furthermore, we demonstrate that the hollow micro- needles together with the applicator are excellent tools for intradermal vaccination and screening of nanoparticulate formulations. 1. Introduction However, most vaccination studies have been performed by intramuscular or subcutaneous injection and no studies have directly compared diﬀerent nanoparticles for in- tradermal vaccine delivery. The aim of this study was to assess the potential of antigen loaded nanoparticles, with or without co-encapsulated adjuvant, to induce humoral and cellular immune responses after hollow microneedle- mediated intradermal immunization. To this end, we prepared four diﬀerent nanoparticulate delivery systems with varying physicochem- ical properties, namely poly (lactic-co-glycolic) acid (PLGA) nano- particles, liposomes, mesoporous silica nanoparticles (MSNs) and ge- latin nanoparticles (GNPs). PLGA nanoparticles [10,20–24] and liposomes [12,18,22,25] have been extensively investigated as bio- compatible and biodegradable nanoparticle vaccine delivery systems. MSNs gain increasing attention for vaccine delivery because of their controlled size and mesostructure, excellent in vivo biocompatibility and high loading capacity [26,27]. Gelatin based nanoparticles have been studied as promising vaccine carriers because of their excellent biocompatibility, stability and aptness for surface modiﬁcation [28–30]. 1. Introduction Skin is an attractive administration site for immunization and may act as an excellent alternative for traditional intramuscular or sub- cutaneous vaccination. Furthermore, intradermal vaccination may en- able dose sparing, since the skin has a rich network of immune cells compared to muscle or subcutaneous tissue [1]. However, the upper- most layer of the skin, the stratum corneum, is the main barrier that prevents the transport of vaccines (> 500 Da) across the skin. There- fore, novel delivery methods need to be developed. Among various methods developed for antigen delivery via the skin, especially micro- needle-based approaches have recently attracted increasing attention [2]. The major advantage of microneedles is their ability to pierce the skin in a minimally invasive manner and subsequently deliver their Several microneedle types have been developed for vaccine de- livery, such as coated or dissolving microneedles which can release the dry antigen into the epidermis and dermis after the piercing of the skin [2]. In contrast, hollow microneedles can be used to deliver antigens or particulate formulations as solutions or suspensions into the skin. To this end, in our group a hollow microneedle device has been developed that allows precise and controlled injections into the epidermis and dermis by using etched fused-silica capillary-based microneedles [4–6]. The advantage of the hollow microneedles compared to dissolving or coated microneedles is that little time is required for modifying the dose, formulation or administration depth. This is particularly G. Du et al. Journal of Controlled Release 266 (2017) 109–118 Aldrich (Zwijndrecht, The Netherlands). Polyvinyl alcohol (PVA) 4–88 (31 kDa) and ethylenediaminetetraacetic acid (EDTA) were purchased from Fluka (Steinheim, Germany). 1-step™ ultra 3,3′,5,5′-tetra- methylbenzidine (TMB) was obtained from Thermo-Fisher Scientiﬁc (Waltham, MA). Endotoxin-free OVA, polyinosinic-polycytidylic acid (poly(I:C)) (low molecular weight) and its rhodamine-labeled version were purchased from Invivogen (Toulouse, France). Egg phosphati- dylcholine (EggPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-[phosphor-L-serine](sodium salt) (DOPS), 1,2- dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were ordered from Avanti Polar Lipids (Alabaster, AL). HRP-conjugated goat anti- mouse total IgG, IgG1 and IgG2a were purchased from Southern Biotech (Birmingham, AL). Fluorescently labeled antibodies speciﬁc for CD4, CD8 and CD45.1 were ordered from eBioscience (San Diego, The Netherlands). Sulfuric acid (95–98%) was obtained from JT Baker (Deventer, The Netherlands). Ethyl acetate and silicone oil (AK350) were ordered from Boom Chemicals (Meppel, The Netherlands). Dimethylsulfoxide (DMSO) was ordered from Biosolve BV (Valkenswaard, The Netherlands). 2.2. Preparation of nanoparticles 2.2. Preparation of nanoparticles 2. Materials and methods To prepare OVA and poly(I:C) co-encapsulated PLGA nanoparticles (PLGA-OVA-PIC), 18.75 μl OVA (40 mg/ml) and 75 μl poly(I:C) (46.7 mg/ml, including 0.03% ﬂuorescently labeled equivalent) were emulsiﬁed with 1 ml PLGA (25 mg/ml) in ethyl acetate to obtain the water-in-oil emulsion. The rest of the procedure was identical to that of PLGA-OVA. 2.1. Materials 2.2.2. Preparation of liposomes Liposomes were prepared by a ﬁlm hydration method [32]. A thin lipid ﬁlm of EggPC: DOPE: DOTAP in a molar ratio of 9:1:2.5 was 110 Journal of Controlled Release 266 (2017) 109–118 G. Du et al. created by evaporating chloroform of lipid stock solutions (25 mg/ml) using a rotary evaporator (Buchi rotavapor R210, Flawil, Switzerland). To prepare OVA loaded liposomes (Lipo-OVA), the lipid ﬁlm was re- hydrated in 10 mM PB (pH 7.4) containing 0.25 mg/ml OVA, and subsequently stabilized at room temperature for 1 h, resulting in ﬁnal lipid concentration of 12.5 mg/ml. In the case of OVA and poly(I:C) co- encapsulated liposomes (Lipo-OVA-PIC), after lipid ﬁlm hydration, 250 μl poly(I:C) solution (1.32 mg/ml, containing 0.5% rhodamine-la- beled poly(I:C)) was added slowly (2 μl/min) by using a syringe pump to the liposome suspension under stirring. Finally, the liposomes were extruded (LIPEX™extruder, Northern Lipids, Burnaby, Canada) four times through a carbonate ﬁlter with a pore size of 400 nm and another four times through a ﬁlter with a pore size of 200 nm (Nucleopore Millipore, Amsterdam, The Netherlands). The obtained suspensions were transferred into VivaSpin 2 centrifuge concentrators (1000 kDa MWCO) and centrifuged (Allegra X-12R, Beckman Coulter, In- dianapolis, IN) twice for 7–8 h (350 g, 22 °C) to remove the excess OVA and poly(I:C) [18]. The liposome suspensions were collected and stored at 4 °C until further use. was added to the suspension to block the unreacted GA and stop the cross-linking reaction. The suspension was stirred for 1 h at room temperature before being centrifuged at 7000 g for 1 h (Avanti™J-20XP centrifuge, Beckman Coulter, Brea, CA) to separate the GNPs from the reaction mixture. The GNPs were rinsed with ultrapure water in three rounds of centrifugation and resuspension. The obtained GNPs were cationized to increase the positive surface charge and consequently enhance the loading of OVA and poly(I:C). Brieﬂy, the pH of GNP suspension was adjusted to 4.5 and the quaternary amine cholamine (10% of the weight of GNPs) was added under constant stirring. After 5 min, EDC (10% of the weight of GNPs) was added to the suspension to activate the carboxylic groups of gelatin which would couple chola- mine. The mixture was stirred for 3 h at room temperature. The ca- tionized GNPs were puriﬁed by three successive centrifugation steps as described above. 2.2.3. Preparation of MSNs Large pore MSNs were synthesized and used for the loading of an- tigen and adjuvant as described earlier [33]. To improve the colloidal stability of antigen loaded MSNs, negatively charged liposomes were fused to the surface of MSNs, as reported previously [34,35]. For this purpose liposomes were prepared by dispensing stock solutions of DOPC (70 μl, 25 mg/ml), DOPS (20 μl, 12.5 mg/ml) and cholesterol (10 μl, 25 mg/ml) in chloroform into scintillation vials. A lipid ﬁlm was created by slow evaporation of chloroform in the vial under a nitrogen ﬂow and dried in vacuum overnight. The lipid ﬁlm was rehydrated by the addition of 1 ml of 1 mM PB (pH 7.4) and the mixture was vortexed for 10 s to form a cloudy lipid suspension. The obtained suspension was sonicated in a water bath for 10 min. The resulting clear liposome dispersions were stored at 4 °C for further use. 2.2.2. Preparation of liposomes Finally, the nanoparticles were resuspended in ultra- pure water by using vortexing and probe sonication [37], and stored at 4 °C for further experiments. To prepare OVA loaded GNPs (GNP-OVA) for the humoral response study, 100 μg OVA in water was added to 2000 μg GNPs in water (total volume 1 ml) and the samples were mixed for 1 h (400 rpm, 25 °C). For OVA and poly(I:C) co-loaded GNPs (GNP-OVA-PIC), after shaking OVA and GNPs for 1 h, 100 μg poly(I:C) (containing 1% rhodamine-labeled poly(I:C)) was added to the GNP suspension and the suspension was mixed for another 1 h. Finally, the loaded nanoparticles were separated by centrifugation at 2800 g for 5 min, followed by re-suspension in de- ionized water. For the cellular response study, a modiﬁcation of the method was required to allow administration of a higher dose. Instead of water, 4 mM HEPES buﬀer (pH 7.4) was used for loading to control the pH. For GNP-OVA the added amounts of GNPs and OVA were 6000 μg and 300 μg (in 1.5 ml), respectively, and for GNP-OVA-PIC, the amounts of GNP, OVA and poly(I:C) were 7000 μg, 200 μg and 200 μg (in 1.5 ml), respectively. The modiﬁcation did not signiﬁcantly change the characteristics of nanoparticles. 2.3.2. Morphological characterization Morphology of PLGA nanoparticles and GNPs was visualized by using scanning electron microscopy (SEM, Nova NanoSEM, FEI, Eindhoven, The Netherlands) with a voltage of 15 kV. Nanoparticles were ﬁrst freeze-dried and coated with a thin layer of carbon. MSNs were visualized by transmission electron microscopy (TEM) using a JEOL 1010 instrument (JEOL Ltd., Peabody, MA) with an accelerating voltage of 70 kV. To prepare the samples, several droplets of MSN suspension (1 mg/ml) were added on a copper grid, dried overnight and coated with carbon. Liposomes were visualized by Cryo-EM. The sam- ples were diluted to 5 mg/ml and drops of 3 μl were applied to 300 mesh EM grids with lacey carbon (Ted Pella, USA). Grids were trans- ferred into an electron microscopy grid plunger (EM GP, Leica, Germany) operated at room temperature and 100% humidity. The sample was vitriﬁed by removing excess liquid immediately followed by plunging into liquid ethane and the plunge-frozen grids were stored in liquid nitrogen until further use. Samples were inserted into a Gatan 626 cryo holder (Gatan, Pleasanton, CA). A Tecnai F20 microscope (Thermo-Fisher, Eindhoven, The Netherlands) was operated at 200 kV and the EM images were recorded at defocus values between 1 and 3 μm underfocus on a Gatan 4 k × 4 k CCD (Gatan, Germany). 2.3.1. Particle size and zeta potential determination The particle size and polydispersity index (PDI), and zeta potential for all formulations were determined by dynamic light scattering and laser doppler velocimetry, respectively, by using a Nano ZS® zetasizer (Malvern Instruments, Worcestershire, U.K.). Particle size measure- ments were performed in 10 mM PB (pH 7.4) (PLGA nanoparticles, li- posomes and MSNs) or ultrapure water (GNPs), while for zeta potential measurements samples were diluted in 5 mM HEPES buﬀer (pH 7.4). 2.3. Characterization of the nanoparticles To prepare lipid bilayer coated and OVA encapsulated MSNs (LB- MSN-OVA), OVA (0.5 ml, 0.25 mg/ml) in 1 mM PB (pH 7.4) was ﬁrst transferred into a 2 ml micro-centrifuge tube, followed by the addition of MSNs (0.5 ml, 1 mg/ml) and liposomes (0.5 ml, 2 mg/ml). For OVA and poly(I:C) co-encapsulated and lipid coated MSNs (LB-MSN-OVA- PIC), 0.5 ml solution containing 0.25 mg/ml OVA and 0.094 mg/ml poly(I:C) (containing 1.2% rhodamine-labeled poly(I:C)) were mixed with MSNs and liposomes similarly to LB-MSN-OVA. The resulting mixtures were incubated for 1.5 h under shaking (400 rpm, 25 °C). The nanoparticles were collected and excess liposomes, OVA and poly(I:C) were removed by centrifuging the sample (9000 g, 5 min) with a Sigma 1–15 centrifuge (Osterode, Germany). The obtained nanoparticles were stored at 4 °C before the use. 2.5. Immunization studies 2.5.2. T cell response study y OT-I (OVA-speciﬁc CD8+) and OT-II (OVA-speciﬁc CD4+) T cell transferred C57BL/6 mice were used for the T cell response study. To obtain OT-I and OT-II T cells, spleens of OT-I and OT-II transgenic mice (CD45.1) were isolated and single cell suspensions were obtained by forcing the spleens through a 70 um strainer. After erythrocyte deple- tion with ammonium chloride, percentage of CD8+/Valpha2+ or CD4+/Valpha2+ cells was determined by ﬂow cytometry (BD FACSCanto-II, San Jose, CA). An equivalent of 8000 OT-I and 56,000 OT-II cells were intravenously transferred through the tail vein into C57BL/6 mice. Next day, the T cell transferred mice were immunized with nanoparticle formulations. OVA and poly(I:C) solutions were used as controls. Before the immunization, mice were anesthetized by iso- ﬂurane inhalation (induction 4–5% and maintenance 1%), which was followed by shaving of the injection site. On the same day, mice (n = 5/group) were immunized by three intradermal injections of 13.3 μl (totally 40 μl) formulation containing 5 μg OVA with or without approximately 5 μg poly(I:C) on the ﬂank of the mouse (two injections on the right side, one injection on the left side) by using the hollow- microneedle applicator as described above. 7 days after the im- munization, venous blood sample was collected from the tail to analyze the T cell response. 2.3.3. Determination of loading eﬃciency of OVA and poly(I:C) 2.3.3. Determination of loading eﬃciency of OVA and poly(I:C) the antibody response and T-cell response study, respectively. The mice were 7–8 weeks old at the beginning of the experiment. All the mice were purchased from Charles Rivers (Maastricht, The Netherlands) and were housed under standardized conditions in the animal facility of Leiden Academic Centre for Drug Research of Leiden University. Experiments were approved by the ethical committee on animal ex- periments of Leiden University (Licence number 14176). To determine the loading eﬃciency of OVA and poly(I:C) in PLGA nanoparticles, the nanoparticles were dissolved in a mixture of 15% (v/ v) DMSO and 85% (v/v) 0.05 M NaOH and 0.5% SDS. The amount of OVA was quantiﬁed by the microBCA method following the manufac- turer's instructions. The amount of poly(I:C) was determined by the ﬂuorescence intensity of rhodamine labeled poly(I:C) (λex 545 nm/λem 576 nm) with a plate reader (Tecan M1000, Männedorf, Switzerland). The loading eﬃciency of OVA in liposomes, MSNs and GNPs was de- termined by measuring its intrinsic ﬂuorescence intensity (λex 280 nm/ λem 320 nm) with the Tecan M1000 plate reader in the supernatant before and after the encapsulation (MSNs and GNPs) or in the pur- iﬁcation ﬁltrates (liposomes). The loading eﬃciency of poly(I:C) in these nanoparticles was quantiﬁed similarly by measuring the ﬂuores- cence of its rhodamine labeled equivalent (λex 545 nm/λem 576 nm). 2.3.4. In vitro release studies To study the release of OVA and poly(I:C), the nanoparticles were dispersed in PBS and shaken by using an Eppendorf thermomixer (Nijmegen, The Netherlands) at 37 °C with a speed of 550 rpm. The concentration for PLGA nanoparticles, liposomes, MSNs and GNPs after the suspension was 3 mg/ml, 5 mg/ml (lipid concentration), 1 mg/ml and 1.3 mg/ml, respectively. At predetermined time intervals, the tubes were taken out of the shaker bath and centrifuged at 10000 g for 10 min (PLGA nanoparticles and MSNs) or at 2800 g for 5 min (GNPs). A re- lease sample of 600 μl was taken from the supernatant and replaced by fresh release medium. In the case of liposomes, 300 μl sample was collected to Vivaspin 500 concentrators. After the centrifuging (350 g, 30 min), the ﬁltrate was collected and replaced with fresh medium. The amount of released OVA and poly(I:C) were determined by intrinsic ﬂuorescence of OVA (λex 280 nm/λem 320 nm) and ﬂuorescence of rhodamine labeled poly(I:C) (λex 545 nm/λem 576 nm), respectively, using a Tecan M1000 plate reader. The amount of released OVA in PLGA nanoparticles was determined by the MicroBCA method. Female BALB/c mice (H2d) and C57BL/6 mice (H2b) were used for 2.5.1. Antibody response study BALB/c mice were anesthetized by intraperitoneal injection of ke- tamine (60 mg/kg) and xylanize (4 mg/kg), which was followed by the shaving of the injection site. At the same day mice (n = 8/group) were immunized by an intradermal injection of 10 μl nanoparticles loaded with 0.31 μg OVA, with or without approximately 0.31 μg poly(I:C), on the ﬂank of the mouse by using the applicator, as described above. Solutions of 0.31 μg OVA, with or without 0.31 μg poly(I:C), were used as controls. The injection depth was set to about 120 μm. In addition, subcutaneous injection of 0.31 μg OVA (100 μl) was used as another control. Mice were immunized on day 0 (prime), day 21 (1st boost) and day 42 (2nd boost), and sacriﬁced on day 49. Before each immuniza- tion, on the same day, a venous blood sample was collected from the tail to measure the antibody responses. Before the sacriﬁce, the blood sample was collected from the femoral artery. The encapsulation eﬃciency (EE) and loading capacity (LC) of OVA and poly(I:C) in the nanoparticles were calculated as below: = × M M EE% 100% loaded OVA poly I C total OVA poly I C ( : ) ( : ) (1) = × + + M M LC% 100% loaded OVA poly I C nanoparticles OVA poly I C ( : ) ( : ) (2) = × M M EE% 100% loaded OVA poly I C total OVA poly I C ( : ) ( : ) (1) = × M M EE% 100% loaded OVA poly I C total OVA poly I C ( : ) ( : ) (1) = × + + M M LC% 100% loaded OVA poly I C nanoparticles OVA poly I C ( : ) ( : ) (2) (1) = × + + M M LC% 100% loaded OVA poly I C nanoparticles OVA poly I C ( : ) ( : ) (2) where Mloaded OVA/poly(I:C) represents the mass of loaded OVA or poly (I:C), Mtotal OVA/poly(I:C) is the total amount of OVA or poly(I:C) added to the formulations and Mnanoparticles + OVA + poly(I:C) is the total weight of nanoparticles, OVA and poly(I:C). 2.2.4. Preparation of GNPs GNPs were prepared by using a two-step desolvation method as previously described [36]. First, 1.25 g gelatin (cationic, pI 7–9) was dissolved in 25 ml ultrapure water at 50 °C while stirring at 600 rpm for 30 min. The ﬁrst desolvation step was carried out by addition of 25 ml acetone. The mixture was left for 1 h until the gelatin precipitated. The supernatant was discarded and the sediment was re-dissolved in 25 ml ultrapure water at 50 °C while stirring at 250 rpm for 30 min. Subse- quently, the pH of the solution was adjusted to 2.5 by using con- centrated HCl and a second desolvation step was performed by drop- wise (0.1 ml/s) addition of 80 ml acetone at 50 °C while stirring at 1200 rpm. The crosslinking of the GNPs was accomplished by adding 25 (w/w)% glutaraldehyde (GA) solution. The amount of added GA was adjusted such that the molar ratio between the NH2 groups of gelatin and GA molecules was 1:1. Calculations were performed based on the assumptions that MWgelatin = 100 kDa and 1 mol gelatin has 37 mol NH2 [36]. The resultant suspension was stirred at 600 rpm for 16 h at room temperature. Next, an equal volume of 100 mM glycine solution 111 G. Du et al. Journal of Controlled Release 266 (2017) 109–118 2.6. Determination of OVA speciﬁc IgG antibodies Hollow microneedles were prepared as described earlier [38]. Brieﬂy, 4-cm pieces of polyimide-coated fused silica capillaries (Poly- micro, Phoenix AZ, 375 μm outer diameter, 50 μm inner diameter) were ﬁrst ﬁlled with silicone oil in a vacuum oven (100 °C) overnight and subsequently etched for 4 h in ≥48% hydroﬂuoric acid. The polyimide coating was removed from the etched ends of capillaries by dipping them into heated (250 °C) sulfuric acid for 5 min. OVA-speciﬁc antibodies were analyzed by a sandwich enzyme- linked immunosorbent assay (ELISA) as described earlier [39]. Brieﬂy, wells of the 96 well-plates were ﬁrst coated with 500 ng OVA for 1.5 h at 37 °C. The plates were blocked by incubation with 1% (w/v) BSA for 1 h at 37 °C. After the blocking, appropriate three-fold serial dilutions of mouse sera were applied to the plates and incubated for 1.5 h at 37 °C. Then the plates were incubated with horseradish peroxidase- conjugated goat antibodies against IgG, IgG1 and IgG2a (1:5000 dilu- tion) for 1 h at 37 °C. Finally, speciﬁc antibodies were detected by TMB. The absorbance was measured at 450 nm (Tecan M1000) and the an- tibody titer was determined as a log10 value of the mid-point dilution of S-shaped dilution-absorbance curve of the diluted serum level. A hollow-microneedle applicator was used to control the injection depth and volume as previously reported [5]. A 100-μl syringe with an inner diameter of 1.46 mm was used in conjunction with a syringe pump (NE-300, Prosense, Oosterhout, The Netherlands) and silica ca- pillaries. High-pressure resistant CapTite™connectors were used to connect the pump, syringe, capillaries and needles. Data are average ± SD of at least 3 independent batches. 3.2. In vitro release of OVA and poly(I:C) from nanoparticles To determine the release properties of OVA or poly(I:C) from the nanoparticles, the particles were dispersed in PBS and the released amount of OVA or poly(I:C) was measured at regular time intervals during one month (Fig. 2). PLGA nanoparticles slowly released OVA and on day 30, approximately 13% and 20% of the encapsulated OVA were released from PLGA-OVA and PLGA-OVA-PIC, respectively. Poly (I:C) release followed the OVA release and approximately 20% of the encapsulated poly(I:C) was released during one month. Liposomes re- leased about 30% OVA on the ﬁrst day, followed by a slow release to 40% during one month. Approximately 12% poly(I:C) was slowly re- leased from liposomes during one month. MSNs showed a burst release of approximately 40% OVA within the ﬁrst 6 h, followed by a slower and linear release phase from 40% to almost 100% in the subsequent two weeks. The release of poly(I:C) was slower and only 30% poly(I:C) was released from LB-MSN-OVA-PIC within 15 days. GNPs showed a burst release of nearly all loaded OVA and poly(I:C) within 2 h, fol- lowed by a slow release until 4 days. 3.1. Physicochemical characterization of nanoparticles Four diﬀerent nanoparticle formulations (PLGA nanoparticles, li- posomes, MSNs and GNPs) were developed and characterized in terms of size, zeta potential, surface morphology, and loading and release properties of encapsulated antigen and adjuvant. Physicochemical characteristics of the nanoparticles are summarized in Table 1. Ac- cording to DLS, PLGA nanoparticles and liposomes had an average diameter between 120 nm and 170 nm with a PDI value below 0.1 (PLGA nanoparticles) and 0.3 (liposomes). MSNs and GNPs had a larger diameter, ranging from 500 nm to 760 nm, and PDI values between 0.1 and 0.3. The electron microscopy images revealed a spherical shape of PLGA nanoparticles, liposomes and GNPs, whereas MSNs had a rec- tangular shape with mesochannels along the short axis (Fig. 1). The estimated size based on electron microscopy images is consistent with the size in Table 1 for PLGA nanoparticles and liposomes. MSNs had a smaller particle size in TEM images than in DLS measurements, in- dicating the presence of aggregates in these nanoparticle suspensions. In the case of GNPs, particles are swelling in aqueous medium [40], which may explain the smaller particle size in SEM images as compared to DLS. 2.8. Statistical analysis All the data of immunization studies were analyzed by one way ANOVA with Bonferoni's post-test by using GraphPad Prism software (version 5.02). The level of signiﬁcance was set at p < 0.05. 2.7. CD4+ and CD8+ T cell responses The erythrocytes of the blood sample (100 μl) were ﬁrst lysed by incubating samples with 3 ml lysis buﬀer for 6 min in ice, followed by Female BALB/c mice (H2d) and C57BL/6 mice (H2b) were used for 112 G. Du et al. Journal of Controlled Release 266 (2017) 109–118 size, the PDI and zeta potential of the nanoparticles (Table 1). More- over, both OVA and poly(I:C) were eﬃciently encapsulated into the nanoparticles. The EE% of OVA reached > 60% for all nanoparticles except LB-MSN-OVA-PIC (34%) (Table 1). Similarly, poly(I:C) had a EE % higher than 60%, except for PLGA nanoparticles (13.9%). During the development of the preparation process of the nanoparticles, the in- troduced amounts of antigen and adjuvant were optimized to obtain similar loading capacities of OVA and poly(I:C) for each delivery system (Table 1). addition of 5 ml cell culture medium. After the centrifugation (5 min, 500 g), the supernatant was discarded and the samples were suspended in 5 ml FACS buﬀer. Next, samples were centrifuged and 200 μl of cell suspension was added to the 96-well plate after discarding the super- natant. The cell surfaces were stained by incubating the cells with 100 μl diluted (1:800) ﬂuorescently labeled antibodies speciﬁc for CD45.1 (eFluor450), CD4 (APC) and CD8α (PerCP) for 30 min (100 μl/ well). After 30 min incubation at 4 °C, the excess antibodies were wa- shed by using FACS buﬀer. The cells were incubated with ﬁxation and permeabilization solution (BD Biosciences) for 10 min at 4 °C. Finally, the cells were washed with FACS buﬀer and analyzed by ﬂow cytometry (BD FACSCanto-II, San Jose, CA). The data were analyzed by using FlowJo software. 3.2. In vitro release of OVA and poly(I:C) from nanoparticles a Size: Z-average in diameter. 3.3. Antibody responses after intradermal immunization First, it was examined whether intradermal vaccination with solu- tions or nanoparticles containing 0.31 μg OVA with or without poly(I:C) (~0.31 μg) (Table 1) can induce antigen speciﬁc antibodies. The dose of antigen was chosen based on a dose response study (data not shown). As shown in Fig. 3, all groups, except the subcutaneous control of OVA solution, showed a detectable total IgG response on day 21, and the highest response was detected for the PLGA-OVA-PIC group (Fig. 3A). The total IgG levels increased after the boost on day 21 (Fig. 3B) and 42 (Fig. 3C). All studied nanoparticle formulations and OVA or OVA-poly (I:C) solutions gave similar total IgG responses, except for PLGA-OVA. These nanoparticles showed signiﬁcant weaker total IgG responses on day 42 and 49, but co-encapsulation of poly(I:C) in PLGA nanoparticles increased signiﬁcantly total IgG titers to similar levels observed with the other nanoparticle suspensions. In conclusion, the nano- PLGA nanoparticles and MSNs had a negative zeta potential, whereas liposomes and GNPs possessed a positive zeta potential. In general, co-encapsulation of poly(I:C) did not substantially aﬀect the Table 1 Physicochemical characteristics of OVA/poly(I:C) loaded nanoparticles. Table 1 Physicochemical characteristics of OVA/poly(I:C) loaded nanoparticles. Nanoparticles Sizea (nm) PDIb ZPc (mV) EEd % LCe % OVA Poly(I:C) OVA Poly(I:C) PLGA-OVA 157 ± 7 0.060 ± 0.028 -18 ± 1 64.7 ± 4.8 – 6.9 ± 0.5 – PLGA-OVA-PIC 160 ± 1 0.052 ± 0.019 −22 ± 4 76.7 ± 2.0 13.9 ± 4.2 2.7 ± 0.7 3.0 ± 0.9 Lipo-OVA 124 ± 15 0.152 ± 0.026 44 ± 2 97.0 ± 2.4 – 1.6 ± 0.1 – Lipo-OVA-PIC 171 ± 9 0.270 ± 0.040 41 ± 1 92.1 ± 5.6 98.6 ± 2.3 1.5 ± 0.1 1.6 ± 0.1 LB-MSN-OVA 656 ± 5 0.280 ± 0.018 −33 ± 3 73.8 ± 5.7 – 15.6 ± 1.2 – LB-MSN-OVA-PIC 603 ± 17 0.318 ± 0.040 −38 ± 3 34.4 ± 3.3 64.9 ± 14.6 7.9 ± 0.8 5.9 ± 1.3 GNP-OVA 507 ± 31 0.131 ± 0.116 21 ± 2 90.9 ± 14.2 – 4.3 ± 0.7 – GNP-OVA-PIC 757 ± 235 0.320 ± 0.179 8 ± 12 96.8 ± 4.3 95.0 ± 4.4 3.5 ± 0.9 3.4 ± 1.0 Table 1 a Size: Z-average in diameter. b b PDI: poly dispersity index. c ZP: zeta potential. d EE: encapsulation eﬃciency. e LC: loading capacity. 113 G. Du et al. Fig. 1. Electron microscope images of nanoparticles. A) Scanning electron microscopy (SEM) image of PLGA na- noparticles; B) Cryo-EM image of liposomes; C) Transmission electron microscopy (TEM) image of MSNs; D) SEM image of GNPs. Fig. 2. Release proﬁles of OVA (A) and poly(I:C) (B) from PLGA nanoparticles (blue/spheres), liposomes (purple/ diamonds), MSNs (green/triangles) and GNPs (brown/ squares) in PBS at 37 °C. Open and closed symbols corre- spond to poly(I:C)-containing and poly(I:C)-free nano- particles, respectively. Data points represent mean ± SD, n = 3. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) G. Du et al. Journal of Controlled Release 266 (2017) 109–118 Fig. 1. Electron microscope images of nanoparticles. A) Scanning electron microscopy (SEM) image of PLGA na- noparticles; B) Cryo-EM image of liposomes; C) Transmission electron microscopy (TEM) image of MSNs; D) SEM image of GNPs. Fig. 2. Release proﬁles of OVA (A) and poly(I:C) (B) from PLGA nanoparticles (blue/spheres), liposomes (purple/ diamonds), MSNs (green/triangles) and GNPs (brown/ squares) in PBS at 37 °C. Open and closed symbols corre- spond to poly(I:C)-containing and poly(I:C)-free nano- particles, respectively. Data points represent mean ± SD, n = 3. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 1. Electron microscope images of nanoparticles. A) Scanning electron microscopy (SEM) image of PLGA na- noparticles; B) Cryo-EM image of liposomes; C) Transmission electron microscopy (TEM) image of MSNs; D) SEM image of GNPs. Fig. 2. Release proﬁles of OVA (A) and poly(I:C) (B) from PLGA nanoparticles (blue/spheres), liposomes (purple/ diamonds), MSNs (green/triangles) and GNPs (brown/ squares) in PBS at 37 °C. Open and closed symbols corre- spond to poly(I:C)-containing and poly(I:C)-free nano- particles, respectively. Data points represent mean ± SD, n = 3. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 3. OVA-speciﬁc total IgG antibody titers measured in BALB/c mice on day 21 (A), day 42 (B) and day 49 (C). Bars represent mean ± SEM, n = 8. p < 0.05, p < 0.01, *p < 0.001. Fig. 2. Release proﬁles of OVA (A) and poly(I:C) (B) from PLGA nanoparticles (blue/spheres), liposomes (purple/ diamonds), MSNs (green/triangles) and GNPs (brown/ squares) in PBS at 37 °C. Open and closed symbols corre- spond to poly(I:C)-containing and poly(I:C)-free nano- particles, respectively. Data points represent mean ± SD, n = 3. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 1. Electron microscope images of nanoparticles. A) Scanning electron microscopy (SEM) image of PLGA na- noparticles; B) Cryo-EM image of liposomes; C) Transmission electron microscopy (TEM) image of MSNs; D) SEM image of GNPs. Journal of Controlled Release 266 (2017) 109–118 Table 1 All the formulations were injected intradermally, except the subcutaneous control of OVA solution (OVA S.C.). Groups without a bar showed titers below the detection limit of the ELISA. 114 Fig. 2. Release proﬁles of OVA (A) and poly(I:C) (B) from PLGA nanoparticles (blue/spheres), liposomes (purple/ diamonds), MSNs (green/triangles) and GNPs (brown/ squares) in PBS at 37 °C. Open and closed symbols corre- spond to poly(I:C)-containing and poly(I:C)-free nano- particles, respectively. Data points represent mean ± SD, n = 3. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the web version of this article.) Fig. 3. OVA-speciﬁc total IgG antibody titers measured in BALB/c mice on day 21 (A), day 42 (B) and day 49 (C). Bars represent mean ± SEM, n = 8. p < 0.05, *p < 0.01, Fig. 3. OVA-speciﬁc total IgG antibody titers measured in BALB/c mice on day 21 (A), day 42 (B) and day 49 (C). Bars represent mean ± SEM, n = 8. p < 0.05, p < 0.01, p < 0.001. All the formulations were injected intradermally, except the subcutaneous control of OVA solution (OVA S.C.). Groups without a bar showed titers below the detection limit of the ELISA. 114 Journal of Controlled Release 266 (2017) 109–118 G. Du et al. Fig. 4 antibo 42 (C, n = 8. formul cutane withou ELISA. G. Du et al. encapsulation of OVA or co-encapsulation of OVA and poly(I:C) did not lead to enhanced total IgG titers. Next, the subtype IgG1 and IgG2a titers were determined (Fig. 4). The IgG1 titers followed the trend of total IgG titers (Fig. 4A, C, E) and (Fig. 4D). After the second boost, on day 49, all the groups, except OVA solution, induced a measurable IgG2a response (Fig. 4F). These results illustrate that encapsulation of OVA, and especially co-encapsulation of OVA and poly(I:C) in nanoparticles is critical for enhancement of IgG2a Fig. 4. OVA-speciﬁc IgG1 (A, C, E) and IgG2a (B, D, F) antibody titers measured in BALB/c mice on day 21 (A, B), 42 (C, D) and 49 (E, F). Bars represent mean ± SEM, n = 8. p < 0.05, p < 0.01, p < 0.001. All the formulations were injected intradermally, except the sub- cutaneous control of OVA solution (OVA S.C). Groups without a bar showed titers below the detection limit of the ELISA. G. Du et al. Table 1 The IgG1 titers followed the trend of total IgG titers (Fig. 4A, C, E) and similarly the encapsulation of OVA or co-encapsulation of OVA and poly (I:C) did not increase the IgG1 response. However, the encapsulation of OVA, and particularly co-encapsulation of OVA and poly(I:C), strikingly increased the IgG2a response compared to OVA and poly(I:C) solution (Fig. 4B, D, F) (except GNP-OVA-PIC). Furthermore, liposomes and PLGA nanoparticles showed higher IgG2a responses than MSNs and GNPs (Fig. 4F). Speciﬁcally, on day 21 only PLGA-OVA-PIC induced an IgG2a response (Fig. 4B). After each boosting on day 21 (Fig. 4D) and 42 (Fig. 4F), there were more groups having an IgG2a response. On day 42, after prime and one boost, all OVA and poly(I:C) co-encapsulated nanoparticles, except GNP-OVA-PIC, showed an IgG2a response Next, the subtype IgG1 and IgG2a titers were determined (Fig. 4). The IgG1 titers followed the trend of total IgG titers (Fig. 4A, C, E) and similarly the encapsulation of OVA or co-encapsulation of OVA and poly (I:C) did not increase the IgG1 response. However, the encapsulation of OVA, and particularly co-encapsulation of OVA and poly(I:C), strikingly increased the IgG2a response compared to OVA and poly(I:C) solution (Fig. 4B, D, F) (except GNP-OVA-PIC). Furthermore, liposomes and PLGA nanoparticles showed higher IgG2a responses than MSNs and GNPs (Fig. 4F). Speciﬁcally, on day 21 only PLGA-OVA-PIC induced an IgG2a response (Fig. 4B). After each boosting on day 21 (Fig. 4D) and 42 (Fig. 4F), there were more groups having an IgG2a response. On day 42, after prime and one boost, all OVA and poly(I:C) co-encapsulated nanoparticles, except GNP-OVA-PIC, showed an IgG2a response Table 1 Journal of Controlled Release 266 (2017) 109118 Fig. 4. OVA-speciﬁc IgG1 (A, C, E) and IgG2a (B, D, F) antibody titers measured in BALB/c mice on day 21 (A, B), 42 (C, D) and 49 (E, F). Bars represent mean ± SEM, n = 8. p < 0.05, p < 0.01, p < 0.001. All the formulations were injected intradermally, except the sub- cutaneous control of OVA solution (OVA S.C). Groups without a bar showed titers below the detection limit of the ELISA. encapsulation of OVA or co-encapsulation of OVA and poly(I:C) did not lead to enhanced total IgG titers. Next, the subtype IgG1 and IgG2a titers were determined (Fig. 4). The IgG1 titers followed the trend of total IgG titers (Fig. 4A, C, E) and similarly the encapsulation of OVA or co-encapsulation of OVA and poly (I:C) did not increase the IgG1 response. However, the encapsulation of OVA, and particularly co-encapsulation of OVA and poly(I:C), strikingly increased the IgG2a response compared to OVA and poly(I:C) solution (Fig. 4B, D, F) (except GNP-OVA-PIC). Furthermore, liposomes and PLGA nanoparticles showed higher IgG2a responses than MSNs and GNPs (Fig. 4F). Speciﬁcally, on day 21 only PLGA-OVA-PIC induced an IgG2a response (Fig. 4B). After each boosting on day 21 (Fig. 4D) and (Fig. 4D). After the second boost, on day 49, all the groups, except OVA solution, induced a measurable IgG2a response (Fig. 4F). These results illustrate that encapsulation of OVA, and especially co-encapsulation of OVA and poly(I:C) in nanoparticles is critical for enhancement of IgG2a response but the magnitude of this eﬀect depends on the type of na- noparticles. 3.4. T cell responses after intradermal immunization The higher IgG2a responses observed with liposomes and PLGA nanoparticles suggested that these formulations may be able to trigger cellular immune responses more eﬀectively. To study the eﬃcacy of the encapsulation of OVA or co-encapsulation of OVA and poly(I:C) did not lead to enhanced total IgG titers. (Fig. 4D). After the second boost, on day 49, all the groups, except OVA solution, induced a measurable IgG2a response (Fig. 4F). These results illustrate that encapsulation of OVA, and especially co-encapsulation of OVA and poly(I:C) in nanoparticles is critical for enhancement of IgG2a response but the magnitude of this eﬀect depends on the type of na- noparticles. Next, the subtype IgG1 and IgG2a titers were determined (Fig. 4). Fig. 4. OVA-speciﬁc IgG1 (A, C, E) and IgG2a (B, D, F) antibody titers measured in BALB/c mice on day 21 (A, B), 42 (C, D) and 49 (E, F). Bars represent mean ± SEM, n = 8. p < 0.05, p < 0.01, p < 0.001. All the formulations were injected intradermally, except the sub- cutaneous control of OVA solution (OVA S.C). Groups without a bar showed titers below the detection limit of the ELISA. Journal of Controlled Release 266 (2017) 109–118 3.4. T cell responses after intradermal immunization The higher IgG2a responses observed with liposomes and PLGA nanoparticles suggested that these formulations may be able to trigger cellular immune responses more eﬀectively. To study the eﬃcacy of the developed nanoparticle formulations to induce T cell mediated im- munity in vivo, OT-I (OVA speciﬁc CD8+ T cells) and OT-II (OVA spe- ciﬁc CD4+ T cells) cells were transferred into C57BL/6 mice before 115 Journal of Controlled Release 266 (2017) 109–118 G. Du et al. Fig. 5. OVA-speciﬁc T cell responses. (A) An example of the ﬂow cytometry gating strategy used to determine the T cell responses. Lymphocytes were gated on forward/sideward scatter, followed by the exclusion of double or adhering cells. After pregating on CD4+ or CD8+ T cells, the percentage of respectively OT-II and OT-I were measured by gating on CD45.1+ cells. OVA speciﬁc CD8+ (B) and CD4+ (C) responses of transferred OT-I and OT-II cells in mouse blood 7 days after the immunization (mean ± SEM, n = 5). p < 0.05, p < 0.01, p < 0.001. Fig. 5. OVA-speciﬁc T cell responses. (A) An example of the ﬂow cytometry gating strategy used to determine the T cell responses. Lymphocytes were gated on forward/sideward scatter, followed by the exclusion of double or adhering cells. After pregating on CD4+ or CD8+ T cells, the percentage of respectively OT-II and OT-I were measured by gating on CD45.1+ cells. OVA speciﬁc CD8+ (B) and CD4+ (C) responses of transferred OT-I and OT-II cells in mouse blood 7 days after the immunization (mean ± SEM, n = 5). p < 0.05, p < 0.01, *p < 0.001. intradermal vaccination. Seven days after the immunization T cell re- sponses in blood were analyzed by ﬂow cytometry with gating strategy shown in Fig. 5A. Lipo-OVA-PIC evoked signiﬁcant higher CD8+ T cell responses than OVA and poly(I:C) solution and the other nanoparticle formulations (Fig. 5B), suggesting eﬃcient induction of CTL responses by liposomes. In general, nano-encapsulation of OVA or co-encapsula- tion of OVA and poly(I:C) increased the CD8+ response compared to OVA or OVA-poly(I:C) solution. In the case of CD4+ T cell response, Lipo-OVA-PIC and LB-MSN-OVA-PIC induced the strongest response (Fig. 5C). OVA loaded nanoparticles induced similar CD4+ response compared to OVA solution. Poly(I:C) co-encapsulation slightly in- creased CD4+ responses compared to OVA-loaded nanoparticles but only in the case of liposomes the improvement was signiﬁcant. 3.4. T cell responses after intradermal immunization Fur- thermore, the Lipo-OVA-PIC formulation induced a signiﬁcantly higher CD4+ response than OVA and poly(I:C) solution. indicated that nanoparticles can be used to modulate the immune re- sponse [9,10,12,15,17,18,20,22,25,39,42]. Owing to its high density of antigen-presenting cells, the skin could be an attractive site of admin- istration of nanoparticulate vaccines. However, relatively little is known about the eﬀect of nanoparticulate vaccines after (microneedle- mediated) intradermal vaccination. Therefore, in this study, we used hollow microneedles together with an applicator to examine the eﬀect of nano-encapsulation of antigen and adjuvant on both the humoral and cellular response in mice. Our results showed that antigen and adjuvant loaded nanoparticles were successfully delivered intradermally in mice by using hollow microneedles together with the applicator, leading to an eﬀective nanoparticle-dependent immune response. Furthermore, after co-encapsulation of OVA and poly(I:C) into nanoparticles, the immune response was modulated towards a Th1 direction. Previously, the in-house developed hollow microneedle/applicator system has been used for immunization with inactivated virus [4–6]. In these studies, we used hollow microneedles with a bore diameter of 20 μm. However, the system has not been used with nanoparticles with larger size (> 100 nm). Therefore, the injection of nanoparticles through the system was tested in vitro (data not shown) prior to the in vivo studies presented here. These pilot studies showed that the hollow microneedles could be blocked due to occasional nanoparticle ag- gregation if the bore diameter was 20 μm. By increasing the bore dia- meter to 50 μm, this problem could be circumvented since increase of the bore diameter decreases particle obstruction in the system. 4. Discussion In recent years, nanoparticles have been intensively investigated as vaccine delivery systems because of their advantages, such as protec- tion of antigen from degradation, increased antigen uptake by dendritic cells and the ability to co-deliver antigen and adjuvant [10,22,41]. Nanoparticles also oﬀer the possibility to adjust the type of immune response by modifying the nanoparticle characteristics such as size, surface charge and antigen release proﬁle [10]. Numerous studies have 116 G. Du et al. Journal of Controlled Release 266 (2017) 109–118 Therefore, the higher IgG2a response induced by the OVA/poly(I:C) loaded PLGA nanoparticles and liposomes could be due to their better uptake by dendritic cells and faster traﬃcking to the lymph nodes. Consequently, there was no blockage or leakage of formulation during the immunization studies. The success of intradermal injection was conﬁrmed by the formation of a bleb at the injection site after each injection. Furthermore, no adverse eﬀects, such as erythema or skin induration, were observed at the injection site during the studies. As IgG2a levels merely give an indication of the extent of IFN-γ induced isotype switching, we also directly assessed the capacity of each type of nanoparticulate formulation to induce CD8+ and CD4+ T cell responses in OT cell transferred mice. The number of transferred OT cells was kept low as an excess of transgenic T-cells have previously been shown to alter the T-cell response [50]. Our results showed that the Lipo-OVA-PIC formulation showed an exceptional high capacity to induce both CD8+ and CD4+ T cell responses, in line with the data from previous studies [18,48]. The DOTAP based cationic liposomes have been shown to be very eﬀective for the induction of CTL responses [9,12,18], as the cationic lipids promote the activation and maturation of dendritic cells and subsequently the T cell priming. Moreover, EggPC, the main lipid component of the present liposomes, has been shown to facilitate antigen presentation by enhancing peptide binding to MHC class II molecules [51]. So, the superior immune responses of liposomes may be caused by the properties of the lipids. j g Intradermally administered OVA/poly(I:C) loaded nanoparticles did not increase the total IgG response compared to administration of an- tigen/adjuvant alone. These results indicate that the encapsulation of OVA or co-encapulation of OVA and poly(I:C) is not required for a strong IgG response following intradermal administration. 4. Discussion This may be caused by the eﬃcient uptake of the free antigen/adjuvant by antigen- presenting cells in epidermis and dermis (Langerhans cells and den- dritic cells) and lymph nodes beneath the skin. Additionally, poly(I:C) has been shown to strongly improve CD8 responses rather than IgG responses [43–45]. In case of PLGA nanoparticles, PLGA-OVA showed even a lower total IgG response than OVA alone. This may be due to a change in the tertiary structure of OVA, either during preparation or in the acidic environment of PLGA nanoparticles during the degradation of the polymer after administration [46,47]. Furthermore, in the cur- rent study the OVA dose was much lower (0.31 μg) compared to the dose used in previous studies (e.g. ≥5 μg) [27,42,48]. The low dose can magnify the detrimental eﬀect of partial OVA degradation in PLGA nanoparticles. As explained above, MSNs and GNPs may not be able to enhance the immune response because of their fast release of OVA/poly(I:C) and large size. In case of PLGA-OVA-PIC group, our data showed that the co- encapsulation of OVA and poly(I:C) did not increase the T cell re- sponses. This is in contrast to previous reports [12–14], which have shown that the co-encapsulation of OVA and poly(I:C) in PLGA nano- particles can induce a strong CTL response after intramuscular or sub- cutaneous vaccination. This indicates that vaccine formulations that provide potent immune responses after intramuscular or subcutaneous administration, may be less suitable for intradermal delivery, re- emphasizing the need for route-speciﬁc optimization of vaccine for- mulations [12,18,42]. Furthermore, targeting of diﬀerent skin layers may also aﬀect immune responses, as shown in previous studies [52,53]. Nowadays, there is an increasing need of eﬃcient Th1/CTL immune response, for example, in therapeutic vaccinations for cancer [13,17,45] and intracellular pathogens [14,21]. Our results indicate that cationic liposomes are very promising nano-carriers to induce a superior Th1/CTL immune response compared to the other nano- particles following hollow microneedle-mediated intradermal admin- istration. nanoparticles. Our results clearly show that co-delivery of the antigen and ad- juvant in nanoparticles, increased signiﬁcantly the IgG2a antibody re- sponse compared to OVA/poly(I:C) solution. This indicates that the nanoparticles skewed the immune response of the antigen more to- wards a Th1 direction [39]. Interestingly, PLGA nanoparticles and li- posomes induced higher IgG2a responses than GNPs and MSNs. There are at least two possible underlying factors that may play a role. 5. Conclusions OVA and poly(I:C) loaded PLGA nanoparticles, liposomes, MSNs and GNPs were successfully developed and compared for hollow mi- croneedle-mediated intradermal immunization in mice. The en- capsulation of OVA and co-encapsulation of OVA and poly(I:C) induced a strikingly higher IgG2a antibody response than OVA/poly(I:C) solu- tion, but the type of nanoparticle has a major eﬀect on response. PLGA nanoparticles and especially cationic liposomes induced the highest IgG2a, CD8+ T cell and CD4+ T cell responses, suggesting their su- periority for intradermal vaccination. Finally, our study demonstrated that the in house developed hollow microneedle/applicator system is an excellent tool for nanoparticle-based intradermal vaccination and to screen diﬀerent intradermal vaccine formulations. 4. Discussion i) The higher IgG2a response is in line with the slower release of OVA and poly (I:C) from PLGA nanoparticles and liposomes. The sustained release can allow the co-processing of adjuvant and antigen within the same an- tigen-presenting cell, which is suggested to be crucial for a higher IgG2a response [15,19,22]. Diﬀerences in release behavior of OVA/poly(I:C) between the nanoparticles may stem from the diﬀerences in the loca- tion of the antigen and adjuvant in nanoparticles and in the strength of the interaction between antigen/adjuvant and the nanoparticle matrix. On the one hand, in PLGA nanoparticles and liposomes the antigen/ adjuvant is mixed with nanoparticle precursors during synthesis, and the antigen/adjuvant is expected to be localized inside the matrix of PLGA nanoparticles or in the aqueous core layer of liposomes. There- fore, it is likely that the antigen/adjuvant is mostly released after the nanoparticles are taken up and processed by antigen-presenting cells or degraded. On the other hand, with GNPs and MSNs the loading of an- tigen/adjuvant is done post-synthesis through adsorption of the an- tigen/adjuvant onto the surface of nanoparticles, presumably based on electrostatic interactions. In addition, in MSNs interactions are ex- pected to occur also between OVA/poly(I:C) and the stabilizing lipid bilayer. Antigen and adjuvant loaded in MSNs and GNPs are sensitive to environmental conditions, such as salts and endogenous proteins pre- sent in the skin tissue, that can accelerate the release. As a result, the release of antigen/adjuvant from MSNs and GNPs can be faster than that from PLGA nanoparticles and liposomes, as suggested by the in vitro release data. Premature release can consequently lead to separate uptake and processing of antigen and adjuvant by diﬀerent antigen- presenting cells. ii) The size of PLGA nanoparticles and liposomes (< 200 nm) is substantially smaller than that of MSNs and GNPs (above 500 nm). Although single MSNs had a size below 200 nm, as shown by TEM images [33], DLS showed a larger diameter, indicating the ag- gregation of MSNs. Smaller particles with a size below 200 nm are expected to be more eﬃciently taken up by dendritic cells than bigger particles [16]. Moreover, large nanoparticles (500–2000 nm) have been shown to be mostly associated with dendritic cells at the injection site after intradermal delivery, while small (20–200 nm) nanoparticles are able to drain to lymph nodes and target there the dendritic cells [49]. References 33 (2016) 2269–2279. [34] E.C. Dengler, J. Liu, A. Kerwin, S. Torres, C.M. Olcott, B.N. Bowman, L. Armijo, K. Gentry, J. Wilkerson, J. Wallace, X. Jiang, E.C. Carnes, C.J. Brinker, E.D. Milligan, Mesoporous silica-supported lipid bilayers (protocells) for DNA cargo delivery to the spinal cord, J. Control. Release 168 (2013) 209–224. d k k d d [6] P. Schipper, K. van der Maaden, S. Romeijn, C. Oomens, G. Kersten, W. Jiskoot, J. Bouwstra, Repeated fractional intradermal dosing of an inactivated polio vaccine by a single hollow microneedle leads to superior immune responses, J. Control. Release 242 (2016) 141–147. [35] J. Tu, G. Du, M. Reza Nejadnik, J. Monkare, K. van der Maaden, P.H.H. Bomans, N. Sommerdijk, B. Slutter, W. Jiskoot, J.A. Bouwstra, A. Kros, Mesoporous silica nanoparticle-coated microneedle arrays for intradermal antigen delivery, Pharm. Res. 34 (8) (2017) 1693–1706. [7] M.L. De Temmerman, J. Rejman, J. Demeester, D.J. Irvine, B. Gander, S.C. De Smedt, Particulate vaccines: on the quest for optimal delivery and immune re- sponse, Drug Discov. Today 16 (2011) 569–582. [36] H. Wang, M.B. Hansen, D.W. Lowik, J.C. van Hest, Y. Li, J.A. Jansen, S.C. Leeuwenburgh, Oppositely charged gelatin nanospheres as building blocks for injectable and biodegradable gels, Adv. Mater. 23 (2011) H119–124. g y [8] S.G. Reed, M.T. Orr, C.B. Fox, Key roles of adjuvants in modern vaccines, Nat. Med. 19 (2013) 1597–1608. [9] N. Benne, J. van Duijn, J. Kuiper, W. Jiskoot, B. Slutter, Orchestrating immune responses: how size, shape and rigidity aﬀect the immunogenicity of particulate vaccines, J. Control. Release 234 (2016) 124–134. [37] S. Azarmi, Y. Huang, H. Chen, S. McQuarrie, D. Abrams, W. Roa, W.H. Finlay, G.G. Miller, R. Lobenberg, Optimization of a two-step desolvation method for preparing gelatin nanoparticles and cell uptake studies in 143B osteosarcoma cancer cells, J. Pharm. Pharm. Sci. 9 (2006) 124–132. [10] T. Akagi, M. Baba, M. Akashi, Biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine, in: S. Kunugi, T. Yamaoka (Eds.), Polymers in Nanomedicine, 2012, pp. 31–64. [10] T. Akagi, M. Baba, M. Akashi, Biodegradable nanoparticles as vaccine adjuvants and delivery systems: regulation of immune responses by nanoparticle-based vaccine, in: S. Kunugi, T. Yamaoka (Eds.), Polymers in Nanomedicine, 2012, pp. 31–64. [11] L. Zhao, A. Seth, N. Wibowo, C.X. Zhao, N. Mitter, C.Z. Yu, A.P.J. Middelberg, Nanoparticle vaccines, Vaccine 32 (2014) 327–337. [38] K. van der Maaden, S.J. Trietsch, H. References References [27] D. Mahony, A.S. Cavallaro, F. Stahr, T.J. Mahony, S.Z. Qiao, N. Mitter, Mesoporous silica nanoparticles act as a self-adjuvant for ovalbumin model antigen in mice, Small 9 (2013) 3138–3146. [1] N. Li, L.H. Peng, X. Chen, S. Nakagawa, J.Q. Gao, Transcutaneous vaccines: novel advances in technology and delivery for overcoming the barriers, Vaccine 29 (2011) 6179–6190. [28] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Protein-based nanocarriers as promising drug and gene delivery systems, J. Control. Release 161 (2012) 38–49. [29] M S S dh h S P V D V K hli N i l b d i i i i [29] M.S. Sudheesh, S.P. Vyas, D.V. Kohli, Nanoparticle-based immunopotentiation via tetanus toxoid-loaded gelatin and aminated gelatin nanoparticles, Drug Deliv. 18 (2011) 320–330. [2] K. van der Maaden, W. Jiskoot, J. Bouwstra, Microneedle technologies for (trans) dermal drug and vaccine delivery, J. Control. Release 161 (2012) 645–655. b l d k k [3] F.J. Verbaan, S.M. Bal, D.J. van den Berg, J.A. Dijksman, M. van Hecke, H. Verpoorten, A. van den Berg, R. Luttge, J.A. Bouwstra, Improved piercing of microneedle arrays in dermatomed human skin by an impact insertion method, J. Control. Release 128 (2008) 80–88. [30] A.O. Elzoghby, Gelatin-based nanoparticles as drug and gene delivery systems: re- viewing three decades of research, J. Control. Release 172 (2013) 1075–1091. [31] B. Slutter, S.M. Bal, I. Que, E. Kaijzel, C. Lowik, J. Bouwstra, W. Jiskoot, Antigen- adjuvant nanoconjugates for nasal vaccination: an improvement over the use of nanoparticles? Mol. Pharm. 7 (2010) 2207–2215. [4] K. van der Maaden, S.J. Trietsch, H. Kraan, E.M. Varypataki, S. Romeijn, R. Zwier, H.J. van der Linden, G. Kersten, T. Hankemeier, W. Jiskoot, J. Bouwstra, Novel hollow microneedle technology for depth-controlled microinjection-mediated dermal vaccination: a study with polio vaccine in rats, Pharm. Res. Dordr. 31 (2014) 1846–1854. [32] A.D. Bangham, M.M. Standish, J.C. Watkins, Diﬀusion of univalent ions across the lamellae of swollen phospholipids, J. Mol. Biol. 13 (1965) 238–252. [33] J. Tu, A.L. Boyle, H. Friedrich, P.H. Bomans, J. Bussmann, N.A. Sommerdijk, W. Jiskoot, A. Kros, Mesoporous silica nanoparticles with large pores for the en- capsulation and release of proteins, ACS Appl. Mater. Interfaces 8 (2016) 32211–32219. [5] P. Schipper, K. van der Maaden, S. Romeijn, C. Oomens, G. Kersten, W. Jiskoot, J. Bouwstra, Determination of depth-dependent intradermal immunogenicity of adjuvanted inactivated polio vaccine delivered by microinjections via hollow mi- croneedles, Pharm. Res. References Kraan, E.M. Varypataki, S. Romeijn, R. Zwier, H.J. van der Linden, G. Kersten, T. Hankemeier, W. Jiskoot, J. Bouwstra, Novel hollow microneedle technology for depth-controlled microinjection-mediated dermal vaccination: a study with polio vaccine in rats, Pharm. Res. 31 (2014) 1846–1854. g y pp [11] L. Zhao, A. Seth, N. Wibowo, C.X. Zhao, N. Mitter, C.Z. Yu, A.P.J. Middelberg, Nanoparticle vaccines, Vaccine 32 (2014) 327–337. [12] E.M. Varypataki, A.L. Silva, C. Barnier-Quer, N. Collin, F. Ossendorp, W. Jiskoot, Synthetic long peptide-based vaccine formulations for induction of cell mediated immunity: a comparative study of cationic liposomes and PLGA nanoparticles, J. Control. Release 226 (2016) 98–106. [39] B. Slutter, S.M. Bal, Z. Ding, W. Jiskoot, J.A. Bouwstra, Adjuvant eﬀect of cationic liposomes and CpG depends on administration route, J. Control. Release 154 (2011) 123–130. [13] S. Hamdy, O. Molavi, Z.S. Ma, A. Haddadi, A. Alshamsan, Z. Gobti, S. Elhasi, J. Samuel, A. Lavasanifar, Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8(+) T cell-mediated anti-tumor immunity, Vaccine 26 (2008) 5046–5057. [40] A.K. Bajpai, J. Choubey, Design of gelatin nanoparticles as swelling controlled delivery system for chloroquine phosphate, J. Mater. Sci. Mater. Med. 17 (2006) 345–358. [41] L.J. Peek, C.R. Middaugh, C. Berkland, Nanotechnology in vaccine delivery, Adv. Drug Deliv. Rev. 60 (2008) 915–928. y [14] C.S.W. Chong, M. Cao, W.W. Wong, K.P. Fischer, W.R. Addison, G.S. Kwon, D.L. Tyrrell, J. Samuel, Enhancement of T helper type 1 immune responses against hepatitis B virus core antigen by PLGA nanoparticle vaccine delivery, J. Control. Release 102 (2005) 85–99. [42] D. Mohanan, B. Slutter, M. Henriksen-Lacey, W. Jiskoot, J.A. Bouwstra, Y. Perrie, T.M. Kundig, B. Gander, P. Johansen, Administration routes aﬀect the quality of immune responses: a cross-sectional evaluation of particulate antigen-delivery systems, J. Control. Release 147 (2010) 342–349. [15] E. Schlosser, M. Mueller, S. Fischer, S. Basta, D.H. Busch, B. Gander, M. Groettrup, TLR ligands and antigen need to be coencapsulated into the same biodegradable microsphere for the generation of potent cytotoxic T lymphocyte responses, Vaccine 26 (2008) 1626–1637. [43] H. Mitsui, T. Watanabe, H. Saeki, K. Mori, H. Fujita, Y. Tada, A. Asahina, K. Nakamura, K. Tamaki, Diﬀerential expression and function of Toll-like receptors in Langerhans cells: comparison with splenic dendritic cells, J. Invest. Dermatol. 122 (2004) 95–102. [16] M.O. Oyewumi, A. Kumar, Z.R. References Cui, Nano-microparticles as immune adjuvants: correlating particle sizes and the resultant immune responses, Expert Rev. Vaccines 9 (2010) 1095–1107. [44] H. Fujita, A. Asahina, H. Mitsui, K. Tamaki, Langerhans cells exhibit low respon- siveness to double-stranded RNA, Biochem. Biophys. Res. Commun. 319 (2004) 832–839. [17] Y. Fan, J.J. Moon, Nanoparticle drug delivery systems designed to improve cancer vaccines and immunotherapy, Vaccines (Basel) 3 (2015) 662–685. [45] R. Ammi, J. De Waele, Y. Willemen, I. Van Brussel, D.M. Schrijvers, E. Lion, E.L.J. Smits, Poly(I:C) as cancer vaccine adjuvant: Knocking on the door of medical breakthroughs, Pharmacol. Ther. 146 (2015) 120–131. [18] E.M. Varypataki, K. van der Maaden, J. Bouwstra, F. Ossendorp, W. Jiskoot, Cationic liposomes loaded with a synthetic long peptide and poly(I:C): a deﬁned adjuvanted vaccine for induction of antigen-speciﬁc T cell cytotoxicity, AAPS J. 17 (2015) 216–226. [46] K. Fu, D.W. Pack, A.M. Klibanov, R. Langer, Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (PLGA) microspheres, Pharm. Res. Dordr. 17 (2000) 100–106. [19] A.L. Silva, R.A. Rosalia, A. Sazak, M.G. Carstens, F. Ossendorp, J. Oostendorp, W. Jiskoot, Optimization of encapsulation of a synthetic long peptide in PLGA nanoparticles: low-burst release is crucial for eﬃcient CD8(+) T cell activation, Eur. J. Pharm. Biopharm. 83 (2013) 338–345. [47] M. van de Weert, W.E. Hennink, W. Jiskoot, Protein instability in poly(lactic-co- glycolic acid) microparticles, Pharm. Res. 17 (2000) 1159–1167. l k l f [48] S.M. Bal, S. Hortensius, Z. Ding, W. Jiskoot, J.A. Bouwstra, Co-encapsulation of antigen and Toll-like receptor ligand in cationic liposomes aﬀects the quality of the immune response in mice after intradermal vaccination, Vaccine 29 (2011) 1045–1052. [20] R.A. Rosalia, L.J. Cruz, S. van Duikeren, A.T. Tromp, A.L. Silva, W. Jiskoot, T. de Gruijl, C. Lowik, J. Oostendorp, S.H. van der Burg, F. Ossendorp, CD40-targeted dendritic cell delivery of PLGA-nanoparticle vaccines induce potent anti-tumor responses, Biomaterials 40 (2015) 88–97. [49] V. Manolova, A. Flace, M. Bauer, K. Schwarz, P. Saudan, M.F. Bachmann, Nanoparticles target distinct dendritic cell populations according to their size, Eur. J. Immunol. 38 (2008) 1404–1413. [21] M. Zaric, O. Lyubomska, O. Touzelet, C. Poux, S. Al-Zahrani, F. Fay, L. Wallace, D. Terhorst, B. Malissen, S. Henri, U.F. Power, C.J. Scott, R.F. Donnelly, A. Kissenpfennig, Skin dendritic cell targeting via microneedle arrays laden with antigen-encapsulated poly-D,L-lactide-co-glycolide nanoparticles induces eﬃcient antitumor and antiviral immune responses, ACS Nano 7 (2013) 2042–2055. [50] V.P. Badovinac, J.S. Haring, J.T. Acknowledgements The research leading to these results has received support from the Innovative Medicines Initiative Joint Undertaking under grant agree- ment n° 115363 resources of which are composed of ﬁnancial con- tribution from the European Union's Seventh Framework Programme (FP7/2007-2013) and EFPIA companies' in kind contribution. G. Du thanks for the part support from Chinese Scholarship Council. Furthermore, we acknowledge K. Pesl for the help with preparation of PLGA nanoparticles and P. Schipper for the help with antibody mea- surements. 117 Journal of Controlled Release 266 (2017) 109–118 G. Du et al. References Harty, Initial T cell receptor transgenic cell pre- cursor frequency dictates critical aspects of the CD8(+) T cell response to infection, Immunity 26 (2007) 827–841. [22] A.M. Hafner, B. Corthesy, H.P. Merkle, Particulate formulations for the delivery of poly(I:C) as vaccine adjuvant, Adv. Drug Deliv. Rev. 65 (2013) 1386–1399. y [51] R.W. Roof, I.F. Luescher, E.R. Unanue, Phospholipids enhance the binding of pep- tides to class II major histocompatibility molecules, Proc. Natl. Acad. Sci. U. S. A. 87 (1990) 1735–1739. [23] A.L. Silva, R.A. Rosalia, E. Varypataki, S. Sibuea, F. Ossendorp, W. Jiskoot, Poly- (lactic-co-glycolic-acid)-based particulate vaccines: particle uptake by dendritic cells is a key parameter for immune activation, Vaccine 33 (2015) 847–854. [52] C. Liard, S. Munier, M. Arias, A. Joulin-Giet, O. Bonduelle, D. Duﬀy, R.J. Shattock, B. Verrier, B. Combadiere, Targeting of HIV-p24 particle-based vaccine into dif- ferential skin layers induces distinct arms of the immune responses, Vaccine 29 (2011) 6379–6391. [24] A.L. Silva, P.C. Soema, B. Slutter, F. Ossendorp, W. Jiskoot, PLGA particulate de- livery systems for subunit vaccines: linking particle properties to immunogenicity, Hum. Vaccines Immunother. 12 (2016) 1056–1069. [53] C. Levin, O. Bonduelle, C. Nuttens, C. Primard, B. Verrier, A. Boissonnas, B. Combadiere, Critical role for skin-derived migratory DCs and langerhans cells in TFH and GC responses after intradermal immunization, J. Invest. Dermatol. 137 (9) (2017) 1905–1913. [25] D. Christensen, K.S. Korsholm, P. Andersen, E.M. Agger, Cationic liposomes as vaccine adjuvants, Expert Rev. Vaccines 10 (2011) 513–521. [26] K.T. Mody, A. Popat, D. Mahony, A.S. Cavallaro, C.Z. Yu, N. Mitter, Mesoporous silica nanoparticles as antigen carriers and adjuvants for vaccine delivery, Nano 5 118
https://openalex.org/W2117079151	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0002883&type=printable	English	null	Preservation of Genes Involved in Sterol Metabolism in Cholesterol Auxotrophs: Facts and Hypotheses	PloS one	2,008	cc-by	8,913	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: Supported by the INSERM and Paris VII university. Funding: Supported by the INSERM and Paris VII university. Competing Interests: The authors have declared that no competing interests exist. * E-mail: veitia@cochin.inserm.fr Competing Interests: The authors have declared that no competing interests exist. * E-mail: veitia@cochin.inserm.fr Giovanna Vinci1, Xuhua Xia2, Reiner A. Veitia1,3* 1 Institut Cochin, De´partement de Ge´ne´tique et De´veloppement. Inserm, U567, CNRS, UMR 8104, Universite´ Paris 5, Faculte´ de Me´decine Paris Descartes, UM 3, Paris, France, 2 CAREG and Biology Department, University of Ottawa, Ottawa, Ontario, Canada, 3 Universite´ Denis Diderot/Paris VII, Paris, France PLoS ONE \| www.plosone.org Abstract Background: It is known that primary sequences of enzymes involved in sterol biosynthesis are well conserved in organisms that produce sterols de novo. However, we provide evidence for a preservation of the corresponding genes in two animals unable to synthesize cholesterol (auxotrophs): Drosophila melanogaster and Caenorhabditis elegans. Principal Findings: We have been able to detect bona fide orthologs of several ERG genes in both organisms using a series of complementary approaches. We have detected strong sequence divergence between the orthologs of the nematode and of the fruitfly; they are also very divergent with respect to the orthologs in organisms able to synthesize sterols de novo (prototrophs). Interestingly, the orthologs in both the nematode and the fruitfly are still under selective pressure. It is possible that these genes, which are not involved in cholesterol synthesis anymore, have been recruited to perform different new functions. We propose a more parsimonious way to explain their accelerated evolution and subsequent stabilization. The products of ERG genes in prototrophs might be involved in several biological roles, in addition to sterol synthesis. In the case of the nematode and the fruitfly, the relevant genes would have lost their ancestral function in cholesterogenesis but would have retained the other function(s), which keep them under pressure. Conclusions: By exploiting microarray data we have noticed a strong expressional correlation between the orthologs of ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding and with Start1 involved in ecdysteroid synthesis. These potential functional connections are worth being explored not only in Drosophila, but also in Caenorhabditis as well as in sterol prototrophs. Citation: Vinci G, Xia X, Veitia RA (2008) Preservation of Genes Involved in Sterol Metabolism in Cholesterol Auxotrophs: Facts and Hypotheses. PLoS ONE 3(8): e2883 doi:10 1371/journal pone 0002883 a X, Veitia RA (2008) Preservation of Genes Involved in Sterol Metabolism in Cholesterol Auxotrophs: Facts and Hypotheses. PLoS ONE ournal.pone.0002883 Editor: Jean-Nicolas Volff, Ecole Normale Supe´rieure de Lyon, France Editor: Jean-Nicolas Volff, Ecole Normale Supe´rieure de Lyon, France Received May 27, 2008; Accepted July 11, 2008; Published August 6, 2008 Received May 27, 2008; Accepted July 11, 2008; Published August 6, 2008 Copyright: 2008 Vinci et al. Looking for ERG orthologs in C. elegans and D. melanogaster Looking for ERG orthologs in C. elegans and D. melanogaster In this section we present our search for ERG gene orthologs in C. elegans and D. melanogaster following their order in the sterol synthesis cascade (as shown in Figure 1). We have taken advantage of the fact that the full genomic sequences of these two animals are available. We have used BLASTp [24] and considered as orthologs the best reciprocal hits [25]. The results are summarized in Figures 1 and 2. Figure 1. Outline of the ergosterol synthesis pathway in yeast. (+) the corresponding gene is present in C. elegans and D. melanogaster, according to our exploration. (2) the corresponding gene is absent. (?) not convincing evidence for the presence of the ortholog. doi:10.1371/journal.pone.0002883.g001 We failed to detect the squalene synthase (ERG9p) homolog, which catalyzes the first committed step in cholesterogenesis. In the search for squalene epoxidase (ERG1p) in a BLAST using ERG1p from yeast, we detected the gene CBG19254 in C. briggsae, with a marginal Score (S = 48 bits, E-value = 10204). This protein, which also exists in C. elegans, contains two functional domains: monooxygenase and UbiH (2-polyprenyl-6-methoxyphenol hy- droxylase and FAD-dependent oxidase). This is also the case in yeast ERG1p. However, in a reverse BLAST, CBG19254 recognized with very high score yeast Coq6 and only marginally ERG1p (S = 48, E = 10206). A similar behavior was observed for GA20231-RA from D. pseudoobscura, so we did not proceed to a further analysis of these sequences. (ERG25p-ERG26p-ERG27p) to the ER [11] and also interacts with the downstream enzyme ERG6p. More recent results have indicated that ERG28p also interacts with ERG11p and ERG1p. Moreover, ERG27p is required for oxidosqualene cyclase (ERG7p) activity. The physical interaction of ERG27p with ERG7p might indeed contribute to yeast sterol biosynthesis regulation [12]. These results altogether suggest that many sterol biosynthetic proteins, if not all, may be tethered to the ER as a large complex [9,10]. Most animals synthesize cholesterol. However, some animals such as D. melanogaster and C. elegans cannot synthesize sterols de novo [13,14]. They are auxotrophic for sterols because they do not possess the enzymatic activities necessary to complete this process [13,14]. C. elegans takes sterols from animal feces or yeast and plant remnants [3]. Drosophila obtains sterols from the diet: ergosterol from yeast and phytosterols from plants [15]. Looking for ERG orthologs in C. elegans and D. melanogaster However, both animals express the homologs of the enzymes that produce the initial intermediates of sterol biosynthesis up to the very early precursor farnesyl pyrophosphate. However, they cannot synthe- size either squalene or lanosterol, key intermediates of sterol biosynthesis [13,14]. For lanosterol synthase (ERG7p), which follows in the classical pathway, we could not gather any convincing evidence for the existence of orthologs in the fruitfly and the nematode. The following enzyme in the pathway is a sterol 14a- demethylase, ERG11p/Cyp51, involved in the biosynthesis of cholesterol, phytosterols and ergosterol. Thus, it is the only cytochrome P450 having an ortholog common to animals, plant and fungi [26]. Similarity BLAST hits with yeast ERG11p/ Cyp51, were obtained in Drosophila (CG2397, CG10247 and other Cyps). However, the Drosophila genes are likely to belong to other Cyp subfamilies (not Cyp51). Cyp51 is probably missing, which is in agreement with the results of Tijet, Helvig & Feyereisen [26] who analyzed 90 sequences of the cytochrome P450 gene superfamily. Cyp51 is also absent in C. elegans [26]. As already noted, sterols in these animals are required as hormone precursors and/or developmental effectors [16]. Not surprisingly, dauer larva formation and molting depend on sterols. Cholesterol, or more likely its derivatives, seem to act as hormones. Indeed, recent papers report the identification of natural steroid ligands for the DAF-12 nuclear receptor [17]. Potential ERG24 orthologs in D. melanogaster (CG17952) and in C. elegans (B0250.9) were easily found. The corresponding proteins contain the ERG4-24 domain. D. melanogaster produces three isoforms that are longer than the yeast ortholog, a peculiarity that they share with the human ortholog. The ortholog of ERG24 in mammals encodes the Lamin B receptor (LBR), a nuclear envelope protein first described in vertebrates. LBR bears extensive structural similarities with the members of the sterol reductase family (ERG24p and ERG4p). Human LBR (hLBR) cannot restore ergosterol biosynthesis in an ERG4 yeast mutant, whereas it is able to restore ergosterol prototrophy in an ERG24 In C. elegans, the distribution and transport of cholesterol in vivo has been studied by using dehydroergosterol (DHE), a fluorescent analog which mimics many cholesterol properties [18,19]. DHE accumulates primarily in the pharynx, nerve ring, excretory gland cell, and gut of L1–L3 larvae [20]. Interestingly, sterols present in the pharynx and in the intestine of feeding animals are distributed in a proximal-to-distal gradient. Cholesterol in C. Introduction ERG7p (lanosterol synthase), respectively. These proteins are essential for aerobic viability and their absence results in an inability to synthesize ergosterol. The third enzyme, ERG7p, converts squalene epoxide into lanosterol, the first cyclic component of the cholesterol biosynthesis cascade (i.e. the first sterol). The remaining enzymes of the pathway metabolize lanosterol into ergosterol. The sequential action of ERG11p (lanosterol demethylase) and ERG24p (C-14 reductase) leads to 4,4-dimethylzymosterol. They are also essential in aerobic conditions [8–10]. Removal of the two C-4 methyl groups of 4,4-dimethylzymosterol is a complex reaction involving the products of genes ERG25, ERG26, and ERG27, in cooperation with ERG28p. Finally, ERG6p (C-24 methylase) converts zymosterol to fecosterol, which is further transformed into ergosterol by ERG2p, ERG3p, ERG5p and ERG4p [8–10] (Figure 1). Cholesterol and other sterols such as ergosterol and phytosterols are universal components of eukaryotic plasma membranes and are absent from the membranes of prokaryotes. These sterols (cholesterol, ergosterol and phytosterol) modulate membrane fluidity [1,2]. In addition to this structural role, cholesterol is essential for signaling processes. In fact, it is a precursor of steroid hormones, oxysterols, ecdysones (in insects) and vitamin D. Moreover, it may influence intercellular signaling through its covalent attachment to proteins such as the protein Hedgehog (Hh) in Drosophila [3–5]. The nematode Caenorhabditis elegans possesses several genes encoding proteins with regions similar to Hh and potentially undergoing cholesterylation [6,7]. Yeast, plants and mammals synthesize sterols through a series of complex reactions that occur in the endoplasmic reticulum (ER) and, therefore, most of the involved enzymes have transmembrane domains (Figure 1). We outline below the series of reactions to produce ergosterol in the yeast Saccharomyces cerevisiae, which involve ERG (i.e. from ERGosterol) genes and the respective ERG proteins. Recent results show that, in yeast, ergosterol biosynthetic enzymes display specific protein-protein interactions and form a functional complex called ergosome. Proteins ERG11p, ERG25p, ERG27p and ERG28p, appear to form a core center of the complex and would interact with other enzymes of the pathway [9,10]. Indeed, the small transmembrane protein ERG28p functions as a scaffold to tether the C-4 demethylation complex The first three steps of sterol biosynthesis are catalized by ERG9p (squalene synthase), ERG1p (squalene epoxidase) and August 2008 \| Volume 3 \| Issue 8 \| e2883 1 August 2008 \| Volume 3 \| Issue 8 \| e2883 Sterologenesis in Auxotrophs Figure 1. Outline of the ergosterol synthesis pathway in yeast. Introduction (+) the corresponding gene is present in C. elegans and D. melanogaster, according to our exploration. (2) the corresponding gene is absent. (?) not convincing evidence for the presence of the ortholog. doi:10.1371/journal.pone.0002883.g001 modulating the activity of signaling molecules (such as Hh-like proteins). A previous comparative genome analysis of D. melanogaster with Anopheles gambiae and prototrophs has suggested that these insects have lost most of the genes involved in sterol synthesis [22]. This makes sense, knowing that Drosophila is unable to synthesize cholesterol. However, in a previous work we have shown that Drosophila contains an ERG28 ortholog that has undergone a process of acceleration in its evolution, and is undetectable using the current techniques for ortholog detection by sequence homology [23]. Thus, here we have revisited this question for both C. elegans and D. melanogaster, not only for ERG28, but most of the genes/enzymes involved in the sterol synthesis pathway, in the light of new genomic and functional data. PLoS ONE \| www.plosone.org Looking for ERG orthologs in C. elegans and D. melanogaster elegans might be involved in the structural and functional organization of the plasma membrane cell types that are richer in this lipid [21] and in PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2883 2 Sterologenesis in Auxotrophs Figure 2. Details of the BLAST analysis that allowed the detection of ERG orthologs in C. elegans and D. melanogaster. The comparisons were performed in the directions indicated by the arrows (i.e. YeastRHuman represents a BLASTp search with an ERG protein from yeast in the division Homo sapiens of Genbank). Low Scores S and high E-values (.1026) are classically considered as non-significant (unrelated or divergent sequences). The small tables display the S and E-values for the comparisons using the species-specific divisions of Genbank. doi:10.1371/journal.pone.0002883.g002 Fi 2 D t il f th BLAST l i th t ll d th d t ti f ERG th l i C l d D l t Th i Figure 2. Details of the BLAST analysis that allowed the detection of ERG orthologs in C. elegans and D. melanogaster. The comparisons were performed in the directions indicated by the arrows (i.e. YeastRHuman represents a BLASTp search with an ERG protein from yeast in the division Homo sapiens of Genbank). Low Scores S and high E-values (.1026) are classically considered as non-significant (unrelated or divergent sequences). The small tables display the S and E-values for the comparisons using the species-specific divisions of Genbank. doi:10.1371/journal.pone.0002883.g002 mutant. This strongly suggests that hLBR is a sterol C14-reductase [27]. Not surprisingly, a mutation in the hLBR gene causes an autosomal recessive disease called hydrops-ectopic calcification- ‘moth-eaten’ (HEM). This mutation leads to high levels of cholesta-8,14-dien-3-beta-ol in cultured skin fibroblasts, which is compatible with a deficiency of the cholesterol biosynthetic enzyme 3-beta-hydroxysterol delta(14)-reductase [28]. transported to the nucleus [31]. This domain might be responsible for the targeting of the hydrophobic domain to the nucleus and for the interaction with lamin B [32]. So far, cholesterol synthesis is supposed to occur in the smooth ER. Since the N-terminal domain of LBR is responsible for its nuclear localization, it would be interesting to investigate whether the LBR transcript, or protein, undergoes some processing leading to the production of a C- terminal domain sorted to the ER. PLoS ONE \| www.plosone.org Sterologenesis in Auxotrophs elegans the paralogs are at less than 3 kb away from each other. F49E12.9 contains 8 exons while F49E12.10 has 5 exons that may have been produced through exon fusion/splitting events. The ortholog of ERG6p in C. elegans (H14E04.1) was easily detected by BLAST with protein sequences from either yeast or A. thaliana (SMT1). We used the plant sequence since no clear ERG6 homolog could be detected in human. The issue with Drosophila turned out to be more complex because, when starting the search with the yeast sequence, we detected CG8067 marginally (E.1). This was even worse when starting with the sequence of A. thaliana. However, when using the C. elegans sequence as a starting point, the first significant hit in Drosophila (E,1026) was CG2453, which proved to be the ortholog of yeast Coq5, but not of ERG6p. Finally, considering i) the similar lengths of the previously marginally detected CG8067, of H14E04.1 and of SMT1 proteins and ii) their similar pI, we have preferred CG8067 as the most likely ortholog. Indeed, the hydrophobic profiles of SMT1 and the protein encoded by CG8067 displayed a strong correlation. Namely, we obtained an R = 0.43 with a p-value 10216. ERG26p (C-3 dehydrogenase) belongs to the 3b-hydroxysteroid dehydrogenase family and convincing evidence for the presence of orthologs in C. elegans (ZC8.1) and D. melanogaster (CG7724) was obtained in BLAST searches taking the sequence from yeast as the starting point (See details in Figure 2). In the case of Drosophila CG7724, similarity with yeast ERG26p extends over the first 250 amino acids while the remaining amino acids are more divergent. Interestingly, when performing a BLAST with the Drosophila sequence, the first hit in C. elegans was C32D5.12, but it was not the best hit either with yeast, or with A. thaliana, or even with the human (NSDHL) orthologs. However, a BLAST with C32D5.12 detected as the first hits NSDHL in man and ERG26 in yeast (Figure 2). Thus, it is tempting to invoke some kind of sequence convergence as an explanation for this behavior. The ortholog of ERG2p in C. elegans (W08F4.3) was marginally detected by BLAST with the yeast and the human protein (opioid sigma-1 receptor, OPRS1). However, W08F4.3 was found to contain a sigma1-receptor domain when compared with the CDD. This strengthens the idea that this gene is the ortholog of OPRS1 and ERG2. The situation in Drosophila was more complex. Sterologenesis in Auxotrophs to the Drosophila lamin B, a function residing in the N-terminal domain. Not unexpectedly, dLBR does not display sterol C14 reductase activity when expressed in the yeast ERG24 mutant. This shows that, during insect evolution, although the enzymatic activity of this protein has been lost, its capacity to bind lamin B has not. However, depletion of dLBR by RNA interference does not lead to any obvious effect on nuclear architecture, or viability, of treated cells and embryos. Thus, although dLBR might be important, it is not a limiting component of the nuclear architecture in Drosophila cells, at least during the first days of development [33]. Our BLAST search shows that sequence B0250.9 is the potential ERG24 ortholog in C. elegans. It would be interesting to experimentally assess if it has kept LBR activity. Metazoa division of Genbank [35]. We included in the iterations the very divergent sequence CG17270 of D. melanogaster, which is the ortholog of ERG28p according to our previous results [23]. This allowed us to detect C14C10.6 as a signinficant hit (S = 120 bits and E = 10227). Reverse Psi-BLAST also suggested orthology (i.e. significant scores). The sequence of C14C10.6 is so divergent that it had no match in the conserved domain database (CDD). Moreover, we also computed the hydrophobic profiles for some of the orthologs [36] and calculated the Pearson correlation coefficient ‘‘R’’ for various pairs of profiles. Significant R-values were obtained for the pair-wise comparisons (Figure 4). This is not a proof of orthology but strengthens the idea of structural relationship at the protein level. Finally, we found that all proteins had similar lengths and were basic, with isoelectric points (pI) .8.5. Sequences homologous to ERG25p (C-4 methyloxidase) were also easily found in both D. melanogaster (CG1998/dERG25A and CG11162/dERG25B) and C. elegans (F49E12.9/ERG25A and F49E12.10/ERG25B) using the sequence of yeast ERG25p as a starting point. In both organisms the duplicated copies of ERG25 are located in the same chromosome (chromosome II for C. elegans and chromosome X for D. melanogaster). The two paralogs in D. melanogaster are separated by 0.25 Mb and contain a different number of predicted exons. Namely, CG1998 contain 6 exons, while CG11162 contains only 2. However, the last intron of both genes interrupts the coding sequence at very similar positions (i.e. between the second and the third positions of a Lys codon, Figure 3). In C. Looking for ERG orthologs in C. elegans and D. melanogaster The hLBR contains two major domains: a ,220-amino-acid N- terminal segment highly charged, and a hydrophobic C-terminal half with eight putative transmembrane segments [29,30]. Interestingly, it has been hypothesized that the region encoding the N-terminal domain of the LBR gene arose from an ancestral gene coding for a soluble nuclear protein (which provides a nuclear localization signal) and that the rest of the protein evolved from another gene, similar to yeast ERG24. Indeed, the C-terminal hydrophobic domain of LBR can be retained in the endoplasmic reticulum when expressed in transfected cells, as expected for the ortholog of ERG24 in mammals. In turn, the N-terminal domain is Recent functional studies show that the Drosophila CG17952 gene is the ortholog of vertebrate LBR [33]. The protein encoded by CG17952 shares some properties with hLBR. The Drosophila LBR (dLBR) possesses a highly charged N-terminal domain of 307 amino acids followed by eight transmembrane segments. Trans- membrane segments 1–6 are similar in length and position to the transmembrane domains 1–6 of hLBR. However, the putative membrane domains 7 and 8 of dLBR are shorter than those of hLBR. Thus, dLBR is expected to have a topological organization similar to that of its vertebrate orthologs [33]. dLBR is able to bind PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2883 3 Sterologenesis in Auxotrophs August 2008 \| Volume 3 \| Issue 8 \| e2883 Sterologenesis in Auxotrophs When starting our BLAST with either yeast or human sequences, we detected the sequence HDC14735 (DAA04220) very marginally (E&1) and no conserved domain was found. However, when starting with the C. elegans sequence, it came as the best hit with E = 0.002 (the reverse was also true, with E = 0.003). Although not significant, this result was taken as suggestive of similarity. The results were improved using Psi-BLAST. Again, we computed the hydrophobic profiles for the various potential orthologs and we found strong correlations (Figure 4). Finally, the pI of the protein encoded by HDC14735 (pI = 6.14) was comparable to those of OPRS1 (5.61) and ERG2 (5.54) (see below). In agreement with Breitling et al. [34] we failed to find any clear homologue of ERG27p (C-3 ketoreductase) in both Drosophila and C. elegans, although several oxidoreductases were detected. As outlined above, ERG28p might tether many other ERG proteins to the ER. The ERG28p ortholog of C. elegans (C14C10.6) was hardly detectable by BLAST starting with yeast sequences. This precludes the use of standard phylogenetics methods to show orthology. However, further evidence of sequence relatedness was gathered using Psi-BLAST with the yeast sequence against the In BLAST searches with yeast ERG3p, we detected again CG1998 and CG11162 in D. melanogaster and F49E12.9 and F49E12.10 in C. elegans, but with worse scores than in BLASTs with ERG25 (S,55 bits versus 85 bits respectively). Therefore, we propose that the orthologs of ERG3 are potentially missing in both organisms. For ERG5p, which belongs to the big Cyp protein family, no clear orthologs could be established. However, three potential candidates were found: CG4321-PA (Cyp4d8), CG3540-PA (Cyp4d14) and CG8859-PA (Cyp6g2). In the reverse BLAST, they all matched ERG5p as the best scoring hit in yeast. Finally, the search for ERG4p orthologs led to the same ERG24 orthologs in the nematode and the fruitfly. Moreover, with ERG4p the BLAST scores were worse than with ERG24p. Thus, either ERG4 orthologs are missing or they have been replaced by ERG24. Figure 3. Segments of the Drosophila paralogs CG1998 and CG11162 (homologs of Erg25) and the corresponding concep- tual translations. The interruption of the open reading frames of both genes, by their last intron, is shown by vertical lines. Sterologenesis in Auxotrophs doi:10.1371/journal.pone.0002883.g003 August 2008 \| Volume 3 \| Issue 8 \| e2883 August 2008 \| Volume 3 \| Issue 8 \| e2883 PLoS ONE \| www.plosone.org 4 Sterologenesis in Auxotrophs Figure 4. Hydrophobic profiles of several potential ERG orthologous proteins. A way to show a structural relationship is to predict the hydrophobic profiles of the relevant proteins. Here we have used the TopPred program [36] as implemented in the server of Pasteur Institute (http:// www.pasteur.fr). Left panels show the results for the potential homologs of ERG2p while the right panels display the profiles for ERG28p homologs (using the Kyte-Doolitle scale, with the default parameters). Negative (positive) values represent hydrophobic (hydrophilic) segments. A way to statistically assess the similarity of two profiles is to calculate their correlation coefficient R. R-values for pairwise comparisons with the human sequence are reported. We tried to maximize the R-value by slightly sliding one profile over the other (that is why the frames of the profiles are not perfectly aligned). doi:10.1371/journal.pone.0002883.g004 Figure 4. Hydrophobic profiles of several potential ERG orthologous proteins. A way to show a structural relationship is to predict the hydrophobic profiles of the relevant proteins. Here we have used the TopPred program [36] as implemented in the server of Pasteur Institute (http:// www.pasteur.fr). Left panels show the results for the potential homologs of ERG2p while the right panels display the profiles for ERG28p homologs (using the Kyte-Doolitle scale, with the default parameters). Negative (positive) values represent hydrophobic (hydrophilic) segments. A way to statistically assess the similarity of two profiles is to calculate their correlation coefficient R. R-values for pairwise comparisons with the human sequence are reported. We tried to maximize the R-value by slightly sliding one profile over the other (that is why the frames of the profiles are not perfectly aligned). doi:10 1371/journal pone 0002883 g004 y doi:10.1371/journal.pone.0002883.g004 to nematodes, the low value of Ka/Ks (dN/dS) ratios show that these genes are under selective pressure (Table 1). Analyzing expression-profiling data to explore the potential function to the divergent ERG orthologs Co-expression is indicative of i) physical interaction between proteins and ii) of membership to the same complex or molecular process [37]. This is called the paradigm of ‘‘guilt by association’’ [38]. Thus, we have used published microarray expression data, downloadable from the Gene Expression Omnibus of the NCBI, to investigate potential co-expression patterns of the ERG orthologs that we have described above for both D. melanogaster and C. elegans. Co- expression can be assessed by determining the correlation coefficient. The correlation coefficient can be artificially inflated by flat profiles (no changes in the expression of the relevant genes). To avoid this, we focused on experiments where the genes of interest display strong variation (see Figure 5A and B). Thus, we gathered data concerning 51 different microarray experiments for D. melanogaster respecting the criterion outlined previously. We performed a similar analysis for C. elegans but, unfortunately, the most interesting genes showed flat profiles (close to 0 in all experiments) and it was not possible to proceed further with the analysis. First, we asked whether the expression profiles of the D. melanogaster ERG orthologs were correlated (Table 3). The strongest correlation was found between the dLBR (ERG24) and CG1998 (ERG25) with an associated p-value of 10216 (after a Bonferroni correction). Such a p-value means that only one correlation coefficient out of 1016 is expected to be as high as 0.89 just by chance (for n = 51 experimental points). Considering the maxi- mum number of possible correlations for the 14000 transcripts in the microarrays (representing the Drosophila genome), such a high R cannot be found by chance. The behavior of dLBR and CG1998 might be reminiscent of the situation in yeast because Table 2. Isoelectric points (pI) of the ERGp orthologs in C. elegans and D. melanogaster. Proteins (n) C. elegans D. melanogaster pI (w/o) mean+/2std pI (w) mean+/2std Erg6 (7) 5.95 (H14E04.1) 5.79 (CG8067) 5.98+/20.38 6.19+/20.83 Erg26 (16) 6.52 (ZC8.1) 8.99 (CG7724) 7.93+/21.23 7.91+/21.22 Erg24 (13) 9.38 (B0250.9) 9.89 (CG17952) 9.19+/20.67 9.25+/20.64 Erg28 (4) 9.02 (C14C10.6) 9.62 (CG17270) 9.31+/20.39 9.31+/20.35 Erg2 (12) 9.39 (W08F4.3) 5.49 (HDC14735) 6.28+/21.02 6.44+/21.28 Erg25 (15) 7.57 (F49E12.9/25A) 8.17 (CG1998/25A) 7.83+/20.63 7.96+/20.73 8.4 (F49E12.10/25B) 9.76 (CG11162/25B) The mean pI values (and standard deviations –std) of the orthologous proteins in cholesterol prototrophs are shown for comparison. ‘‘pI (w/o)’’ stands for means +/2 std calculated without taking into account the orthologs in C. elegans and D. Evolution of the ERG orthologs in C. elegans and D. melanogaster pseudobscura ERG Ka/Ks dN/dS Ka/Ks dN/dS 2 0.127 0.100 0.080 0.029 6 0.044 0.029 0.041 0.016 24 0.055 0.055 0.122 0.085 25A 0.041 0.039 0.064 0.041 25B 0.055 0.031 0.138 0.087 28 0.080 0.064 0.033 0.013 doi:10.1371/journal.pone.0002883.t001 nematode and the fly lineages in spite of primary sequence divergence (Table 2). Similarity in pI is expected, when the orthologs are detected as the best reciprocal significant hits in BLAST searches. However, the existence of similarity in the hydrophobic profiles and the pI between the potential orthologs, for which the BLAST comparisons failed to be significant, is more surprising. It means that, in spite of important sequence divergence, which renders BLAST unreliable in many cases, the proteins have maintained substantial structural and physico- chemical similarity. nematode and the fly lineages in spite of primary sequence divergence (Table 2). Similarity in pI is expected, when the orthologs are detected as the best reciprocal significant hits in BLAST searches. However, the existence of similarity in the hydrophobic profiles and the pI between the potential orthologs, for which the BLAST comparisons failed to be significant, is more surprising. It means that, in spite of important sequence divergence, which renders BLAST unreliable in many cases, the proteins have maintained substantial structural and physico- chemical similarity. Figure 5. Expression profiles of several genes expressionally correlated with dLBR and CG1998. Panels A and B show schematic profiles displaying very strong R. However, in panel A ‘‘co-variation’’ is not very informative, in this case either the red and blue genes are not expressed or do not change their expression in the conditions analyzed. To avoid this artificial inflation of R, we have focused on experiments where the genes of interest display strong variation (as in panel B). Panel C: Expression profiles of several genes involved in protein trafficking and folding, which co-vary with the expression of dLBR and CG1998. The profile of Start1 is also represented. doi:10.1371/journal.pone.0002883.g005 Evolution of the ERG orthologs in C. elegans and D. melanogaster This is called the paradigm of ‘‘guilt by asso [38]. Thus, we have used published microarray expressi downloadable from the Gene Expression Omnibus of the N investigate potential co-expression patterns of the ERG ortho we have described above for both D. melanogaster and C. ele expression can be assessed by determining the correlation co The correlation coefficient can be artificially inflated by fla (no changes in the expression of the relevant genes). To avoid focused on experiments where the genes of interest displa variation (see Figure 5A and B). Thus, we gathered data co 51 different microarray experiments for D. melanogaster respe criterion outlined previously. We performed a similar analy elegans but, unfortunately, the most interesting genes sho Table 1. Ka/Ks and dN/dS values for several ortholog genes in the fruitfly and the worm. Orthologs C. elegans vs C. briggsae D. melanogaster D. pseudobscura ERG Ka/Ks dN/dS Ka/Ks dN/d 2 0.127 0.100 0.080 0.029 6 0.044 0.029 0.041 0.016 24 0.055 0.055 0.122 0.085 25A 0.041 0.039 0.064 0.041 25B 0.055 0.031 0.138 0.087 28 0.080 0.064 0.033 0.013 doi:10.1371/journal.pone.0002883.t001 Table 2. Isoelectric points (pI) of the ERGp ortholog Proteins (n) C. elegans D. Erg6 (7) 5.95 (H14E04.1) 5. Erg26 (16) 6.52 (ZC8.1) 8. Erg24 (13) 9.38 (B0250.9) 9. Erg28 (4) 9.02 (C14C10.6) 9. Erg2 (12) 9.39 (W08F4.3) 5.4 Erg25 (15) 7.57 (F49E12.9/25A) 8. 8.4 (F49E12.10/25B) 9. The mean pI values (and standard deviations std) of the orthologou Figure 5. Expression profiles of several genes expressionally correlated with dLBR and CG1998. Panels A and B show schematic profiles displaying very strong R. However, in panel A ‘‘co-variation’’ is not very informative, in this case either the red and blue genes are not expressed or do not change their expression in the conditions analyzed. To avoid this artificial inflation of R, we have focused on experiments where the genes of interest display strong variation (as in panel B). Panel C: Expression profiles of several genes involved in protein trafficking and folding, which co-vary with the expression of dLBR and CG1998. The profile of Start1 is also represented. doi:10.1371/journal.pone.0002883.g005 Table 1. Ka/Ks and dN/dS values for several orthologs of ERG genes in the fruitfly and the worm. Orthologs C. elegans vs C. briggsae D. melanogaster vs. D. Evolution of the ERG orthologs in C. elegans and D. melanogaster It is natural to ask what protein properties have been conserved by the purifying selection. We have investigated a protein-level property, namely, the isoelectric point (pI). This property is important for enzymes because protein-protein and enzyme- substrate interactions are often electrostatic in nature. Thus, we should expect the pI of an enzyme to be similar in different organisms if protein-protein and/or enzyme-substrate interactions are conserved. In order to test this idea we gathered the protein sequences of the orthologs of ERG2, 6, 24, 25, 26 and 28. Next, we asked whether the corresponding Drosophila and Caenorhabditis sequences displayed outlier pI values. None of the orthologous proteins in the auxotrophs was detected as outlier, suggesting conservation of this physico-chemical property within the To assess whether some of the ERG orthologs mentioned above undergo a selective pressure, we have examined the ratio of the number of non-synonymous substitutions per non-synonymous site (Ka) and the number of synonymous substitutions per synonymous site (Ks). The ratio Ka/Ks is indicative of the mode of evolution operating on the sequences. If selection is dominantly purifying, then we expect few non-synonymous substitutions per background synonymous changes and hence, a low ratio. If selection is absent, then a ratio of unity is expected (Table 1). To obtain the Ka/Ks values for the fly we compared the sequences from D. melanogaster and D. pseudoobscura while for the nematode we compared the sequences from C. elegans and C. briggsae. In spite of the divergence of the various ERG orthologs in the lineages leading to insects and PL August 2008 \| Volume 3 \| Issue 8 \| e2883 August 2008 \| Volume 3 \| Issue 8 \| e2883 PLoS ONE \| www.plosone.org 5 Sterologenesis in Auxotrophs nematode and the fly lineages in spite of primary s divergence (Table 2). Similarity in pI is expected, w orthologs are detected as the best reciprocal significan BLAST searches. However, the existence of similarity hydrophobic profiles and the pI between the potential o for which the BLAST comparisons failed to be significant surprising. It means that, in spite of important s divergence, which renders BLAST unreliable in many c proteins have maintained substantial structural and chemical similarity. Analyzing expression-profiling data to explore th potential function to the divergent ERG ortholog Co-expression is indicative of i) physical interaction proteins and ii) of membership to the same complex or m process [37]. Analyzing expression-profiling data to explore the potential function to the divergent ERG orthologs However, no unifying theme emerged from this analysis (data not shown, available upon request). All in all, the strong expressional correlation between dLBR and CG1998 with proteins involved in intracellular protein trafficking or folding, and the absence of such correlation with other Erg orthologs (that also require chaperons) suggest that the involvement of dLBR and CG1998 in both processes is worth exploring. At first, we were expecting good expressional correlation among the ERG orthologs in Drosophila. However, it seems that only dLBR and CG1998 still ‘‘remember’’ their ancestral belonging to the sterol biosynthesis pathway. Poor expressional correlation among the rest of the ERG orthologs also suggests that the corresponding proteins either have lost their ability to physically interact in order to form stable complexes, or they do so in conditions/moments not covered by the microarray experiments explored here. Then, we focused our attention on dLBR and CG1998 by determining which other genes were expressionally correlated with them. For this, we gathered 84 genes displaying R$0.875 with respect to both genes. For a correlation involving 51 data points, this R cut-off is associated with a safe p-value of 10213 after correction (Figure 5C). In order to get insights about these 84 genes, we used the functional classification tool of the DAVID database (http://david.abcc.ncifcrf.gov/ [40]). This software provides a rapid means to organize large lists of genes into functionally related groups and to unravel biological relationships. In a previous paper, given the structural similarity between cholesterol and ecdysteroids, we had proposed that divergent ERGp orthologs might somehow participate in the synthesis of the latter [23]. We have therefore assessed the expressional correlation between candidate genes involved in this process: Dare1/CG12390 [43], Jhamt [44] and Start1 [45], with dLBR and CG1998. While for Jahmt and Dare1 the values of R are below 0.6, a very strong correlation (R= 0.8) was found for Start1. Interestingly, Start1, which is involved in intra-mitochondrial sterol transport, is expressed ubiquitously. However, in situ hybridization demonstrates a stronger expression in the prothoracic gland, where ecdysteroids are synthesized from cholesterol. These and other observations are consistent with the idea that Start1 plays a key role in the regulation of ecdysteroid synthesis [45]. The potential functional link between dLBR, CG1998 and Start1 is also worth exploring. Analyzing expression-profiling data to explore the potential function to the divergent ERG orthologs y g p g p In the analysis using DAVID, the most overrepresented class included genes encoding membrane proteins, often targeted to the ER, where sterol biosynthesis takes place in prototrophs (Group 1, in Table 4). Interestingly, several of these proteins are supposed to be involved in co-translational protein targeting to membranes, signal peptide recognition, heat shock protein-binding, as well as unfolded protein binding, or to be elements of the translocon (a complex of proteins associated with the translocation of nascent polypeptides into the ER [41]). The following functional category (Group 2) contained four transporter proteins while the last functional group involved chaperones (i.e. peptidyl-prolyl cis-trans isomerase), chaperone cofactors or unfolded protein-binding factors. A similar analysis conducted using g:profiler [42] confirmed that genes encoding protein folding actors were overrepresented among the genes displaying strong expressional correlation with dLBR and CG1998 (p,1025). The existence of expressional correlation does not imply any causality. In fact, from this exploration it is not possible to determine whether dLBR and CG1998 somehow interact with other partners to participate in intracellular protein trafficking or folding, or on the contrary, they undergo the action of the latter. Since dLBR has been shown to be a nuclear protein [31,32], it would be interesting to investigate whether the dLBR transcript or protein are somehow processed to produce a C-terminal polypeptide that might be sorted to the ER. That would explain the expressional correlation between dLBR and ER proteins observed above. On the other hand, we have p g In conclusion, we detected a preservation of ERG genes in Drosophila melanogaster and Caenorhabditis elegans. In spite of their sequence divergence with respect to the corresponding orthologs in sterol prototrophs, they still are under selective pressure. Since insects are unable to synthesize cholesterol de novo, an appealing way to explain this evolutionary acceleration is that ERGp orthologs have other biological functions in addition to sterol synthesis. This is clearly the case of the LBR, which is also a reductase in sterol prototrophs. Shut-down of cholesterogenesis in insects and nematodes would have allowed these proteins to evolve as much as their other functions were not compromised [23]. Another, less parsimonious, explanation would be the evolution of different novel functions. Our microarray meta-analysis shows strong expressional correlation between the orthologs of ERG24 and ERG25 in D. melanogaster and genes encoding factors involved in intracellular protein trafficking and folding. Analyzing expression-profiling data to explore the potential function to the divergent ERG orthologs melanogaster while ‘‘pI (w)’’ stands for means +/2 std calculated including the orthologs in C. elegans and D. melanogaster. ‘‘n’’ is the number of orthologs in cholesterol prototrophs used to calculate the means. doi:10.1371/journal.pone.0002883.t002 Table 2. Isoelectric points (pI) of the ERGp orthologs in C. elegans and D. melanogaster. The mean pI values (and standard deviations –std) of the orthologous proteins in cholesterol prototrophs are shown for comparison. ‘‘pI (w/o)’’ stands for means +/2 std calculated without taking into account the orthologs in C. elegans and D. melanogaster while ‘‘pI (w)’’ stands for means +/2 std calculated including the orthologs in C. elegans and D. melanogaster. ‘‘n’’ is the number of orthologs in cholesterol prototrophs used to calculate the means. doi:10.1371/journal.pone.0002883.t002 August 2008 \| Volume 3 \| Issue 8 \| e2883 PLoS ONE \| www.plosone.org 6 Sterologenesis in Auxotrophs Table 3. Expressional correlation among D. melanogaster ERG orthologs. CG1998 ERG25A CG11162 ERG25B LBR ERG24 CG17270 ERG28 CG8067 ERG6 CG2453 ERG6? CG7724 0.22 0.40 0.18 0.14 0.32 0.27 CG1998 0.03 0.89 20.08 0.01 0.14 CG11162 0.07 0.27 0.28 0.34 LBR 20.06 20.02 0.17 CG17270 0.31 0.41 CG8067 0.55 The analyses were performed using data downloaded from the Gene expression-Omnibus database (http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?CMD = search&DB = geo). We considered the datasets GDS192 (wing imaginal disc spatial gene expression), GDS653 (neurotransmitter-specific neuronal gene expression), GDS664 (splicing factor mutant at permissive and restrictive temperatures) and GDS667 (mRNA splicing factor knock-down) which contain 51 data points for 14 000 transcripts. doi:10.1371/journal.pone.0002883.t003 med using data downloaded from the Gene expression-Omnibus database (http://www.ncbi.nlm.nih.gov/entrez/query. ) W id d h d GDS192 ( i i i l di i l i ) GDS653 ( i ifi The analyses were performed using data downloaded from the Gene expression-Omnibus database (http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?CMD = search&DB = geo). We considered the datasets GDS192 (wing imaginal disc spatial gene expression), GDS653 (neurotransmitter-specific neuronal gene expression), GDS664 (splicing factor mutant at permissive and restrictive temperatures) and GDS667 (mRNA splicing factor knock-down) which contain 51 data points for 14 000 transcripts. doi:10.1371/journal.pone.0002883.t003 p doi:10.1371/journal.pone.0002883.t003 ERG24p and ERG25p are supposed to interact, according to Epistatic MiniArray Profiling experiments [39]. CG7724 and CG11162 displayed the second highest R (R = 0.4, p = 0.03), but this R is not relevant in genomic terms. also explored the annotation of the genes expressionally correlated with the rest of the ERG orthologs. Ortholog search We used BLASTp [24] and considered as orthologs the best reciprocal hits. In short, a BLAST search is performed starting with a protein sequence from organism A to detect the matching sequences in organism B. Then, the sequence from B displaying the highest score is compared (reverse BLAST) against the all sequences of A and the highest scoring hit must be the initial sequence. This is a widely accepted criterion of orthology [25]. We also exploited the significance of matches using Psi-BLAST [35] or with the conserved domain database (CDD), where the scoring of the protein alignment uses very sensitive ‘‘tailor made’’ scoring matrices. Analyzing expression-profiling data to explore the potential function to the divergent ERG orthologs This is compatible with our idea that ERGp might be involved in other biological roles in addition to sterol synthesis. The potential link between ERG proteins and intracellular protein trafficking and folding deserves experimental exploration not only in Drosophila and Caenorhabditis but also in sterol prototrophs. Moreover, the potential link between dLBR, CG1998 and Start1 is to be explored in D. melanogaster. This is compatible with our previous idea of a potential implication of these proteins in the synthesis of ecdysteroids. We hope PLoS August 2008 \| Volume 3 \| Issue 8 \| e2883 PLoS ONE \| www.plosone.org 7 Sterologenesis in Auxotrophs Gene Group 1 Enrichment Score: 4.16 Gene Name Key words 2 CG7872 HSP binding 3 FKBP13 peptidyl-prolyl cis-trans isomerase activity, protein folding, ER 4 CG14715 peptidyl-prolyl cis-trans isomerase activity, protein folding, ER 5 GP93 glycoprotein 93, unfolded protein binding, response to stress, ER ER: endoplasmic reticulum, HSP: heat shock protein, SRP: signal recognition particle. At high stringency, only the first functional cluster is obtained. doi:10.1371/journal.pone.0002883.t004 Table 4. cont. Table 4. cont. Table 4. Functional clustering of genes whose expression profiles stronlgy correlate with those of dLBR and CG1998 (using the DAVID classification tool at Medium stringency). Computation of hydrophobic profiles as further evidence of structural relationship between potentially orthologous proteins For some distant homologues we computed the hydrophobic profiles and calculated the Pearson correlation coefficient ‘‘R’’ for various pairs of profiles. The values of R range between 1 (perfect match between the profiles) and 21 (profiles are mirror images). Strong positive correlation is not a proof of orthology but strengthens the idea of structural relationship at the protein level. Analyzing expression-profiling data to explore the potential function to the divergent ERG orthologs Gene Group 1 Enrichment Score: 4.16 Gene Name Key words 1 CG5885 cotranslational protein targeting to membrane, integral to ER membrane, SRP, translocon complex 2 SURF4 surfeit 4, receptor signaling protein activity, asymmetric protein localization, ER membrane 3 CG8583 HSP binding, SRP binding, unfolded protein binding, SRP receptor complex 4 SEC61ALPHA protein translocase activity, SRP-dependent cotranslational protein targeting to membrane, translocon complex 5 CG32700 oxidoreductase activity 6 CG33162 SRP receptor 7 GP210 transporter activity, protein targeting, integral to membrane 8 SSRBETA signal sequence receptor 9 PROMININ-LIKE prominin-like protein, intracellular protein transport, integral to membrane, 10 CG33105 intracellular protein transport, Golgi apparatus, integral to membrane 11 CG1967 intracellular transporter activity, post-Golgi vesicle- mediated transport, coated vesicle,integral to membrane 12 CG11857 vesicle-mediated transport 13 GFR GDP-fucose transport, Golgi- associated vesicle 14 CG1751 Probable signal peptidase complex subunit 2 15 CG33214 FGF binding, receptor binding, cell adhesion, intracellular protein transport Gene Group 2 Enrichment Score: 1.84 1 CG5594 amino acid-polyamine transporter activity, cation transporter activity 2 CG15094 high affinity inorganic phosphate:sodium symporter activity,serine-type endopeptidase inhibitor activity 3 CG8291 neurotransmitter transporter activity 4 CG6293 L-ascorbate:sodium symporter activity, nucleotide and nucleic acid transport Gene Group 3 Enrichment Score: 1.64 1 TORP4A torsin-like protein precursor, unfolded protein binding, protein folding ER: endoplasmic reticulum, HSP: heat shock protein, SRP: signal recognition particle. At high stringency, only the first functional cluster is obtained. doi:10.1371/journal.pone.0002883.t004 ER: endoplasmic reticulum, HSP: heat shock protein, SRP: signal recognition particle. At high stringency, only the first functional cluster is obtained. doi:10.1371/journal.pone.0002883.t004 that this genomic exploration and the hypotheses prompted here might open new avenues of experimental research. Calculation of Ka/Ks The protein-coding nucleotide sequences were first translated into amino acid sequences and aligned. Then, this alignment was used to define the alignment of the corresponding nucleotide sequences to avoid frame-shifting indels as alignment artifacts [46]. Ka and Ks were estimated in two ways: i) by using the PBL method [47,48] implemented in DAMBE [46,49], and ii) by the likelihood-based method implemented in the YN00 program in the PAML package with the resulting Ka and Ks designated by dN and dS, respectively in table 1 [50]. pI estimation and outlier detection We have investigated whether the pI of presumably orthologous proteins were similar. In order to test this, we gathered the protein sequences of the orthologs of ERG2, 6, 24, 25, 26 and 28 listed in the HomoloGene division of the NCBI database and estimated their pI (http://www.expasy.ch/tools/pi_tool.html). Then, we August 2008 \| Volume 3 \| Issue 8 \| e2883 August 2008 \| Volume 3 \| Issue 8 \| e2883 8 Sterologenesis in Auxotrophs asked whether the corresponding Drosophila and Caenorhabditis sequences displayed outlier pI values. In short, we performed an extreme studentized deviate test to determine whether one of the values in the pI list was a significant outlier from the rest. References 26. Tijet N, Helvig C, Feyereisen R (2001) The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylog- eny. Gene 262: 189–198. 1. Mouritsen OG, Zuckermann MJ (2004) What’s so special about cholesterol? Lipids 39: 1101–1113. 2. Haines TH (2001) Do sterols reduce proton and sodium leaks through lipid bilayers? Prog Lipid Res 40: 299–324. 27. Silve S, Dupuy PH, Ferrara P, Loison G (1998) Human lamin B receptor exhibits sterol C14-reductase activity in Saccharomyces cerevisiae. Bioch Biophys Acta 1392: 233–44. 3. Kurzchalia TV, Ward S (2003) Why do worms need cholesterol? Nature Cell Biol 5: 684–688. 28. Waterham HR, Koster J, Mooyer P, Noort G, Kelley RI, et al. (2003) Autosomal recessive HEM/Greenberg skeletal dysplasia is caused by 3b- hydroxysterol D14-reductase deficiency due to mutations in the lamin B receptor gene. Am J Hum Genet 72: 1013–1017. 4. Porter JA, Young KE, Beachy PA (1996) Cholesterol modification of hedgehog signaling proteins in animal development. Science 274: 255–259. 5. Mann RK, Beachy PA (2000) Cholesterol modification of proteins. Bioch Bioph Acta 1529: 188–202. 29. Ye Q, Barton RM, Worman HJ (1998) Nuclear lamin binding proteins. In: Subcellular Biochemistry: Intermediate Filaments. H Hermann, J Robin Harris, eds. London, UK: Plenum Publishing. pp 587–610. 6. Aspock G, Kagoshima H, Niklaus G, Burglin TR (1999) Caenorhabditis elegans has scores of hedgehog-related genes: sequence and expression analysis. Genome Res 9: 909–923. 30. Ye Q, Callebaut I, Pezhman A, Courvalin JC, Worman HJ (1997) Domain- specific interactions of human HP1-type chromodomain proteins and inner nuclear membrane protein LBR. J Biol Chem 272: 14983–14989. 7. Wendler F, Franch-Marro X, Vincent JP (2006) How does cholesterol affect the way Hedgehog works? Development 133: 3055–3061. 8. Lees ND, Skaggs B, Kirsch DR, Bard M (1995) Cloning of the late genes in ergosterol biosynthetic pathway of Saccharomyces cereviasiae – A Review. Lipids 30: 221–226. p J 31. Soullam B, Worman HJ (1993) The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal. J Cell Biol 120: 1093–1100. 32. Schuler E, Lin F, Worman HJ (1994) Characterization of the human gene encoding LBR, an integral protein of the nuclear envelope inner membrane. J Biol Chem 269: 11312–11317. p 9. Mo C, Bard M (2005a) A systematic study of yeast sterol biosynthetic protein- protein interactions using the split-ubiquitin system. Bioch Bioph Acta 1737: 152–160. J 33. References Wagner N, Weber D, Seitz S, Krohne G (2004) The lamin B receptor of Drosophila melanogaster. J Cell Sci 117: 2015–2028. 10. Mo C, Bard M (2005b) Erg28p is a key protein in the yeast sterol biosynthetic enzyme complex. J Lipid Res 46: 1991–1998. p g J 34. Breitling R, Krazeisen A, Moller G, Adamski J (2001) 17beta-hydroxysteroid dehydrogenase type 7–an ancient 3-ketosteroid reductase of cholesterogenesis. Mol Cell Endo 171: 199–204. 11. Mo C, Valachovic M, Bard M (2004) The ERG28-encoded protein, Erg28p, interacts with both the sterol C-4 demethylation enzyme complex as well as the late biosynthetic protein, the C-24 sterol methyltransferase (Erg6p). Bioch Bioph Acta 1686: 30–36. 35. Altschul SF, Madden TL, Scha¨ffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. 12. Mo C, Milla P, Athenstaedt K, Ott R, Balliano G, Daum G, Bard M (2003) In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity. Bioch Bioph Acta 1633: 68–74. p g 36. Claros MG, von Heijne G (1994) TopPred II: An Improved Software For Membrane Protein Structure Predictions. CABIOS 10: 685–686. 13. Hieb WF, Rothstein M (1968) Sterol requirement for reproduction of a free- living nematode. Science 160: 778–780. 37. Jansen R, Greenbaum D, Gerstein M (2002) Relating whole-genome expression data with protein-protein interactions. Genome Res 12: 37–46. g 14. Chitwood DJ, Lusby WR (1991) Metabolism of plant sterols by nematodes. Lipids 26: 619–627. p p 38. Quackenbush J (2003) Genomics. Microarrays-guilt by association. Science 302: 240–241. 15. Rosenfeld JM, Osborne TF (1998) HLH106, a Drosophila sterol regulatory element-binding protein in a natural cholesterol auxotroph. J Biol Chem 273: 16112–16121. 39. Schuldiner M, Collins SR, Thompson NJ, Denic V, Bhamidipati A, et al. (2005) Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123: 507–519. 16. Merris M, Wadsworth WG, Khamrai U, Bittman R, Chitwood DJ, Lenard J (2003) Sterol effects and sites of sterol accumulation in Caenorhabditis elegans: developmental requirement for 4alpha-methyl sterols. J Lipid Res 44: 172–181. p y g p y p 40. Dennis G Jr, Sherman BT, Hosack DA, Yang J, Gao W, et al. (2003) DAVID: Database for Annotation, Visualization, and Integrated Discovery. Genome Biol 4: P3. p q p y p 17. References Beckstead RB, Thummel CS (2006) Indicted: worms caught using steroids. Cell 124: 1137–1140. 41. Eichler J, Duong F (2004) Break on through to the other side–the Sec translocon. Trends Biochem Sci 29: 221–223. 18. Schroeder F, Jefferson JR, Kier AB, Knittel J, Scallen TJ, et al. (1991) Membrane cholesterol dynamics: cholesterol domains and kinetic pools. Proc Socr Exp Biol Med 196: 235–252. 42. Reimand J, Kull M, Peterson H, Hansen J, Vilo J (2007) g:Profiler — a web- based toolset for functional profiling of gene lists from large-scale experiments. Nucleic Acids Res 35: W193–200. 19. Schroeder F, Woodford JK, Kavecansky J, Wood WG, Joiner C (1995) Cholesterol domains in biological membranes. Mol Membrane Biol 12: 113–119. 43. Freeman MR, Dobritsa A, Gaines P, Segraves WA, Carlson JR (1999) The dare gene: steroid hormone production, olfactory behavior, and neural degeneration in Drosophila. Development 126: 4591–4602. 20. Matyash V, Geier C, Henske A, Mukherjee S, Hirsh D, et al. (2001) Distribution and transport of cholesterol in Caenorhabditis elegans. Mol Biol Cell 12: 1725–1736. 44. Shinoda T, Itoyama K (2003) Juvenile hormone acid methyltransferase: a key regulatory enzyme for insect metamorphosis. Proc of the Natl Acad Sci U S A 100: 11986–11991. 21. Scheel J, Srinivasan J, Honnert U, Henske A, Kurzchalia TV (1999) Involvement of caveolin-1 in meiotic cell-cycle progression in Caenorhabditis elegans. Nat Cell Biol 1: 127–129. 45. Roth GE, Gierl MS, Vollborn L, Meise M, Lintermann R, et al. (2004) The Drosophila gene Start1: a putative cholesterol transporter and key regulator of ecdysteroid synthesis. Proc of the Natl Acad Sci U S A 101: 1601–1606. ecdysteroid synthesis. Proc of the Natl Acad Sci U S A 101: 1601–16 22. Zdobnov EM, von Mering C, Letunic I, Torrents D, Suyama M, et al. (2002) Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science 298: 149–159. 46. Xia X (2001) Data analysis in molecular biology and evolution. Boston: Kluwer Academic Publishers. pp 277, 38–39. pp 47. Li WH (1993) Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J Mol Evol 36: 96–99. 23. Veitia RA, Hurst LD (2001) Accelerated molecular evolution of insect orthologues of ERG28/C14orf1: a link with ecdysteroid metabolism? J Genet 80: 17–21. y y 48. Pamilo P, Bianchi NO (1993) Evolution of the ZFX and ZFY genes: Rates and interdependence between the genes. Mol Biol Evol 10: 271–281. 24. Author Contributions Conceived and designed the experiments: GV XX RAV. Performed the experiments: RAV. Analyzed the data: GV XX RAV. Wrote the paper: GV XX RAV. References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410. 49. Xia X, Xie Z (2001) DAMBE: Software package for data analysis in molecular biology and evolution. J Hered 92: 371–373. 25. Hulsen T, de Vlieg J, Groenen PM (2006) PhyloPat: phylogenetic pattern analysis of eukaryotic genes. BMC Bioinformatics 7: 398. 50. Yang Z, Nielsen R (2000) Estimating synonymous and nonsynonymous substitution rates under realistic evolutionary models. Mol Biol Evol 17: 32–43. PLoS ONE \| www.plosone.org August 2008 \| Volume 3 \| Issue 8 \| e2883 9
https://openalex.org/W4394850428	https://www.frontiersin.org/articles/10.3389/fcvm.2024.1406370/pdf?isPublishedV2=False	English	null	Editorial: Community series in epigenetic regulation in cardiovascular diseases, volume III	Frontiers in cardiovascular medicine	2,024	cc-by	1,568	Editorial on the Research Topic Editorial on the Research Topic Community series in epigenetic regulation in cardiovascular diseases, volume III Editorial: Community series in epigenetic regulation in cardiovascular diseases, volume III EDITED AND REVIEWED BY Neil Morgan, University of Birmingham, United Kingdom CORRESPONDENCE Zhihua Wang treerwang@163.com †These authors have contributed equally to this work RECEIVED 25 March 2024 ACCEPTED 29 March 2024 PUBLISHED 16 April 2024 CITATION Wang Z, Miao QR, Xu S, Pillai ICL and Rau CD (2024) Editorial: Community series in epigenetic regulation in cardiovascular diseases, volume III. Front. Cardiovasc. Med. 11:1406370. doi: 10.3389/fcvm.2024.1406370 COPYRIGHT © 2024 Wang, Miao, Xu, Pillai and Rau. 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. EDITED AND REVIEWED BY Neil Morgan, University of Birmingham, United Kingdom CORRESPONDENCE Zhihua Wang treerwang@163.com †These authors have contributed equally to this work RECEIVED 25 March 2024 ACCEPTED 29 March 2024 PUBLISHED 16 April 2024 CITATION Wang Z, Miao QR, Xu S, Pillai ICL and Rau CD (2024) Editorial: Community series in epigenetic regulation in cardiovascular diseases, volume III. Front. Cardiovasc. Med. 11:1406370. doi: 10.3389/fcvm.2024.1406370 COPYRIGHT © 2024 Wang, Miao, Xu, Pillai and Rau. 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 Frontiers in Cardiovascular Medicine KEYWORDS Front. Cardiovasc. Med. 11:1406370. doi: 10.3389/fcvm.2024.1406370 Front. Cardiovasc. Med. 11:1406370. doi: 10.3389/fcvm.2024.1406370 cardiovascular diseases, epigenetics, chromatin remodelling, histone modiﬁcations, DNA methylation COPYRIGHT © 2024 Wang, Miao, Xu, Pillai and Rau. 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. Editorial on the Research Topic Community series in epigenetic regulation in cardiovascular diseases, volume III University of Birmingham, United Kingdom Zhihua Wang 1,2*†, Qing Robert Miao 3†, Suowen Xu 4†, Indulekha C. L. Pillai 5† and Christoph D. Rau 6† 1Shenzhen Key Laboratory of Cardiovascular Disease, Fuwai Hospital Chinese Academy of Medical Sciences, Shenzhen, China, 2State Key Laboratory of Cardiovascular Disease, National Center for Cardiovascular Disease, Fuwai Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China, 3Grossman Long Island School of Medicine, New York University, Mineola, NY, United States, 4Institute of Endocrine and Metabolic Diseases, The First Afﬁliated Hospital of USTC, University of Science and Technology of China, Hefei, China, 5Stem Cells and Regenerative Biology Laboratory, School of Biotechnology, Amrita Vishwa Vidyapeetham, Kollam, India, 6Computational Medicine Program and Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States 6Computational Medicine Program and Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States TYPE Editorial PUBLISHED 16 April 2024 DOI 10.3389/fcvm.2024.1406370 Main Frontiers in Cardiovascular Medicine Epigenetic mechanisms contribute to the gene expression abnormality underlying multiple cardiovascular diseases (CVDs). Unveiling novel epigenetic players and their interplays governing gene reprogramming during the pathogenesis of CVDs remains a cutting-edge topic in the ﬁeld. In the third volume of our research topic series, we collected four high-quality papers, including one research article and three reviews, discussing the roles of transcription factors, epigenetic modiﬁers, non-coding RNAs, and RNA modiﬁcations in different types of CVDs. These novel ﬁndings and insights in this collection represent on-time instructions to renew our understanding of cardiovascular epigenetics under pathophysiological conditions. The increasing global prevalence of obesity and diabetes leads to an epidemic rise of diabetic cardiomyopathy, which has become a prominent medical challenge, particularly in the context of global population ageing. The molecular links between metabolic disorders and cardiovascular complications have not yet been fully resolved. Transcription factor Tumor Protein p53-inducible Nuclear Protein 2 (Trp53inp2) has previously been identiﬁed as a candidate gene linking glucose utilization and cardiomyocyte hypertrophy through an unbiased systems genetics study in mice. However, direct evidence still needs to be included. Here, Liu et al. developed an inducible Trp53inp2 knockout mouse line and investigated the impact of Trp53inp2 inactivation on the pathogenesis of heart failure under mechanical and/or metabolic stresses. Surprisingly, Trp53inp2 inactivation in the heart led to the accelerated onset of Frontiers in Cardiovascular Medicine 01 frontiersin.org Wang et al. 10.3389/fcvm.2024.1406370 emergence of genetic variations that exhibit gender bias, and variations in hormonal proﬁles between the sexes, with organized perspectives from DNA methylation, histone marks, and chromatin structure. This review raises critical concerns about the long-standing neglect of gender differences in cardiovascular disease research. Elucidating how sex-speciﬁc genetic, hormonal, and physiological factors contribute to the development and progression of cardiovascular diseases would not only advance our overall understanding of disease mechanisms but also open up new avenues for personalized medicine approaches. heart failure with reduced ejection fraction (HFrEF) in response to pressure overload, but it ameliorated cardiac dysfunction induced by combined stresses of high-fat diet and moderate pressure overload (so-called Cardiometabolic Disorder Model) due to the differential regulation of glucose metabolism genes. This study provides a biological basis to bridge metabolic stresses and cardiovascular outcomes. Obesity is a major risk factor for heart failure with preserved ejection fraction (HFpEF), which attracts more and more attention in current cardiovascular medicine. Author contributions ZW: Conceptualization, Writing – original draft, Writing – review & editing. QM: Writing – review & editing. SX: Writing – review & editing. IP: Writing – review & editing. CR: Writing – review & editing. 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. Sexual differences in heart disease are a signiﬁcant area of interest within the realm of personalized medicine. Despite clear variations in how heart conditions manifest and progress between genders, a historical lack of attention to gender-speciﬁc factors has meant that patient treatment has only sometimes been tailored accordingly. This oversight can be attributed to intensive emphasis on male participants in research studies and a failure to categorize ﬁndings by sex. Epigenetics has emerged as a key player in shedding light on the differences between males and females in terms of both heart health and disease. A review by Bridges et al. thoroughly examines the biological variances between men and women in the development of various cardiac complications, particularly focusing on uncovering the genetic and epigenetic mechanisms at play. The authors digested current ﬁndings regarding unique sex-chromosome compositions, the The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the ﬁnal decision. Main Despite decades of research, the treatment option for HFpEF still needs to be improved due to our incomplete understanding of the underlying molecular mechanisms. In a review written by Jalink et al. the authors summarized the recent advances in the role of non- coding RNAs, including miRNAs, lncRNAs, and circRNAs, in the pathophysiology of HFpEF by interrogating clinical evidence and dissecting the molecular mechanisms of the ncRNAs, the authors proposed the potential of ncRNAs as biomarkers and potential novel therapeutic targets for future HFpEF treatments. The papers we have gathered in conjunction with the two preceding volumes of this research topic comprehensively address the various aspects we initially aimed to explore when we embarked on this endeavor three years ago. They collectively provide a current overview of the cutting-edge research in the ﬁeld of epigenetics related to diverse cardiovascular diseases. These studies have yielded valuable ﬁndings that shed new light on the understanding of cardiovascular pathologies and undoubtedly hold promise for advancing the development of innovative therapeutic approaches to tackle these speciﬁc cardiovascular conditions. Although RNA modiﬁcations have long been studied on housekeeping RNAs, like tRNAs and rRNAs, we only recently recognized the important physiological function of the modiﬁcations, particularly methylation, on mRNAs. N6- methyladenosine (m6A) methylation is the most common epigenetic modiﬁcation on mRNAs that has been extensively investigated in diverse disease models. Zhang et al. provide a comprehensive review, summarizing recent advances in the role of mRNA m6A modiﬁcation in CVDs. The authors digested the regulations and actions by m6A writers, erasers, and readers in different biological aspects, including calcium homeostasis, endothelial function, cell death, autophagy, endoplasmic reticulum stress, macrophage response and inﬂammation, in CVDs, especially in heart failure and ischemic heart disease. Current evidence suggests that targeting m6A modiﬁers could hold translational therapeutic value in treating CVDs. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Frontiers in Cardiovascular Medicine 02 frontiersin.org
https://openalex.org/W2010509418	https://europepmc.org/articles/pmc2581734?pdf=render	English	null	Male Genitoplasty for Intersex Disorders	Advances in urology	2,008	cc-by	3,823	Hindawi Publishing Corporation Advances in Urology Volume 2008, Article ID 685897, 5 pages doi:10.1155/2008/685897 Hindawi Publishing Corporation Advances in Urology Volume 2008, Article ID 685897, 5 pages doi:10.1155/2008/685897 Hindawi Publishing Corporation Advances in Urology Volume 2008, Article ID 685897, 5 pages doi:10.1155/2008/685897 Shilpa Sharma and Devendra K. Gupta Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi 110029, India Correspondence should be addressed to Devendra K. Gupta, profdkgupta@gmail.com Received 10 May 2008; Accepted 5 August 2008 Recommended by Miroslav L. Djordjevic Aim. To evaluate surgical procedures adopted for male genitoplasty in intersex disorders. Patients and Methods. Case records of intersex patients undergoing male genitoplasty from Pediatric Intersex clinic were studied. Results. Of 356 intersex cases undergoing urethroplasty from 1989–2007, the hypospadias was penoscrotal (68%), scrotal (17%) and perineal (15%). 351 patients underwent chordee correction for mild: moderate: severe chordee in 24 : 136 : 191 cases. Byars ﬂaps were ﬁxed upto the corona in 267 cases. Urethroplasty performed was Theirsch duplay in 335 cases, Snodgrass in 16 cases and Ducketts onlay graft in 5 cases that did not require chordee correction. Age at urethroplasty was 2.5 years—22 years (mean 11.5 years, median— 5.6 years). Penoscrotal transposition correction and testicular prosthesis insertion were performed independently. Complications included ﬁstula (45), recurrent ﬁstula (11), stricture (12), baggy urethra (8) and recurrent infection due to persistent vaginal pouch (5). Additional distal urethroplasty was required in 15 patients for previous urethroplasty done upto the corona 5–15 years earlier. Conclusion. Hypospadias in intersex disorders is associated with severe chordee in most cases and requires an early chordee correction to allow phallic growth, staged urethroplasty and multiple surgeries to achieve good cosmetic and functional results. Copyright © 2008 S. Sharma and D. K. Gupta. 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. Purpose This work aims to study the surgical procedures performed for male genitoplasty in cases of intersex disorders. To perform urethroplasty for cases of intersex disorders, now designated as disorders of sex diﬀerentiation, it requires far sight into the problems that are faced even many years after the urethroplasty. A number of surgical procedures need to be performed before the ﬁnal outcome is attained. These will be discussed in detail in this article along with the authors experience in this ﬁeld. 2. PATIENTS AND METHODS Case records of 356 intersex patients undergoing male genitoplasty from 1989–2007 from the Pediatric Intersex clinic of the department of pediatric surgery, AIIMS, were studied. Preoperative investigations included chromosomal analysis, ascending urethrogram, abdominal ultrasonogra- phy, and hormonal work up to establish the diagnosis of intersexuality. The vital decision in cases of intersex disorders is the accurate diagnosis and the gender assignment. The gender assignment takes into account the prevalent social factors in a community and the parent’s desire [1]. There are still some countries where the parents would prefer to have an inadequate male rather than an incomplete female. 4. DISCUSSION Out of 356 cases, 351 patients had chordee and under- went chordee correction between 2 months—7.6 years age (mean 23.6 months). Five patients did not have chordee. The chordee was mild: moderate: severe in 24 : 136 : 191 cases. Byars ﬂaps for the chordee correction were ﬁxed upto the corona in 267 cases. Testosterone (5–10%) cream was advised for local application daily for 3 months at one time. The patients were called for follow up after that, and the response was assessed. If response was noted, it was continued for other three months. The treatment was stopped if any side eﬀects were noted. To manage disorders of sexual diﬀerentiation, a growing need is felt for extensive counseling, informed consent, and adherence to ethical and legal norms that protect the rights of the child as outlined in respective constitutions and a multidisciplinary approach [2]. The foremost step is the sex assignment. This should be based upon what is judged to be the most likely adult gender identity, diagnosis, genital appearance and surgical options, fertility, cultural pressures, as well as family dynamics and social circumstance, with preference given to psychosocial factors when the outcome is unpredictable [3]. However, the complex management of these patients must be individualized [3]. In this series, seven cases of CAH were reared as males as they had already been reared as males for a long time before they came for treatment Periodic local treatment with testosterone was also advised later, during childhood till puberty, to help in penile growth in patients with low testosterone level and those having a small-sized phallus. However, systemic injections were given only postpuberty to prevent bone maturation if given earlier. Gender assignment procedures have been questioned by intersex activists opposed to early genital surgery [4]. Western societies have a binary perspective on gender and this leads to a stigma on intersex cases [4]. Also, the current data challenges the past practice of sex reassignment, thus, a careful judgment is warranted [5]. The debate whether surgical genitoplasty aﬀects gender identity in the intersex infant centres around which is more vital for development of gender identity, that is, the biological sex of a child or the sex in which a child is reared [6]. 4. DISCUSSION g Theirsch Duplay urethroplasty was performed in 335 cases, Snodgrass urethroplasty was made in 16 cases, and Ducketts onlay graft was performed in 5 cases for whom chordee correction was not required. The age at urethro- plasty was 2.5–22 years (mean 11.5 years, median—5.6 years). Scrotal transpositions for cases with penoscrotal transposition as well as testicular prosthesis were performed as an independent operation so as not to jeopardize the perineal and preputial ﬂaps (Figure 3). The complications included ﬁstula formation in 45 cases, recurrent ﬁstula formation in 11 cases, stricture formation in 12 cases, baggy urethra in 8 cases, and recurrent infection due to persistent vaginal pouch in 5 cases. Postoperative infection was more in the boys that were operated after ten years of age. Additional surgery was performed for diverticuli in 3 cases. An additional distal urethroplasty was requested by 15 patients, where the urethral tube was constructed earlier only upto the corona 5–15 years following the previous urethroplasty. Five patients out of 356 (1.4%) developed hair growth in the region of the scrotal neourethral tube that led to infection and urinary obstruction. They had to undergo epilation or redourethroplasty with removal of the skin patch. Hormonal and genetic factors may have a more impor- tant role in gender identity and sexual satisfaction than previously recognized, whereas the importance of phallus size to male gender identity and sexual satisfaction may have been overestimated [7]. The impact of androgen imprinting on the developing brain has also been debated. A neutral upbringing may induce psychosocial consequences that are more damaging than carefully considered neonatal sex attribution and concordant surgical genitoplasty [6]. The spectrum of iatrogenic harm to children with inter- sex characteristics has now become a legal issue [8]. Thus, a multidisciplinary approach involving pediatric surgeons, endocrinologist, and psychiatry is necessary, along with educational programmes that promote tolerance in society to variations in gender [4]. The urethroplasty has been completed in 324 patients and 27 patients are still awaiting the urethroplasty. Of the 46 cases that had attained puberty, 23 (50%) of them had to milk their urethra after voiding. Intramuscular testosterone was administered every month for cases having a low testosterone level and those having a small-sized phallus. None had residual chordee on interrogation of the patients. 3. RESULTS With lot of global discussion on the patient’s wishes when they attain puberty, it has now become important to keep a possibility of a change in gender in some cases and wait a little longer before a deﬁnitive surgery unless there is parental pressure. These controversial issues have long been debated and vary from community to community. Of 526 patients that had undergone urethroplasty, 356 were cases of intersex disorders. These included 298 cases of male pseudohermaphrodite, 24 cases of True Hermaphrodite, 27 cases of mixed gonadal dysgenesis and dysgenetic male Advances in Urology 2 pseudohermaphrodite, and 7 cases of congenital adrenal hyperplasia. had a relatively small phallus than those in whom chordee correction had been done earlier. The postoperative ure- thrography revealed few urethral diverticulae and pouches in 5 cases that were otherwise asymptomatic. The uroﬂowmetry revealed low mean ﬂow in 20 out of 46 cases. This was due to the fact that there is poor outﬂow resistance in these cases due to underdeveloped corpus spongiosum. Ten out of twenty cases interviewed were able to ejaculate, though the amount was less and 8 had to milk the ejaculate. The hypospadias was penoscrotal in 242 (68%) cases, scrotal in 61 (17%), and perineal in 53 (15%) cases (Figures 1, 2). The mullerian duct opening was present in the perineum along with the urethral meatus in 35 (10%) cases. A genitogram performed in all cases was suﬃcient to outline a vaginal pouch and establish the diagnosis of male pseudohermaphrodite in the presence of unilateral- or bilateral-descended gonads and absent uterus with XY karyotype. 4. DISCUSSION The cases in which the chordee correction had been delayed The most common disorder of sexual diﬀerentiation is male pseudohermaphroditism that comprises about 55–60% of all cases [9]. However, some series from endocrine centres have the largest number of cases as congenital adrenal hyperplasia [10]. The causes of male pseudohermaphroditism include 17 beta-hydroxysteroid S. Sharma and D. K. Gupta 3 (a) (b) (c) Figure 1: (a) A case of mixed gonadal dysgenesis with severe chordee, perineal hypospadias, and visible urethral and vaginal openings. (b) A case of intersex disorder with small phallus, ﬁsh mouth urethra, and mucosa-lined urethral plate. (c) Post chordee correction case of intersex disorder for second stage reconstruction with long urethroplasty. (c) (a) (a) (a) (c) (b) Figure 1: (a) A case of mixed gonadal dysgenesis with severe chordee, perineal hypospadias, and visible urethral and vaginal openings. (b) A case of intersex disorder with small phallus, ﬁsh mouth urethra, and mucosa-lined urethral plate. (c) Post chordee correction case of intersex disorder for second stage reconstruction with long urethroplasty. (a) (a) (a) (b) Figure 2: A case of True Hermaphrodite with (a) well-developed phallus and right scrotal ovotestis gonad, and (b) penoscrotal hypospadias and severe chordee. (b) stricture, and wound dehiscence. The reason is that the phallus in these cases is inadequate to support the formation of a 6Fr urethral tube formed from the inadequate preputial skin at a tender age. In this series, the number of urethroplasties in intersex cases is higher than that in hypospadias patients as being a tertiary-level hospital, the cases are selected for operations. Only the diﬃcult cases or those with more proximal hypospadias are dealt with after ruling out intersex disorders. In this series, most of the cases were operated with a two- stage procedure. In the ﬁrst stage, the chordee is corrected, followed by urethroplasty from the neopenile skin ﬂaps after an interval of at least 6 months. The Byars ﬂaps for the chordee correction are ﬁxed upto the corona in most cases as the glans is very underdeveloped at the time of chordee correction. The parents are then advised to apply local testosterone. For adequate chodee correction, the urethral plate was divided during the ﬁrst stage. The ﬁbrous tissue was excised. Total mobilisation was done upto the base of the phallus. As a result, the meatus moved proximally 3–6 cms. 4. DISCUSSION Some authors have performed a single-stage procedure using a transverse pedicled preputial island ﬂap as an onlay, tubulariza- tion of the mucosa in the perineal area, and end-to-end anastomosis to a tube made from the pedicled prepuce [11]. There may be lack of ejaculation related to frequent intrautricular termination of the vas deferens [15]. Some patients with androgen insensitivity syndrome, that is, the other end of male pseudohermaphrodites who are initially assigned a female gender, may seek help for conversion into a male at a later date. For these cases, the neophallus may be created from sensate-free forearm ﬂaps or regional abdominal ﬂaps that exist [16]. The corporal tissue is preserved for sensations and placed at a suitable place in the perineum. However, a one-stage male genitoplasty for male pseudo- hermaphroditism is accompanied by a reasonable incidence of major complications [11]. Thus, the two-stage technique for male genitoplasty is preferable [12]. The two-stage approach for severe hypospadias without intersex disorders results in excellent function, cosmesis, and patient satisfac- tion after puberty, with no chordee in patients though minor voiding and ejaculatory problems are to be expected [14]. Most series are satisﬁed with their functional and cosmetic outcome of masculinising genitoplasty in patients with ambiguous genitalia raised as males [17]. Good results may be expected if the initial phallus size is adequate, especially those that have responded to local testosterone. The results are poor in cases with micropenis and minimal virilisation [13]. Intramuscular testosterone is administered every month to most cases with a low testosterone level. The uroﬂowmetry revealed low mean ﬂow in 20 out of 46 cases in this series due to the fact that there is poor outﬂow resistance in these cases due to underdeveloped corpus spongiosum. Spontaneous puberty may be observed in true hermaphrodites raised as males [13]. Most cases have to undergo multiple genital surgeries to correct the appearance of their genitalia and/or to enable sexual functioning [18, 19]. However, cases with intersex disorders have a deﬁcient spongiosum and thus the urethral tube is poorly supported. About 50% of them have to milk their urethra after voiding to keep themselves dry. In intersex patients, Mullerian duct remnants are not an unusual occurrence. The presence of a vaginal pouch (utriculus prostaticus) does not aﬀect the urethroplasty. A genitogram performed in all cases is usually suﬃcient to outline a vaginal pouch and establish the diagnosis. 4. DISCUSSION (b) However, there are others who prefer to remove a big or symptomatic utriculus prostaticus [15]. Due to the location of the pouch, a surgical removal from the perineal or sacral route is always risky. If at all required as in symptomatic cases, a laparoscopic removal is much safe. Authors prefer to include the opening of the utriculus in the urethroplasty. If the opening joining the urethra is narrow, the opening is widened endoscopically. (a) (b) Figure 3: (a) A case of true hermaphrodite with right scrotal ovotestis and left undescended gonad needing a prosthesis. (b) Indeginized (DK Gupta) testicular prosthesis made of Teﬂon. In a series of 47 boys, based on the symptoms and the size of the remnants, the structures were removed in 32 patients by extirpation done by perineal approach in younger asymp- tomatic children, by transperitoneal approach, by posterior sagittal pararectal approach, or by combined abdominal and perineal approach [15]. Complications like rectal or bladder injuries and temporary impotence after abdominoperineal extirpation may occur during attempted removal. Dihydrotestosterone (DHT) is beneﬁcial for patients with 5 alphareductase deﬁciency that are unable to convert testosterone into DHT due to absence/default of receptors. Some of these patients respond with an increase in penile size when 25 mg/d of 2% DHT cream is applied to the external genitalia. However, anabolic eﬀects may enhance hypoglycaemia and bone maturation. Adverse reactions include pruritus, erythema, allergic contact dermatitis, and burning. The presence of Mullerian duct remnants may occasion- ally lead to symptoms of urinary infection, urinary retention, or epididymitis. However, these are manageable with courses of antibiotics during acute episodes and preventable by exe- cuting a habit of milking out the contents after micturation. Thus, on comparing the risks with the beneﬁts, it is wiser to leave the vaginal pouch as such. In this series, only 5 patients had problems due to the vaginal pouch. The rest remained asymptomatic. These were more frequent in the cases of congenital adrenal hyperplasia that preferred to be assigned a male gender. Once the phallus size is adequate, urethroplasty is performed. Recurrence of chordee was assessed by artiﬁcial penile erection test of Gittes and McLaughlin at the time of stage 2 reconstruction. The most common procedure adopted is Theirsch Duplay urethroplasty. 4. DISCUSSION The excision of all ﬁbrous strands assures the complete removal of chordee. The intervening deﬁciency after adequate chordee correction was covered with preputial skin that was mobilised during degloving of the phallus. The authors used to perform Gittes’ test to assess chordee in the intial cases but soon realised that it is not necessary in cases where there is no doubt of residual chordee in cases where the urethral plate has been cut. (b) (a) Figure 2: A case of True Hermaphrodite with (a) well-developed phallus and right scrotal ovotestis gonad, and (b) penoscrotal hypospadias and severe chordee. dehydrogenase deﬁciency, 3 beta-hydroxysteroid dehydroge- nase deﬁciency, 5 alpha-reductase deﬁciency, and idiopathic male pseudohermaphroditism [11]. The timing of performing the masculinisation proce- dures is still a controversy. However, most feel that the optimal time for external genital correction is 2 years of age [3, 9, 12]. However, in this series, most of the patients presented late after running from pillar to post for accurate diagnosis. Moreover, in recent years, traditional views regarding the management of infants with intersex conditions have been challenged [7]. The severe chordee that is present in most of the cases prevents adequate growth of the phallus. The deﬁciency of testosterone, either systemically or locally due to various enzyme defects present in these cases, prevents adequate growth of the phallus both in length and girth. The appli- cation of steroid cream after chordee correction facilitates proper growth of the phallus that forms a good base for the neourethra. Also, the penile skin in these cases is thin and lacks strength and good vascularity for proper healing. Some authors perform urethroplasty only after obliga- tory testosterone treatment [10]. The application of testos- terone helps to make the skin supple besides increasing the length and girth of the phallus. Though prior to 1980s, single-stage reconstruction was in vogue, with the passing time, it has been realised that single-stage reconstruction is associated with more complications in cases with intersex disorders [13]. These include ﬁstula formation, complete Advances in Urology 4 (a) (b) Figure 3: (a) A case of true hermaphrodite with right scrotal ovotestis and left undescended gonad needing a prosthesis. (b) Indeginized (DK Gupta) testicular prosthesis made of Teﬂon. (a) Due to potential injury to continence mechanisms and for preservation of fertility (vas deferens often joins the utricle), it is better to reserve surgical treatment only to symptomatic cases. REFERENCES [1] D. K. Gupta and P. S. N. Menon, “Ambiguous genitalia—an Indian perspective,” Indian Journal of Pediatrics, vol. 64, no. 2, pp. 189–194, 1997. [19] S. Sharma and D. K. Gupta, “Gender assignment and hormonal treatment for disorders of sexual diﬀerentiation,” Pediatric Surgery International, 2008. [2] N. R. Maharaj, A. Dhai, R. Wiersma, and J. Moodley, “Intersex conditions in children and adolescents: surgical, ethical, and legal considerations,” Journal of Pediatric and Adolescent Gynecology, vol. 18, no. 6, pp. 399–402, 2005. [3] Z. M. Nabhan and P. A. Lee, “Disorders of sex development,” Current Opinion in Obstetrics and Gynecology, vol. 19, no. 5, pp. 440–445, 2007. [4] N. D. Duncan, L. Gabay, E. Williams, S. E. Dundas, N. Plummer, and P. A. Leake, “Hermaphroditism: cytogenetics, gonadal pathology and gender assignment: a case report,” West Indian Medical Journal, vol. 55, no. 6, pp. 430–433, 2006. [5] D. Frimberger and J. P. Gearhart, “Ambiguous genitalia and intersex,” Urologia Internationalis, vol. 75, no. 4, pp. 291–297, 2005. [6] C. Nihoul-F´ek´et´e, “Does surgical genitoplasty aﬀect gender identity in the intersex infant?” Hormone Research, vol. 64, supplement 2, pp. 23–26, 2005. [7] C. P. Nelson and J. P. Gearhart, “Current views on evaluation, management, and gender assignment of the intersex infant,” Nature Clinical Practice Urology, vol. 1, no. 1, pp. 38–43, 2004. [8] K. Gurney, “Sex and the surgeon’s knife: the family court’s dilemma... informed consent and the specter of iatrogenic harm to children with intersex characteristics,” American Journal of Law and Medicine, vol. 33, no. 4, pp. 625–661, 2007. ˇ [9] Z. Krsti´c, S. Perovic, S. Radmanovi´c, S. Necli´c, ˇZ. Smoljani´c, and P. Jevti´c, “Surgical treatment of intersex disorders,” Journal of Pediatric Surgery, vol. 30, no. 9, pp. 1273–1281, 1995. [10] A. G. Coran and T. Z. Polley Jr., “Surgical management of ambiguous genitalia in the infant and child,” Journal of Pediatric Surgery, vol. 26, no. 7, pp. 812–820, 1991. [11] B. Chertin, D. Koulikov, I. Hadas-Halpern, and A. Farkas, “Masculinizing genitoplasty in intersex patients,” The Journal of Urology, vol. 174, part 2, no. 4, pp. 1683–1686, 2005. [12] W. C. Hecker, “Timing and technic of the surgical correction of childhood intersex genitals including the procedure for partial vaginal aplasia,” Zeitschrift f¨ur Kinderchirurgie, vol. 37, no. 3, pp. 93–99, 1982. [13] C. Nihoul-F´ek´et´e, E. Thibaud, S. Lortat-Jacob, and N. 4. DISCUSSION In this series, the Mullerian duct remnants were removed, if present, in all patients assigned a male gender, only the vaginal pouch was preserved. The uterus, fallopian tubes, and upper part of the vagina were removed in all, and the lower part of the pouch was left as such in all cases. The vaginal pouch was not removed in any case of male pseudohermaphrodite. To summarize, hypospadias in intersex disorders is asso- ciated with severe chordee in most of the cases and requires an early chordee correction to allow phallic growth. The issue There is no evidence that removal of utriculus and Mullerian remnants, which are asymptomatic, is necessary. S. Sharma and D. K. Gupta 5 of genital surgery in infancy remains controversial although many adult patients do concur that infancy is the best time for such procedures. Good anatomical and functional results are achieved better with the two-stage repair and intervening period of local testosterone application. [16] M. Sohn and H. A. G. Bosinski, “Gender identity disor- ders: diagnostic and surgical aspects,” The Journal of Sexual Medicine, vol. 4, no. 5, pp. 1193–1208, 2007. [17] A. Farkas, D. Koulikov, and B. Chertin, “Masculinizing genito- plasty in male pseudohermaphroditism,” Pediatric Endocrinol- ogy Reviews, vol. 2, no. 1, pp. 15–20, 2004. [18] L. Brinkmann, K. Schuetzmann, and H. Richter-Appelt, “Gender assignment and medical history of individuals with diﬀerent forms of intersexuality: evaluation of medical records and the patients’ perspective,” The Journal of Sexual Medicine, vol. 4, no. 4, pp. 964–980, 2007. REFERENCES Josso, “Long-term surgical results and patient satisfaction with male pseudohermaphroditism or true hermaphroditism: a cohort of 63 patients,” The Journal of Urology, vol. 175, no. 5, pp. 1878–1884, 2006. [14] P. N. Lam, S. P. Greenﬁeld, and P. Williot, “2-stage repair in infancy for severe hypospadias with chordee: long-term results after puberty,” The Journal of Urology, vol. 174, no. 4, part 2, pp. 1567–1572, 2005. [15] Z. Krsti´c, ˇZ. Smoljani´c, ˇZ. Mi´covi´c, V. Vukadinovi´c, A. Sreten- ovi´c, and D. Varinac, “Surgical treatment of the M¨ullerian duct remnants,” Journal of Pediatric Surgery, vol. 36, no. 6, pp. 870– 876, 2001.
https://openalex.org/W2585631685	https://www.matec-conferences.org/10.1051/matecconf/20179701046/pdf	English	null	Low Power CMOS Operational Amplifier with Integrated Common-Mode Feedback for Data Converter	MATEC web of conferences	2,017	cc-by	2,526	* Corresponding author: sohiful@unimap.edu.my DOI: 10.1051/ 79701046 matecconf/201 DOI: 10.1051/ 79701046 matecconf/201 , 01046 (2017) 97 MATEC Web of Conferences ETIC 2016 © 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/). Low Power CMOS Operational Amplifier with Integrated Common-Mode Feedback for Data Converter S.A.Z Murad1,, Muhammad M. Ramli1 , A. Azizan1 , M.N.M Isa1 , and I.S Ishak1 1School of Microelectronic Engineering, University Malaysia Perlis, Pauh Putra Campus, 02600 Arau, Perlis, Malaysia Sciences Abstract. The development a high-performance design of analog circuits becomes increasingly challenging with the continuous trend towards reducing the voltage supply and low power consumption without neglecting the trade-off among other performance parameters. This paper presents the design and implementation of CMOS operational amplifier (op-amp) with integrated common-mode feedback (CMFB) circuit for data converter using 0.13-µm Silterra CMOS technology. The folded cascode topology is employed as a main op-amp design because it provides high gain and high bandwidth besides low power consumption. The simulation results indicate that the DC gain of 64.5 dB along 133.1 MHz unity gain bandwidth (UGB) is achieved for a 1 pF load capacitor. The slew rate of 22.6 V/µs, the phase margin (PM) of 68.4 ° with settling time of 72.4 ns are obtained. The power consumption of this op-amp is 0.3 mW through voltage supply of 1.8 V. 1 Introduction Wireless Operational amplifier is a core element and integral part for most analog and mixed-signal systems. The behavioral of op-amp such as high gain, high input impedances, low output impedance, high bandwidth and fast settling makes this device often used amongst multiplicity of applications like in pipeline ADC. Traditionally, op-amp can be classified into several topologies which are telescopic, folded cascode, two-stage and gain- boosted [1]. Each topology has their own compensation, but they can be applied in any design op-amp circuit by considering the performance parameter. A telescopic op-amp is a simple topology and provided a high gain as well faster performance [2, 3]. This topology is called as ‘telescopic’ because the cascades are attached between the voltage supplies with the transistor in the differential pair, occurs in a structure where each branch of the transistor connected directly together in a straight line [4]. Commonly the telescopic topology has a slighter swing because of lesser current legs and produces a small power consumption and low noise. 2.2 Unity gain bandwidth The unity gain bandwidth state that the frequency at which the open loop gain of the amplifier is unity with the maximum capacitance at the output node. Thus the UBW is obtained as: UBW = 2π f CL (2) (2) UBW = 2π f CL 2.1 Gain Ideally, the gain is the product of the transconductance structure over the output resistances of the load structure that express as: Gain = Gm Rout (1) (1) where the gain extremely depends on the frequency of the input signal of an amplifier. where the gain extremely depends on the frequency of the input signal of an amplifier. Corresponding author: sohiful@unimap.edu.my , 01046 (2017) 97 MATEC Web of Conferences ETIC 2016 DOI: 10.1051/ 79701046 matecconf/201 In a folded cascode op-amp, the topology is normally customized from the telescopic op-amp and it issues higher gain and performance [5,6] compared to the telescopic because it consumes more currents legs. This topology is called ‘folded cascode’ because of small signal current is folded up or to down [1]. Generally, this op-amp allows the particular input common-mode level of being near to the voltage supplies as well as performing a high output swing, wide input common-mode range and preferably steering in low voltage supply circuits [4]. However, this topology contributed greater noise that effect from the more currents legs. 2 Design constraints A design constraint is a key element in preparing the best performance of op-amp circuit. As a requirement of a high speed and high accuracy in pipeline ADC, there are numerous constraint parameters that should be considered such as gain, unity gain bandwidth, phase margin, slew rate and also output swing. Each parameter constraints are explained as follows. 2.3 Phase margin The purpose of phase margin (PM) is to determine the stability of the amplifier where the higher values of PM will allow the output signal to achieve a stable state without much swing. Noted that the PM is depends on the applications [1]. Phase margin (PM) = tan-1 [gm / (2π f CL)] (3) Phase margin (PM) = tan-1 [gm / (2π f CL)] (3) Phase margin (PM) = tan-1 [gm / (2π f CL)] (3) (3) 2 2 DOI: 10.1051/ 79701046 matecconf/201 , 01046 (2017) 97 MATEC Web of Conferences ETIC 2016 2.5 Output swing This constraint relates to the output of the op-amp where the saturation voltage of load structure mainly defines the output swing of the op-amp. Commonly, most systems employing op amps require large voltage swings to accommodate a wide range of signal amplitude [1]. Output swing = (VDD – Vmax/min) / 2 (5) Output swing = (VDD – Vmax/min) / 2 (5) 2.4 Slew rate Slew rate is defined as the rate of change in the output voltage that caused by a step change on the input. It can be determines by the output capacitance and the current across the output branch. Slew rate = Iout / CL (4) Slew rate = Iout / CL (4) (4) 3 Design implementation According to the op-amp specifications as presented in Table 1, the desired op-amp topology was determined. Folded cascode topology idyllically to be principle op-amp for this work since the design has been used in [7] for obtaining fast settling, high gain and high unity gain bandwidth besides low power consumption. Table 1. Op-amp Specifications for Pipeline ADC. Table 1. Op-amp Specifications for Pipeline ADC. Parameter Value Voltage supply 1.8 V Vin 1.2 V Vout 1.2 V DC gain > 70 dB Phase margin > 60º Unity gain bandwidth > 130 MHz The architecture of folded cascode op-amp with common-mode feedback is illustrated as Fig. 1. Traditionally, the folded cascode op-amp has been designed by using a pair of PMOS type or NMOS type for the input range of op-amp through the limits of input common mode range [7]. For this work, the NMOS type is chosen to be an input of the differential amplifier since this input type can assign more large output gain compare to PMOS input type. 3 3 DOI: 10.1051/ 79701046 matecconf/201 , 01046 (2017) 97 MATEC Web of Conferences ETIC 2016 Fig. 1. Folded cascode op-amp with CMFB architecture. Fig. 1. Folded cascode op-amp with CMFB architecture. Fig. 1. Folded cascode op-amp with CMFB architecture. As shown in Fig. 1, the folded cascode topology consists of two different structures which are NMOS differential amplifier (M1-M3) and folded cascode structure (M4-M11). The open loop voltage gain can be determined as: Av = gm Ro (6) (6) where gm is a short-circuit transconductances of the output current gain across the transistor of M6 and Ro is the output impedance of folded cascode by looking into the drains of M6 and M8. Ro = gm6.ro6(ro1\|\|ro4) \|\| (gm8.ro8.ro10) (7) (7) Ro = gm6.ro6(ro1\|\|ro4) \|\| (gm8.ro8.ro10) Therefore, the gain is expressed as: Av = gm{[gm6.ro6.(ro1\|\|ro4)] \|\| [gm8.ro8.ro10] (8) (8) Meanwhile, the gain bandwidth of the folded cascode circuit is: Meanwhile, the gain bandwidth of the folded cascode circuit is: Meanwhile, the gain bandwidth of the folded cascode circuit is: GBW = gm1 / CL (9) (9) where gm1 is a transconductance of M1 and CL is the capacitance at the output node where gm1 is a transconductance of M1 and CL is the capacitance at the output node. 3 Design implementation A CMFB circuit (M12-M16) is designed in order to fix the voltages at high impedances node to the desired voltage value of CMRR performance while ensuring the stability of common-mode voltage for fully differential op-amp [8]. As referred in Fig. 2, M12 is assigned to be a feedback to the folded cascode op-amp while M13 and M14 is an input of CMFB that attaches to the output of folded cascode op-amp and M15 represents as a reference voltage. This CMFB architecture is modified from the conventional amplifier as 4 4 , 01046 (2017) 97 MATEC Web of Conferences ETIC 2016 DOI: 10.1051/ 79701046 matecconf/201 in Fig. 1(a) in [9]. The modification is based on M12 because its need a stable voltage to fed the folded cascode op-amp. Subsequently, these two circuit blocks are implemented in 1.5-bit per stage pipeline ADC as shown in Fig. 2 in order to simulate the circuit performances. The result is discussed in section below. in Fig. 1(a) in [9]. The modification is based on M12 because its need a stable voltage to fed the folded cascode op-amp. Subsequently, these two circuit blocks are implemented in 1.5-bit per stage pipeline ADC as shown in Fig. 2 in order to simulate the circuit performances. The result is discussed in section below. Fig. 2. Implementation circuit in 1.5-bit per stage pipeline ADC. Folded cascode CMFB S/H out+ incm S/H incm+ out Fig. 2. Implementation circuit in 1.5-bit per stage pipeline ADC. 4 Results and discussion The proposed folded cascode op-amp was designed using Cadence Software and implemented in 0.13-μm process technology with 1.8 V supply voltage whereas the simulation of circuit via Cadence Virtuoso spectre. The circuit design is analyzed in two methods which are AC analysis and Transient analysis. Fig. 3 shows the AC analysis result of the proposed circuit. The simulated DC gain demonstrated 64.5 dB with 68.4 degrees of phase margin (PM) as shown in Fig. 3 (a) and Fig. 3 (b), respectively. Meanwhile, the transient analysis result is depicted in Fig. 4. The slew rate performs 22.6 V/μs among 72.4 ns of settling times. (a) (b) Fig. 3. AC analysis: (a) Gain, (b) Phase margin. (a) (b) Fig. 3. AC analysis: (a) Gain, (b) Phase margin. 5 DOI: 10.1051/ 79701046 matecconf/201 , 01046 (2017) 97 MATEC Web of Conferences ETIC 2016 Fig. 4. Transient analysis. Table 1 Fig. 5 illustrates the result for implementation of the circuit in 1.5 bit per-stage pipeline ADC. The performances of the circuit are referring to the process information as tabulated in Table 2. Table 1 Fig. 5 illustrates the result for implementation of the circuit in 1.5 bit per-stage pipeline ADC. The performances of the circuit are referring to the process information as tabulated in Table 2. Table 1 Fig. 5 illustrates the result for implementation of the circuit in 1.5 bit per-stage pipeline ADC. The performances of the circuit are referring to the process information as tabulated in Table 2. Fig. 5. Implementation results. Fig. 5. Implementation results. Table 2. Data converter information for 1.5-bit per stage pipeline ADC. Vin B1 B0 DAC output Residue output Vin > Vref/4 1 0 +Vref 0.18 -Vref/4 < Vin < Vref/4 0 1 0 1.0 Vin < -Vref/4 0 0 -Vref 2.4 Vin > Vref/4 1 0 +Vref 9.0 Fig. 5. Implementation results. Table 2. Data converter information for 1.5-bit per stage pipeline ADC. Table 2. Data converter information for 1.5-bit per stage pipeline ADC. Vin B1 B0 DAC output Residue output Vin > Vref/4 1 0 +Vref 0.18 -Vref/4 < Vin < Vref/4 0 1 0 1.0 Vin < -Vref/4 0 0 -Vref 2.4 Vin > Vref/4 1 0 +Vref 9.0 Table 2. Data converter information for 1.5-bit per stage pipeline ADC. 4 Results and discussion 6 , 01046 (2017) 97 MATEC Web of Conferences ETIC 2016 DOI: 10.1051/ 79701046 matecconf/201 The comparison for folded cascode op-amp performances between previous works is summarized as in Table 3. From the table, this circuit design attains low power consumption with smallest load capacitance; therefore the chip size can be reduced. 5 Conclusion The design and implementation of CMOS op-amp with integrated common-mode feedback circuit for data converter using 0.13-μm Silterra technology is presented. The DC gain of the op-amp is obtained about 64.5 dB along with unity gain bandwidth (UGB) of 133.1 MHz for a 1 pF load and 68.4 degrees. The circuit in this work consumes a low power consumption which is around 0.3 mW at 1.8 V supply supply. The simulation result shows that the proposed design is suitable for data converter applications. Table 3. Folded cascode op-amp performances. Reference [10] [11] [12] [6] This wok Technology (μm) 0.18 0.18 0.13 0.13 0.13 VDD (V) 1.8 1.8 3.0 1.8 1.8 DC gain (dB) 90.30 50.97 94.9 91.5 64.5 Unity gain Bandwidth (MHz) 700.7 944.0 41 714.5 133.1 Phase margin (º) 63.85 75.9 82.3 62.0 68.4 Slew rate (V/μs) N/A 712.5 N/A N/A 22.6 Settling time (ns) N/A N/A 6.17 40.0 72.4 Power consumption (mW) 3.24 172.8 11.0 9.0 0.3 Load capacitance (pF) 0.5 2.4 2.0 7.5 1.0 Table 3. Folded cascode op-amp performances. This work was financially supported by the Fundamental Research Grant Scheme (9003-00387). References 7. W. Gu, W. Gao, World Acad. Sci Eng. Technol., 67, 284 (2012). 12. S.A.E. Ab Rahim, I.M. Azmi, International Symposium on Integrated Circuit, (2011 8. I.S. Ishak, S.A.Z. Murad, M.F. Ahmad, S.C. Neoh, Proceedings of Malaysian Technical Universities Conference on Engineering and Technology (MUCET), (2013) 11. Z. Hao, F. Xiangning, S. Yutao, International Conference on Wireless Communications and Signal Processing (WCSP2011), (2011). 7. W. Gu, W. Gao, World Acad. Sci Eng. Technol., 67, 284 (2012). 8. I.S. Ishak, S.A.Z. Murad, M.F. Ahmad, S.C. Neoh, Proceedings of Malaysian Technical Universities Conference on Engineering and Technology (MUCET), (2013) 9. M.F. Ahmad, S.A.Z. Murad, M.M. Shahimim, S.A.A. Rais, A.F. Hasan, Applied Mechanics and Materials, 446, 992 (2014). 10. M.K. Hati, T.K. Bhattacharyya, IEEE Computer Society Annual Symposium on VLSI, (2011) 11. Z. Hao, F. Xiangning, S. Yutao, International Conference on Wireless Communications and Signal Processing (WCSP2011), (2011). 12. S.A.E. Ab Rahim, I.M. Azmi, International Symposium on Integrated Circuit, (2011). 9. M.F. Ahmad, S.A.Z. Murad, M.M. Shahimim, S.A.A. Rais, A.F. Hasan, Applied Mechanics and Materials, 446, 992 (2014). 7. W. Gu, W. Gao, World Acad. Sci Eng. Technol., 67, 284 (2012). 8. I.S. Ishak, S.A.Z. Murad, M.F. Ahmad, S.C. Neoh, Proceedings of Malaysian Technical Universities Conference on Engineering and Technology (MUCET), (2013) 10. M.K. Hati, T.K. Bhattacharyya, IEEE Computer Society Annual Symposium on VLSI, (2011) References 1. Razavi, B. Design of Analog CMOS Integrated Circuits, (McGraw-Hill Higher Education, Singapore, 2001). 1. Razavi, B. Design of Analog CMOS Integrated Circuits, (McGraw-Hill Higher Education, Singapore, 2001). g p ) 2. Nageshwarrao, D., Venkata Chalam, S. Malleswara Rao V.,International Journal Electronic Engineering Research, 2, 159 (2010). 3. S. Kant, O.P. Sahu, International Journal of Engineering Research & Technology, 1 (2012). ( ) 4. I.S. Ishak, S.A.Z. Murad, M.F. Ahmad, J. Eng. Appl. Sci., 11, 20 (2016). 5. S.A.E. Ab Rahim, M.A. Ismail, A.I. Rahim, M.R. Yahya, A.F.A. Mat, International Conference on Electronic Devices, Systems and Applications, (2010) 6. X. Liu, J.F. Mcdonald J.F., International Conference on IEE, (2012). 7 7 7 DOI: 10.1051/ 79701046 matecconf/201 , 01046 (2017) 97 MATEC Web of Conferences ETIC 2016 ( ) 11. Z. Hao, F. Xiangning, S. Yutao, International Conference on Wireless Communications and Signal Processing (WCSP2011), (2011). 8 8
https://openalex.org/W2990247177	https://nv.nltu.edu.ua/index.php/journal/article/view/2042/2096	Ukrainian	null	Біолого-екологічні особливості поширення омели білої (Viscum album) в умовах міста Вінниця	Naukovij vìsnik NLTU Ukraïni	2,019	cc-by	3,254	М. В. Матусяк у Вінницький національний аграрний університет, м. Вінниця, Україна Науковий вісник НЛТУ України, 2019, т. 29, № 8 Scientific Bulletin of UNFU, 2019, vol. 29, no 8 66 Інформація про авторів: Матусяк Михайло Васильович, канд. с.-г. наук, ст. викладач, кафедра садово-паркового господарства, садівництва та виноградарства. Email: mikhailo1988@gmail.com; https://orcid.org/0000-0001-8099-7290 Цитування за ДСТУ: Матусяк М. В. Біолого-екологічні особливості поширення омели білої (Viscum album) в умовах міста Вінниця. Науковий вісник НЛТУ України. 2019, т. 29, № 8. С. 66–69. Citation APA: Matusiak, M. V. (2019). Biological and environmental characteristics of distribution of Viscum album in the conditions of Vinnytsa. Scientific Bulletin of UNFU, 29(8), 66–69. https://doi.org/10.36930/40290810 БІОЛОГО-ЕКОЛОГІЧНІ ОСОБЛИВОСТІ ПОШИРЕННЯ ОМЕЛИ БІЛОЇ (VISCUM ALBUM) В УМОВАХ МІСТА ВІННИЦЯ У межах населених пунктів, зокрема й у Вінниці, омела біла набула інтенсивного поширення. Ураження дерев омелою призводить як до погіршення стану дерев, їх подальшого всихання, так і до зниження естетичної оцінки насаджень. Незва- жаючи на інтенсивне пошкодження деревних насаджень омелою у Вінниці, достатньо стійкими виявилися дерева більшості хвойних видів, зокрема: сосна, ялина, дугласія. Із листяних видів достатньо високою стійкістю відрізняється дуб, граб, бук. Незначною мірою пошкоджується омелою каштан, дуб червоний, горіх та ін. Поряд із цим, більшість алейних насаджень, зокрема тополі та липи, істотно пошкоджені цим напівпаразитом. Це зумовлено не лише особливістю деревних порід, але й і їхнім віком. Зокрема, більшість дерев старшого віку із наявними пошкодженнями стовбурової частини та гілок інтенсивні- ше уражаються омелою. За результатами проведених досліджень виявлено, що вік дерев, які найбільш уражені омелою бі- лою, становить 50-60 років і з віком ступінь ураження зростає, а частка ураження дерев віком 70-80 років становить 25- 29 %. Ступінь ураження дерев омелою білою визначено за 5-бальною шкалою, згідно з якою найвищий бал ураження вияв- лено у тополі чорної та дельтовидної (5 балів), найнижчий – у ялини звичайної, липи серцелистої (1-2 бали). Розглядаючи види Populus за інтенсивністю заселення їх Viscum album, встановлено, що низький і високий ступені ураження мають 26 % дерев, середній – 28 %, а дуже високий – 30 %. Найбільшою часткою дерев з високим і дуже високим ступенем ураження відзначаються P. nigra та P. deltoids – 22 і 32 %. Ключові слова: омела біла; стійкість насаджень; інтенсивність пошкодження; фітонциди; рослина-напівпара Ключові слова: омела біла; стійкість насаджень; інтенси Н. Ю. Таран та ін. (Vasylenko, Filipova & Fuchylo, 2013; Rybalka, 2016; Rumyankov, 2010; Taran et al., 2008; Ivchenko et al., 2014). Вступ. У межах населених пунктів, зокрема й у Він- ниці, омела біла набула інтенсивного поширення. Ура- ження дерев омелою призводить як до погіршення ста- ну дерев, їх подальшого всихання, так і до зниження ес- тетичної оцінки насаджень, особливо в осінньо-зимо- вий та у зимово-весняний періоди. Зростання інтенсив- ності ураження омелою вимагає застосування заходів щодо обмеження поширення цього напівпаразита. До основних заходів можна віднести: обрізування гілок і крон дерев; видалення окремих дерев і створення на їх місці насаджень, стійких до ураження; формування груп насаджень за участю хвойних порід, які менш пошкоджуються омелою (Rumyankov, 2010; Taran et al., 2008). Мета і завдання дослідження. Дослідити сучасний стан деревних насаджень Вінниці, вивчити особливості ураження омелою білою насаджень та її поширення в умовах міста. УДК 582.728.22-024.84(477.44) УДК 582.728.22-024.84(477.44) https://nv.nltu.edu.ua https://doi.org/10.36930/40290810 Article received 18.09.2019 р. Article accepted 31 10 2019 р M. V. Matusiak mikhailo1988@gmail.com Article received 18.09.2019 р. Article accepted 31.10.2019 р. БІОЛОГО-ЕКОЛОГІЧНІ ОСОБЛИВОСТІ ПОШИРЕННЯ ОМЕЛИ БІЛОЇ (VISCUM ALBUM) В УМОВАХ МІСТА ВІННИЦЯ № вид. № рос- лини Назва рослини Пошкодження рослини, бал 28 1 1 Fraxinus exceicior 3 28 1 2 Acer platanoides 4 28 1 3 Fraxinus exceicior "Pendula" 1 28 1 4 Tilia cordata 2 28 1 5 Acer platanoides 2 28 1 6 Tilia cordata 2 28 1 7 Acer platanoides 4 28 1 8 Fraxinus exceicior 2 28 1 9 Fraxinus exceicior 2 28 1 10 Acer platanoides 3 28 1 11 Fraxinus exceicior 2 28 1 12 Fraxinus exceicior 2 28 1 13 Fraxinus exceicior 2 28 1 14 Fraxinus exceicior 1 28 1 15 Fraxinus exceicior 1 28 2 16 Pіcea abies 1 28 2 17 Acer compestre 4 28 2 18 Aesculus hippocastanum 1 28 2 19 Populus alba 5 28 2 20 Acer platanoides 4 28 2 21 Acer platanoides 2 28 2 22 Acer platanoides 3 28 2 23 Populus Deltoids 5 28 2 24 Acer compestre 3 28 2 25 Populus nigra 5 28 2 26 Acer platanoides 3 28 2 27 Acer platanoides 3 Рис. 2. Розподіл загальної кількості дерев за віковими групами (у %) Табл. 1. Перелік видів дерев, обстежених Табл. 1. Перелік видів дерев, обстежених Табл. 1. Перелік видів дерев, обстежених на предмет ураження омелою білою № з/п Українська назва рослин Латинська назва рослин 1 Клен гостролистий Acer platanoides 2 Клен польовий Acer campestre 3 Тополя біла Populus alba 4 Осика Populus tremula 5 Верба біла Salix alba 6 Липа серцелиста Tilia cordata 7 Тополя чорна Populus nigra 8 Ясен звичайний Fraxinus excelsior Рис. 1. Розподіл обстежуваних дерев на предмет пошкодження омелою р р на предмет ураження омелою білою № з/п Українська назва рослин Латинська назва рослин 1 Клен гостролистий Acer platanoides 2 Клен польовий Acer campestre 3 Тополя біла Populus alba 4 Осика Populus tremula 5 Верба біла Salix alba 6 Липа серцелиста Tilia cordata 7 Тополя чорна Populus nigra 8 Ясен звичайний Fraxinus excelsior на предмет ураження омелою білою Табл. 2. Ступінь пошкодження дерев Табл. 2. Ступінь пошкодження дерев Табл. 2. Ступінь пошкодження дерев Центрального парку Вінниці омелою білою № кв. № вид. БІОЛОГО-ЕКОЛОГІЧНІ ОСОБЛИВОСТІ ПОШИРЕННЯ ОМЕЛИ БІЛОЇ (VISCUM ALBUM) В УМОВАХ МІСТА ВІННИЦЯ Вивчити біолого-екологічні особливості омели, оцінити вплив омели білої на санітарно-гігієніч- ний стан насаджень. Результати дослідження та їх обговорення. Viscum album L. – це вічнозелений кущ кулястої форми родини Loranthaceae, який має стійкі гаусторії у дереві-живите- лі. Рослина асимілює свій власний вуглець завдяки фо- тосинтезу, що зумовлює її зелене забарвлення, при цьому повністю залежить від водних і мінеральних ре- сурсів дерева, на якому оселяється (Vasylenko, Filipova & Fuchylo, 2013; Rybalka, 2016). Аналіз останніх досліджень і публікацій. Численні публікації у науковій та популярній літературі свідчать, що особливості поширення омели білої широко вивча- ють як вітчизняні, так і зарубіжні науковці. Велику ува- гу поширенню, вивченню екологічних та біологічних особливостей росту та розвитку омели білої приділили у своїх наукових працях І. Д. Василенко, Л. М. Філіпо- ва, Я. Д. Фучило, І. О. Рибалка, Ю. О. Рум'янков, Ми обстежили паркові насадження та алеї на пред- мет ураження дерев омелою білою. Всього обстежено близько 600 дерев різних видів, враховуючи хвойні та листяні породи. Дані щодо обстежених дерев наведено у табл. 1. Науковий вісник НЛТУ України, 2019, т. 29, № 8 Scientific Bulletin of U Науковий вісник НЛТУ України, 2019, т. 29, № 8 За даними рис. 1, більшість обстежених дерев, які мали той чи інший ступінь ураження омелою, – це де- рева тополі білої. Загальна частка дерев тополі станови- ла 58 %. Частка дерев інших видів була значно нижчою. Зокрема, обстежено близько 10 % дерев липи серцелис- тої, 7-8 % дерев клена гостролистого та верби білої, 5 % клена польового та інших видів (Shlapak et al., 2010; Hawksworth & Scharpf, 1986). джених дерев за віковими групами відображено на рис. 2. За даними рис. 2, більшість дерев, які пошкоджені омелою, мають вік 50-60 років та більше. У дерев віком до 30 років пошкодження омелою є мінімальним. Почи- наючи із віку 50-60 років, інтенсивність ураження оме- лою значно зростає. Частка дерев 60-80-річного віку, уражених омелою, становить 25-29 %. З огляду на це можна зазначити те, що у старшому віці стійкість дерев до ураження значно знижується, що залежить від дерев- ної породи, особливостей її росту та розвитку та інших чинників, які пов'язані із біолого-екологічними особли- востями дерев та умовами середовища. можна зазначити те, що у старшому віці стійкість дерев до ураження значно знижується, що залежить від дерев- ної породи, особливостей її росту та розвитку та інших чинників, які пов'язані із біолого-екологічними особли- востями дерев та умовами середовища. Табл. 2. Ступінь пошкодження дерев Центрального парку Вінниці омелою білою № кв. Науковий вісник НЛТУ України, 2019, т. 29, № 8 Scientific Bulletin of UNFU, 2019, vol. 29, no 8 67 БІОЛОГО-ЕКОЛОГІЧНІ ОСОБЛИВОСТІ ПОШИРЕННЯ ОМЕЛИ БІЛОЇ (VISCUM ALBUM) В УМОВАХ МІСТА ВІННИЦЯ 29, № 8 Обстежено дерева тополі вздовж основних вулиць Вінниці, пошкоджених омелою за стадіями розвитку та стадіями пошкодження. Для оцінювання ступеня пош- кодження дерев омелою нами використано відповідну градацію дерев. Дані щодо класифікації дерев за ступе- нем пошкодження наведено у табл. 3. Табл. 3. Оцінювання ступеня ураження дерев омелою білою насаджень тополі Ступінь ураження/"кущів" омели на 1-му дереві (шт.) низький середній високий дуже високий Варі- ант категорія кількість категорія кількість категорія кількість категорія кількість 1 перша до 25 - - - - - - 2 перша друга разом 7-8 6-7 13-15 перша друга 9-15 7-15 16-30 - - - - - - - - - - - - 3 перша друга третя разом 5-6 4 4-5 13-15 перша друга тертя 6-10 5-10 5-10 16-30 перша друга третя 11-25 11-30 11-20 33-75 перша друга третя 26-70 31-90 21-60 78-220 4 друга третя разом 6-7 7-8 13-15 друга третя 10-16 6-14 16-30 друга третя 17-40 15-35 32-75 друга третя 41-70 36-100 77-170 5 перша третя разом 7-8 6-7 13-15 перша третя 8-15 8-15 16-30 перша третя 16-40 16-35 32-75 перша третя 41-100 36-60 77-160 Примітка: категорія за діаметром "кущів" омели (см): І – до 30, ІІ – 31-30, ІІІ – 61-90. 3. Здебільшого бал пошкодження становить 3-5 бали. При цьому уражаються верхні частини крон, гілки 2-5-річ- ної давності. Скелетні гілки, а також пристовбурова частина, пошкоджуються незначною мірою. Зазначимо, що пошкодження скелетних гілок і стов- бурів трапляється досить рідко. Це зумовлено особли- вістю росту омели (насамперед вона надає перевагу мо- лодим гілкам, а в міру розростання на пошкодженому дереві, лише надалі пошкоджує його скелетні гілки і стовбур). Розглядаючи види Populus за інтенсивністю заселення їх Viscum album, відзначимо, що низький і ви- сокий ступені ураження мають 26 % дерев, середній – 28 %, а дуже високий – 30 %. Найбільшою часткою де- рев з високим і дуже високим ступенем ураження від- значаються P. nigra та P. deltoids – 22 і 32 %. Неушко- дженість дерев P. pyramidalis, очевидно, спричинена тим, що птахам, які розповсюджують омелу, досить незручно сідати на гілки, що розташовані вертикально. Це ж стосується і P. bolleana. У Р. alba, P. tremula, P. balsamifera і P. laurifolia низький ступінь заселення де- рев напівпаразитом, на нашу думку, що збігається з висновками деяких інших дослідників, зумовлений тим, що ці тополі виділяють фітонциди, які відлякують пер- натих. БІОЛОГО-ЕКОЛОГІЧНІ ОСОБЛИВОСТІ ПОШИРЕННЯ ОМЕЛИ БІЛОЇ (VISCUM ALBUM) В УМОВАХ МІСТА ВІННИЦЯ № рос- лини Назва рослини Пошкодження рослини, бал 28 1 1 Fraxinus exceicior 3 28 1 2 Acer platanoides 4 28 1 3 Fraxinus exceicior "Pendula" 1 28 1 4 Tilia cordata 2 28 1 5 Acer platanoides 2 28 1 6 Tilia cordata 2 28 1 7 Acer platanoides 4 28 1 8 Fraxinus exceicior 2 28 1 9 Fraxinus exceicior 2 28 1 10 Acer platanoides 3 28 1 11 Fraxinus exceicior 2 28 1 12 Fraxinus exceicior 2 28 1 13 Fraxinus exceicior 2 28 1 14 Fraxinus exceicior 1 28 1 15 Fraxinus exceicior 1 28 2 16 Pіcea abies 1 28 2 17 Acer compestre 4 28 2 18 Aesculus hippocastanum 1 28 2 19 Populus alba 5 28 2 20 Acer platanoides 4 28 2 21 Acer platanoides 2 28 2 22 Acer platanoides 3 28 2 23 Populus Deltoids 5 28 2 24 Acer compestre 3 28 2 25 Populus nigra 5 28 2 26 Acer platanoides 3 28 2 27 Acer platanoides 3 Табл. 2. Ступінь пошкодження дерев Центрального парку Вінниці омелою білою у д д р Центрального парку Вінниці омелою білою Рис. 1. Розподіл обстежуваних дерев на предмет пошкодження омелою Для кожного дерева встановлювали бал інтенсив- ності пошкодження крони. При цьому використовували шкалу, яка враховує ступінь ураження від 1 до 5. Мак- симальний бал ураження крони відображався балом 5, мінімальний – 1. У табл. 2 наведено дані стосовно ін- тенсивності пошкодження дерев у Центральному місь- кому парку Вінниці. За наведеними даними більшість дерев має середній та незначний бал пошкодження кро- ни омелою. Поряд із цим є достатня кількість дерев, які істотно уражені омелою. Рис. 2. Розподіл загальної кількості дерев за віковими групами (у %) За даними табл. 2, найбільшого ураження у межах Центрального міського парку зазнають такі види дерев, як тополя чорна та дельтовидна, ясен звичайний, клен гостролистий, клен-явір, клен польовий. Ці види знач- ною мірою пошкоджуються омелою. Здебільшого бал пошкодження становить 3-5 бали. При цьому уража- ються верхні частини крон, гілки 2-5-річної давності. Скелетні гілки, а також пристовбурова частина, пошко- джуються незначною мірою. Здійснено групування дерев за віковими групами. Таке групування важливе з огляду на оцінювання впли- ву омели на різні вікові групи дерев. Розподіл пошко- Рис. 2. Розподіл загальної кількості дерев за віковими групами (у %) Scientific Bulletin of UNFU, 2019, vol. 29, no 8 67 Науковий вісник НЛТУ України, 2019, т. БІОЛОГО-ЕКОЛОГІЧНІ ОСОБЛИВОСТІ ПОШИРЕННЯ ОМЕЛИ БІЛОЇ (VISCUM ALBUM) В УМОВАХ МІСТА ВІННИЦЯ Чорна та дельтовидна тополі з розлогою кроною і розгалуженою системою гілок різних порядків є найпривабливішими для птахів, а отже, і для появи на їхніх деревах омели. 4. Більшість дерев, які пошкоджені омелою, мають вік 50- 60 років та більше. У дерев віком до 30 років пошко- дження омелою є мінімальним. Починаючи із віку 50- 60 років, інтенсивність ураження омелою значно зрос- тає. Частка дерев 60-80-річного віку, уражених оме- лою, становить 25-29 %. З огляду на це можна зазначи- ти, що у старшому віці стійкість дерев до ураження значно знижується, це залежить від деревної породи, особливостей її росту та розвитку та інших чинників, які пов'язані із біолого-екологічними особливостями дерев та умовами середовища. 4. Більшість дерев, які пошкоджені омелою, мають вік 50- 60 років та більше. У дерев віком до 30 років пошко- дження омелою є мінімальним. Починаючи із віку 50- 60 років, інтенсивність ураження омелою значно зрос- тає. Частка дерев 60-80-річного віку, уражених оме- лою, становить 25-29 %. З огляду на це можна зазначи- ти, що у старшому віці стійкість дерев до ураження значно знижується, це залежить від деревної породи, особливостей її росту та розвитку та інших чинників, які пов'язані із біолого-екологічними особливостями дерев та умовами середовища. Перелік використаних джерел Hawksworth, F. G., & Scharpf, R. F. (1986). Spread of European mistletoe (Viscum album) in California, U.S.A. European Journal of Plant Pathology, 16, 1–5. Ivchenko, A. I., Bozhok, O. P., Paczura, I. M., Kolyada, L. B., Boz- hok, V. O., & Ivchenko, A. I. (2014). Features of the organization of effective struggle against mistletoe. Scientific Bulletin of UNFU, 24(5), 12–18. [In Ukrainian]. Rumyankov, Y. O. (2010). Degree of damage to Viscum album L. species of the genus Celtis L. in the plantations of the National Dendrological Park "Sofiyivka" of NAS of Ukraine. Indigenous and introduced plants, 6, 42–45. [In Ukrainian]. Науковий вісник НЛТУ України, 2019, т. 29, № 8 68 Scientific Bulletin of UNFU, 2019, vol. 29, no 8 BIOLOGICAL AND ENVIRONMENTAL CHARACTERISTICS OF DISTRIBUTION OF VISCUM ALBUM IN THE CONDITIONS OF VINNYTSA Within the settlements located in Vinnytsia, European mistletoe has gained intense influence. Tree damage by Viscum album le- ads to both the deterioration of the trees, their further drying and the reduction of aesthetic evaluation of the plantations. Despite the intensive damage of tree plantations in Vinnytsya caused by Viscum album, the trees of most coniferous species including pine, spru- ce and douglasia, appeared to be quite stable. Oak, hornbeam and beech are distinguished from deciduous species by high resistance. Chestnut, red oak, walnut and other deciduous species are found to be less damaged. At the same time, most of the alley cropping, in particular poplar and linden trees are largely damaged by this hemiparasite plant. This is due not only to the peculiarity of the tree species, but also their age. In particular, most of older trees with existing damage to the trunk and branches are more severely affec- ted by mistletoe. As a result of our research we have found that the age of the trees mostly affected by Viscum album is 50-60 years and with age the degree of damage increases, as well as the proportion of damage to trees aged 70-80 years is 25-29 %. The degree of damage to trees with European mistletoe is determined by a 5-point scale, according to which the highest score of damage is found in black poplar and eastern cottonwood stands (5 points), the lowest score is found in European spruce and small-leaved linden stands (1-2 points). Populus species are considered to have low and high level of damage by Viscum album (26 % of trees), average (28 % of trees), and a very high (30 % of trees) respectively. P. nigra and P. deltoids account for the highest proportion of trees with high and very high levels of damage, 22 % and 32 %, respectively. The intactness of P. pyramidalis trees is apparently caused by the fact that the mistletoe birds find it uncomfortable to sit on vertically arranged branches. The same applies to P. bolleana. In P. alba, P. tremula, P. balsamifera, and P. laurifolia, the low population density of trees by hemiparasite plant is consistent with the findings of some other researchers, due to the fact that these poplar trees secrete volatiles that deter birds. Scientific Bulletin of UNFU, 2019, vol. 29, no 8 69 Науковий вісник НЛТУ України, 2019, т. 29, № 8 Науковий вісник НЛТУ України, 2019, т. 29, № 8 Scientific Bulletin of UNFU, 2019, vol. 29, no 8 69 Keywords: European mistletoe; Viscum album; planting resistance; damage intensity; volatile matter; hemiparasite plant. Науковий вісник НЛТУ України, 2019, т. 29, № 8 Scien Висновки: 1. Загальне обстеження зелених насаджень Вінниці пока- зало, що найбільш інтенсивним пошкодженням харак- теризуються деревостани Центрального міського пар- ку. Менш інтенсивне пошкодження характерне для на- саджень парку "Дружби народів" та Ботанічного саду "ВНАУ". Інтенсивним пошкодженням відрізняються алейні посадки за переважанням тополі білої та чорної. Rybalka, I. O. (2016). Viscum album L. in the urban landscape: a ret- rospective study of population changes in the future. Scientific ba- ses of biodiversity conservation, 7(14), 1, 211–228. [In Ukrainian]. Shlapak, V. P., Muzyka, G. I., Sobchenko, V. F., Vitenko, V. A., Mar- no, L. I., & Pasichnyj, O. P. (2010). Features of determining the degree of damage to the Viscum album of plantations in the histori- cal part of the Sofiyivka Arboretum. Scientific Bulletin of UNFU, 20(7), 8–14. [In Ukrainian]. 2. Дерева тополі значною мірою пошкоджуються омелою білою. Із обстежених дерев тополі близько половини була уражена омелою. Загальна частка дерев тополі становила 58 %. Частка дерев інших видів була значно нижчою. Зокрема, виявлено близько 10 % пошкодже- них дерев липи серцелистої, 7-8 % дерев клена гостро- листого та верби білої, 5 % клена польового та інших видів. Taran, N. Y., Svyetlova, N. B., Baczmanova, L. M., Ulynecz, V. Z., & Ganchurin, V. V. (2008). Biologiya rozvytku Viscum album L. ta ekologichnyj monitoryng yiyi poshyrennya v lisoparkovyx bioce- nozax. Ukrainian Botanical Journal, 2, 242–251. [In Ukrainian]. Vasylenko, I. D., Filipova, L. M., & Fuchylo, Y. D. (2013). Fighting mistletoe on poplar trees in the green zone of White Church. Scien- tific Bulletin of UNFU, 23(12), 31–38. [In Ukrainian]. Науковий вісник НЛТУ України, 2019, т. 29, № 8 68 Scientific Bulletin of UNFU, 2019, vol. 29, no 8 Scientific Bulletin of UNFU, 2019, vol. 29, no 8 M. V. Matusiak Vinnytsia National Agrarian University, Vinnytsia, Ukraine BIOLOGICAL AND ENVIRONMENTAL CHARACTERISTICS OF DISTRIBUTION OF VISCUM ALBUM IN THE CONDITIONS OF VINNYTSA Black poplar and eastern cottonwood trees with a spreading crown and a spreading branch system of different orders are most attractive to birds, and therefore to the appe- arance of mistletoe on their trees. Keywords: European mistletoe; Viscum album; planting resistance; damage intensity; volatile matter; hemiparasite plant.
https://openalex.org/W4361936211	https://figshare.com/articles/journal_contribution/Supplementary_Table_2_from_Correlative_Analyses_of_the_SARC028_Trial_Reveal_an_Association_Between_Sarcoma-Associated_Immune_Infiltrate_and_Response_to_Pembrolizumab/22473812/1/files/39925298.pdf	English	null	Supplementary Table 2 from Correlative Analyses of the SARC028 Trial Reveal an Association Between Sarcoma-Associated Immune Infiltrate and Response to Pembrolizumab	null	2,023	cc-by	242	Supplemental Table 2. SARC028 tumor biopsies (that passed QC) obtained at baseline and after 8 weeks treatment and associated patient response to pembrolizumab Supplemental Table 2. SARC028 tumor biopsies (that passed QC) obtained at baseline and after 8 weeks treatment and associated patient response to pembrolizumab passed QC) obtained at baseline and after 8 weeks treatment and associated patient Supplemental Table 2. SARC028 tumor biopsies (that passed QC) obtained at baseline and after 8 weeks treatment and associated patient response to pembrolizumab Baseline biopsies On-treatment biopsies Histology of biopsied tumor CR PR SD PD CR PR SD PD Soft tissue sarcoma (n=31 baseline, 35 on-treatment) Leiomyosarcoma (n=9 baseline, 15 on-treatment) 0 0 5 4 0 0 9 6 Undifferentiated pleomorphic sarcoma (n= 8 baseline, 3 on-treatment) 1 3 2 2 0 0 1 2 Dedifferentiated liposarcoma (n= 8 baseline, 10 on-treatment) 0 2 2 4 0 2 6 2 Synovial sarcoma (n= 6 baseline, 7 on-treatment) 0 0 2 4 0 1 2 4 Bone sarcoma (n= 35 baseline, 36 on-treatment) Chondrosarcoma (n= 5 baseline, 5 on-treatment) 0 1 1 3 0 1 1 3 Ewing’s sarcoma (n= 13 baseline, 10 on-treatment) 0 0 2 11 0 0 2 8 Osteosarcoma (n= 17 baseline, 21 on-treatment) 0 1 4 12 0 1 9 11 CR, complete response; PD, progressive disease; PR, partial response; QC, quality control; SD, stable disease se; PD, progressive disease; PR, partial response; QC, quality control; SD, stable disease
https://openalex.org/W3016783226	https://cris.unibo.it/bitstream/11585/758446/1/Articolo_2020_IRISok_Zhu_Metabolites_GiraffeUrine.pdf	English	null	First Insights into the Urinary Metabolome of Captive Giraffes by Proton Nuclear Magnetic Resonance Spectroscopy	Metabolites	2,020	cc-by	9,583	Received: 13 March 2020; Accepted: 15 April 2020; Published: 17 April 2020 Abstract: The urine from 35 giraﬀes was studied by untargeted 1H-NMR, with the purpose of obtaining, for the ﬁrst time, a ﬁngerprint of its metabolome. The metabolome, as downstream of the transcriptome and proteome, has been considered as the most representative approach to monitor the relationships between animal physiological features and environment. Thirty-nine molecules were unambiguously quantiﬁed, able to give information about diet, proteins digestion, energy generation, and gut-microbial co-metabolism. The samples collected allowed study of the eﬀects of age and sex on the giraﬀe urinary metabolome. In addition, preliminary information about how sampling procedure and pregnancy could aﬀect a giraﬀe’s urinary metabolome was obtained. Such work could trigger the setting up of methods to non-invasively study the health status of giraﬀes, which is utterly needed, considering that anesthetic-related complications make their immobilization a very risky practice. Keywords: captive giraﬀes; urine; metabolomics; 1H-NMR Received: 13 March 2020; Accepted: 15 April 2020; Published: 17 April 2020 metabolites H OH OH metabolites H OH OH metabolites H OH OH metabolites H OH OH metabolites Metabolites 2020, 10, 157; doi:10.3390/metabo10040157 Article First Insights into the Urinary Metabolome of Captive Giraﬀes by Proton Nuclear Magnetic Resonance Spectroscopy Chenglin Zhu 1,† , Sabrina Fasoli 2,†, Gloria Isani 2 and Luca Laghi 1,* 1 Department of Agro-Food Science and Technology, University of Bologna, 47521 Cesena, Italy; chenglin.zhu2@unibo.it 2 Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, 40064 Bologna, Italy; sabrina.fasoli2@unibo.it (S.F.); gloria.isani@unibo.it (G.I.) * Correspondence: l.laghi@unibo.it; Tel.: +39-0547-338105 † These authors contributed equally to this work. 1 Department of Agro-Food Science and Technology, University of Bologna, 47521 Cesena, Italy; chenglin.zhu2@unibo.it 2 Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, 40064 Bologna, Italy; sabrina.fasoli2@unibo.it (S.F.); gloria.isani@unibo.it (G.I.) * Correspondence: l.laghi@unibo.it; Tel.: +39-0547-338105 † These authors contributed equally to this work. 1 Department of Agro-Food Science and Technology, University of Bologna, 47521 Cesena, Italy; chenglin.zhu2@unibo.it 2 Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia, 40064 Bologna, Ita sabrina.fasoli2@unibo.it (S.F.); gloria.isani@unibo.it (G.I.) † These authors contributed equally to this work. 1. Introduction According to the International Union for Conservation of Nature (IUCN), giraﬀe (Giraﬀa camelopardalis) is declared a vulnerable species [1]. Moreover, diﬀerent measures have been taken to monitor and protect giraﬀe population. For example, the International Union for Conservation of Nature (IUCN) Species Survival Commission (SSC) Giraﬀe and Okapi Specialist Group (GOSG) was established with the aim of studying and guaranteeing the conservation needs of this species (https://www.giraﬃdsg.org/). In addition, from November 26, 2019, giraﬀes are included in Appendix II of the CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) to improve its protection, subjecting it to strict regulation (https://www.cites.org/). Zoos represent a signiﬁcant part of the protection strategy for giraﬀes, with projects explicitly aimed at protecting endangered species and pursuing high standards of animal welfare [2]. In these structures, however, giraﬀes may be subjected to sources of stress that reverberate negatively on individual and social behaviors [3]. Causes of stress could be represented by the presence of visitors and attendants [4]. Among the eﬀorts that have been made to reduce the stressors, some are devoted to developing protocols to evaluate their general health status that do not involve immobilization, but are based on indirect methods [3]. In fact, giraﬀes are particularly prone to anesthetic-related complications and death, due to their unique cardiovascular system, making immobilization a risky practice [5,6]. Metabolites 2020, 10, 157; doi:10.3390/metabo10040157 www.mdpi.com/journal/metabolites www.mdpi.com/journal/metabolites 2 of 13 a risky Metabolites 2020, 10, 157 complications and d The possibility of obtaining information from urine collected from the ground seems particularly attractive from this point of view, but the literature on this type of sampling is absent for giraﬀes and it has been only reported in okapi [7]. Indeed giraﬀes have been studied more for their iconic height and the mechanisms existing at the cardiovascular level to counterbalance the consequent state of primary hypertension [8–10]. p [ , ] The possibility of obtaining information from urine collected from the ground seems particularly attractive from this point of view, but the literature on this type of sampling is absent for giraffes and it has been only reported in okapi [7]. Indeed giraffes have been studied more for their iconic height and the mechanisms existing at the cardiovascular level to counterbalance the consequent state of primary hypertension [8–10]. 1. Introduction Among the completely unexplored characteristics of giraﬀe urine is its metabolome, the ensemble of low weight molecules produced by the cellular metabolism. Studies carried out by liquid chromatography–mass spectrometry (LC/MS) or by proton nuclear magnetic resonance spectroscopy (1H-NMR) on humans and other animals suggest that the giraﬀe’s urinary metabolome may be particularly informative about the general health of the animal. In horse urine, molecules revealing the action of the intestinal microbiota were found in micromolar concentrations [11,12]. Molecular patterns of the urinary metabolome linked to inﬂammatory processes have been identiﬁed in humans [13]. Urinary proﬁle responses to the calorie content of the diet were identiﬁed in rat [14]. The eﬀects of heat stress were studied in cattle by metabolomic proﬁling of urine [15]. Indeed, the use of urine as a source of biological data in giraﬀes could be a suitable alternative, due to its non-invasive approach that could avoid the immobilization of animals. q p y yp [ ] Among the completely unexplored characteristics of giraffe urine is its metabolome, the ensemble of low weight molecules produced by the cellular metabolism. Studies carried out by liquid chromatography–mass spectrometry (LC/MS) or by proton nuclear magnetic resonance spectroscopy (1H-NMR) on humans and other animals suggest that the giraffe's urinary metabolome may be particularly informative about the general health of the animal. In horse urine, molecules revealing the action of the intestinal microbiota were found in micromolar concentrations [11,12]. Molecular patterns of the urinary metabolome linked to inflammatory processes have been identified in humans [13]. Urinary profile responses to the calorie content of the diet were identified in rat [14]. The effects of heat stress were studied in cattle by metabolomic profiling of urine [15]. Indeed, the use of urine as a source of biological data in giraffes could be a suitable alternative, due to its non-invasive approach that could avoid the immobilization of animals. Among the analytical platforms capable of fulﬁlling the requirements, proton nuclear magnetic resonance spectroscopy (1H-NMR) has been widely used for the investigation of urine metabolomes, taking advantage of its high reproducibility and minimal sample preparation. pp Among the analytical platforms capable of fulfilling the requirements, proton nuclear magnetic resonance spectroscopy (1H-NMR) has been widely used for the investigation of urine metabolomes, taking advantage of its high reproducibility and minimal sample preparation. 1. Introduction In the present study, we wanted to verify the feasibility of 1H-NMR based metabolomic studies focusing on the urine of giraﬀes. For this purpose, we characterized the molecular proﬁle of healthy giraﬀes held in captivity to obtain preliminary quantitative values that could be applied for the diagnosis of diseases aﬀecting this animal. Moreover, the samples collected gave the opportunity to have a ﬁrst insight about the inﬂuence of important physiological factors, such as the sex and age of the subjects, on the urinary metabolomic proﬁle. In the present study, we wanted to verify the feasibility of 1H-NMR based metabolomic studies focusing on the urine of giraffes. For this purpose, we characterized the molecular profile of healthy giraffes held in captivity to obtain preliminary quantitative values that could be applied for the diagnosis of diseases affecting this animal. Moreover, the samples collected gave the opportunity to have a first insight about the influence of important physiological factors, such as the sex and age of the subjects, on the urinary metabolomic profile. 2.2. Eﬀects of Sampling Procedure and Location 2.2. Effects of Sampling Procedure and Location Representative sections of two spectra obtained from analyzing urine from the same giraffe (Ronny), collected directly (blue line) and from the ground (red line) during one urination, Figure 2. Representative sections of two spectra obtained from analyzing urine from the same giraﬀe (Ronny), collected directly (blue line) and from the ground (red line) during one urination, respectively. respectively. To obtain hints about the potential effects of location on the metabolome of giraffe urine, we selected the samples from the locations BG (Parco Faunistico Le Cornelle) and FA (Zoosafari Fasanolandia) , where most of the samples had been collected, and we set up a three-way ANOVA analysis aiming at excluding any effect related to gender or age. None of the molecules quantified d i ifi tl diff t i l ti t thi i bl t id d i th To obtain hints about the potential eﬀects of location on the metabolome of giraﬀe urine, we selected the samples from the locations BG (Parco Faunistico Le Cornelle) and FA (Zoosafari Fasanolandia), where most of the samples had been collected, and we set up a three-way ANOVA analysis aiming at excluding any eﬀect related to gender or age. None of the molecules quantiﬁed appeared as signiﬁcantly diﬀerent in relation to zoo, so this variable was not considered in the subsequent analyses. respectively. To obtain hints about the potential effects of location on the metabolome of giraffe urine, we selected the samples from the locations BG (Parco Faunistico Le Cornelle) and FA (Zoosafari Fasanolandia) , where most of the samples had been collected, and we set up a three-way ANOVA analysis aiming at excluding any effect related to gender or age. None of the molecules quantified To obtain hints about the potential eﬀects of location on the metabolome of giraﬀe urine, we selected the samples from the locations BG (Parco Faunistico Le Cornelle) and FA (Zoosafari Fasanolandia), where most of the samples had been collected, and we set up a three-way ANOVA analysis aiming at excluding any eﬀect related to gender or age. None of the molecules quantiﬁed appeared as signiﬁcantly diﬀerent in relation to zoo, so this variable was not considered in the subsequent analyses. 2.1. Urinary Metabolites Identiﬁcation by Untargeted 1H-NMR 2.1. Urinary Metabolites Identification by Untargeted 1H-NMR A representative spectrum of the metabolites identiﬁed in the giraﬀe’s urine is reported in Figure 1. In this study, we identiﬁed 39 molecules (Table S1). These molecules mainly pertain to the classes of amino acids and derivatives and organic acids and derivatives. Hippurate (30.63%), creatinine (25.17%), and phenylacetylglycine (12.64%) were the most represented metabolites. A representative spectrum of the metabolites identified in the giraffe’s urine is reported in Figure 1. In this study, we identified 39 molecules (Table S1). These molecules mainly pertain to the classes of amino acids and derivatives and organic acids and derivatives. Hippurate (30.63%), creatinine (25.17%), and phenylacetylglycine (12.64%) were the most represented metabolites. Figure 1. Portions of 1H-NMR spectra, representative of all the spectra obtained in this study. Each molecule’s name appears over the NMR peak used for its quantification. To ease the visual Figure 1. Portions of 1H-NMR spectra, representative of all the spectra obtained in this study. Each molecule’s name appears over the NMR peak used for its quantiﬁcation. To ease the visual inspection of each portion, a diﬀerent spectrum with a convenient signal-to-noise ratio has been selected. Figure 1. Portions of 1H-NMR spectra, representative of all the spectra obtained in this study. Each molecule’s name appears over the NMR peak used for its quantification. To ease the visual Figure 1. Portions of 1H-NMR spectra, representative of all the spectra obtained in this study. Each molecule’s name appears over the NMR peak used for its quantiﬁcation. To ease the visual inspection of each portion, a diﬀerent spectrum with a convenient signal-to-noise ratio has been selected. 3 of 13 en Metabolites 2020, 10, 157 inspection of eac 2.2. Eﬀects of Sampling Procedure and Location 2.2. Effects of Sampling Procedure and Location 2.2. Eﬀects of Sampling Procedure and Location 2.2. Effects of Sampling Procedure and Location To check the potential inﬂuence of the diﬀerent sampling methods, we wanted to collect pairs of samples during the same voiding, one directly and one from the ground. Unfortunately, we only succeeded in this task for one individual (Ronny). Among 39 quantiﬁed compounds, four molecules showed a variation of concentration higher than 50%, namely p-cresol sulfate, citrate, glycine, and benzoate. 1H-NMR signals for these compounds are reported in Figure 2. In detail, benzoate and glycine were more concentrated in the urine collected from the ground, while citrate and p-cresol sulfate showed the opposite trend. Overall, the 39 molecules showed a median diﬀerence between the two samples of 4.8%. As these observations were based only on one pair of samples from a single individual, we decided not to exclude these molecules from the subsequent analyses. To check the potential influence of the different sampling methods, we wanted to collect pairs of samples during the same voiding, one directly and one from the ground. Unfortunately, we only succeeded in this task for one individual (Ronny). Among 39 quantified compounds, four molecules showed a variation of concentration higher than 50%, namely p-cresol sulfate, citrate, glycine, and benzoate. 1H-NMR signals for these compounds are reported in Figure 2. In detail, benzoate and glycine were more concentrated in the urine collected from the ground, while citrate and p-cresol sulfate showed the opposite trend. Overall, the 39 molecules showed a median difference between the two samples of 4.8%. As these observations were based only on one pair of samples from a single individual, we decided not to exclude these molecules from the subsequent analyses. Figure 2. Representative sections of two spectra obtained from analyzing urine from the same giraffe (Ronny), collected directly (blue line) and from the ground (red line) during one urination, Figure 2. Representative sections of two spectra obtained from analyzing urine from the same giraﬀe (Ronny), collected directly (blue line) and from the ground (red line) during one urination, respectively. Figure 2. Representative sections of two spectra obtained from analyzing urine from the same giraffe (Ronny), collected directly (blue line) and from the ground (red line) during one urination, Figure 2. Representative sections of two spectra obtained from analyzing urine from the same giraﬀe (Ronny), collected directly (blue line) and from the ground (red line) during one urination, respectively. Figure 2. appeared as significantly different in relation subsequent analyses. 2.3. Sex Aﬀects the Giraﬀe Urine Molecular Proﬁle rPCA model calculated on the concentration of the significantly different molecules between male and female giraffes. The scoreplot (A) represents with squares and circles females and males, respectively. The median of each sample group is represented by wide circles. The loading plot (B) reports the correlation between the importance of each substance over principal component 1 and its concentration. Gray bars highlight significant correlations (p < 0.05). Figure 3. rPCA model calculated on the concentration of the signiﬁcantly diﬀerent molecules between male and female giraﬀes. The scoreplot (A) represents with squares and circles females and males, respectively. The median of each sample group is represented by wide circles. The loading plot (B) reports the correlation between the importance of each substance over principal component 1 and its concentration. Gray bars highlight signiﬁcant correlations (p < 0.05). Three principal components (PCs) were accepted by the algorithm to depict the overall data features. PC 1, accounting for 59% of the variance thus represented, indeed significantly summarized the peculiarities connected to sex (p < 0.05), with female and male individuals appearing respectively at low and high PC scores. Among these molecules, hippurate, phenylacetylglycine, and thymine were more abundant in the urine of male individuals, while lactate, acetate, and succinate were more concentrated in the females’ urine. Three principal components (PCs) were accepted by the algorithm to depict the overall data features. PC 1, accounting for 59% of the variance thus represented, indeed signiﬁcantly summarized the peculiarities connected to sex (p < 0.05), with female and male individuals appearing respectively at low and high PC scores. Among these molecules, hippurate, phenylacetylglycine, and thymine were more abundant in the urine of male individuals, while lactate, acetate, and succinate were more concentrated in the females’ urine. appeared as significantly different in relation subsequent analyses. 2.3. Sex Aﬀects the Giraﬀe Urine Molecular Proﬁle To have an overall view of the data, a robust principal component analysis (rPCA) model was calculated on their concentration, as shown in Figure 3. y ( ) ( ) ↑ To have an overall view of the data, a robust principal component analysis (rPCA) model was calculated on their concentration, as shown in Figure 3. To have an overall view of the data, a robust principal component analysis (rPCA) model was calculated on their concentration, as shown in Figure 3. y ( ) ( ) To have an overall view of the data, a robust principal component analysis (rPCA) model was calculated on their concentration, as shown in Figure 3. Figure 3. rPCA model calculated on the concentration of the significantly different molecules between male and female giraffes. The scoreplot (A) represents with squares and circles females and males, respectively. The median of each sample group is represented by wide circles. The loading plot (B) reports the correlation between the importance of each substance over principal component 1 and its concentration. Gray bars highlight significant correlations (p < 0.05). Figure 3. rPCA model calculated on the concentration of the signiﬁcantly diﬀerent molecules between male and female giraﬀes. The scoreplot (A) represents with squares and circles females and males, respectively. The median of each sample group is represented by wide circles. The loading plot (B) reports the correlation between the importance of each substance over principal component 1 and its concentration. Gray bars highlight signiﬁcant correlations (p < 0.05). Figure 3. rPCA model calculated on the concentration of the significantly different molecules between male and female giraffes. The scoreplot (A) represents with squares and circles females and males, respectively. The median of each sample group is represented by wide circles. The loading plot (B) reports the correlation between the importance of each substance over principal component 1 and its concentration. Gray bars highlight significant correlations (p < 0.05). Figure 3. rPCA model calculated on the concentration of the signiﬁcantly diﬀerent molecules between male and female giraﬀes. The scoreplot (A) represents with squares and circles females and males, respectively. The median of each sample group is represented by wide circles. The loading plot (B) reports the correlation between the importance of each substance over principal component 1 and its concentration. Gray bars highlight signiﬁcant correlations (p < 0.05). Figure 3. appeared as significantly different in relation subsequent analyses. 2.3. Sex Aﬀects the Giraﬀe Urine Molecular Proﬁle q y 2.3. Sex Affects the Giraffe Urine Molecular Profile To obtain preliminary data on the effect of sex on the urinary metabolome, we focused on To obtain preliminary data on the eﬀect of sex on the urinary metabolome, we focused on samples collected from adult, non-pregnant individuals. Six molecules were found to be signiﬁcantly (p < 0.05) aﬀected by sex, as shown in Table 1. 4 of 13 Metabolites 2020, 10, 157 Table 1. Metabolite concentrations (mmol/L, median (IQR)) in the adult group were signiﬁcantly (p < 0.05) aﬀected by sex, as assessed by t-test. Table 1. Metabolite concentrations (mmol/L, median (IQR)) in the adult group were significantly (p < 0 05) affe ted by e a a e ed by t te t Females (6) Males (7) Trend p Value Acetate 2.04 (5.23 × 10−1) 1.33 (9.04 × 10−1) ↓ 0.034 Hippurate 13.50 (10.70) 19.30 (19.50) ↑ 0.047 Lactate 2.77 × 10−1 (8.90 × 10−2) 1.28 × 10−1 (7.35 × 10−2) ↓ 0.003 Phenylacetylglycine 7.82 (2.41) 15.20 (5.53) ↑ 0.014 Succinate 2.48 × 10−1 (3.00 × 10−2) 1.66 × 10−1 (8.80 × 10−2) ↓ 0.006 Thymine 1.77 × 10−1 (4.94 × 10−2) 2.86 × 10−1 (1.79 × 10−1) ↑ 0.043 To have an overall view of the data, a robust principal component analysis (rPCA) model was calculated on their concentration, as shown in Figure 3. 0.05) affected by sex, as assessed by t test. Females (6) Males (7) Trend P value Acetate 2.04 (5.23 × 10−1) 1.33 (9.04 × 10−1) ↓ 0.034 Hippurate 13.50 (10.70) 19.30 (19.50) ↑ 0.047 Lactate 2.77 × 10−1 (8.90 × 10−2) 1.28 × 10−1 (7.35 × 10−2) ↓ 0.003 Phenylacetylglycine 7.82 (2.41) 15.20 (5.53) ↑ 0.014 Succinate 2.48 × 10−1 (3.00 × 10−2) 1.66 × 10−1 (8.80 × 10−2) ↓ 0.006 Thymine 1.77 × 10−1 (4.94 × 10−2) 2.86 × 10−1 (1.79 × 10−1) ↑ 0.043 To have an overall view of the data, a robust principal component analysis (rPCA) model was l l t d th i t ti h i Fi 3 To have an overall view of the data, a robust principal component analysis (rPCA) model was calculated on their concentration, as shown in Figure 3. y ( ) ( ) ↑ To have an overall view of the data, a robust principal component analysis (rPCA) model was calculated on their concentration, as shown in Figure 3. 2.4. Effect of Age on the Urinary Metabolome 2.4. Eﬀect of Age on the Urinary Metabolome Age was found to significantly affect (p < 0.05) the concentration of three urinary metabolites, namely formate, alanine, and valerate, (Figure 4). To understand if their evolution was part of a trend spanning over the entire life of the giraffe, these molecules were used as a base for an rPCA model (Figure 5). Age was found to signiﬁcantly aﬀect (p < 0.05) the concentration of three urinary metabolites, namely formate, alanine, and valerate, (Figure 4). To understand if their evolution was part of a trend spanning over the entire life of the giraﬀe, these molecules were used as a base for an rPCA model (Figure 5). 5 of 13 5 of 13 Metabolites 2020, 10, 157 Metabolites 2020, 10, x FO Figure 4. Boxplots showing the concentration of molecules significantly (p < 0.05) affected by age, as assessed by two-way ANOVA followed by Tukey post-hoc test. Figure 4. Boxplots showing the concentration of molecules signiﬁcantly (p < 0.05) aﬀected by age, as assessed by two-way ANOVA followed by Tukey post-hoc test. Figure 4. Boxplots showing the concentration of molecules significantly (p < 0.05) affected by age, as assessed by two-way ANOVA followed by Tukey post-hoc test. i f l l i ifi Fi 4 B l h i h Figure 4. Boxplots showing the concentration of molecules significantly (p < 0.05) affected by age, as assessed by two-way ANOVA followed by Tukey post-hoc test. Figure 4. Boxplots showing the concentration of molecules signiﬁcantly (p < 0.05) aﬀected by age, as assessed by two-way ANOVA followed by Tukey post-hoc test. Figure 4. Boxplots showing the concentration of molecules significantly (p < 0.05) affected by age, as assessed by two-way ANOVA followed by Tukey post-hoc test. Figure 5. rPCA model of the concentration of the molecules showing a significant difference among the giraffes grouped by age. The scoreplot (A) shows the samples from the three groups with squares (Young), circles (Adult), and triangles (Old). The median of each sample group is represented by wide circles. The boxplot (B) summarizes the positions of the samples along PC1 and compares them by two-way ANOVA, followed by Tukey post-hoc test. The loading plot (C) reports the correlation between the importance of each substance over PC 1 and its concentration. Gray bars highlight significant correlations (p < 0.05). Figure 5. 2.4. Effect of Age on the Urinary Metabolome 2.4. Eﬀect of Age on the Urinary Metabolome rPCA model of the concentration of the molecules showing a signiﬁcant diﬀerence among the giraﬀes grouped by age. The scoreplot (A) shows the samples from the three groups with squares (Young), circles (Adult), and triangles (Old). The median of each sample group is represented by wide circles. The boxplot (B) summarizes the positions of the samples along PC1 and compares them by two-way ANOVA, followed by Tukey post-hoc test. The loading plot (C) reports the correlation between the importance of each substance over PC 1 and its concentration. Gray bars highlight signiﬁcant correlations (p < 0.05). Three PCs were accepted by the algorithm to depict the overall data features. PC 1, accounting for 44.1% of the variance thus represented, summarized effectively the peculiarities connected to age (p < 0.05), with Young, Adult, and Old individuals appearing respectively at low, intermediate, and high PC scores. Among these molecules, formate and alanine were more abundant in young i di id l hil l h d i d Three PCs were accepted by the algorithm to depict the overall data features. PC 1, accounting for 44.1% of the variance thus represented, summarized effectively the peculiarities connected to age (p < 0.05), with Young, Adult, and Old individuals appearing respectively at low, intermediate, and high PC scores. Among these molecules, formate and alanine were more abundant in young individuals, while valerate showed an opposite trend. Three PCs were accepted by the algorithm to depict the overall data features. PC 1, accounting for 44.1% of the variance thus represented, summarized eﬀectively the peculiarities connected to age (p < 0.05), with Young, Adult, and Old individuals appearing respectively at low, intermediate, and high PC scores. Among these molecules, formate and alanine were more abundant in young individuals, while valerate showed an opposite trend. 2.4. Effect of Age on the Urinary Metabolome 2.4. Eﬀect of Age on the Urinary Metabolome rPCA model of the concentration of the molecules showing a significant difference among the giraffes grouped by age. The scoreplot (A) shows the samples from the three groups with squares (Young), circles (Adult), and triangles (Old). The median of each sample group is represented by wide circles. The boxplot (B) summarizes the positions of the samples along PC1 and compares them by two-way ANOVA, followed by Tukey post-hoc test. The loading plot (C) reports the correlation between the importance of each substance over PC 1 and its concentration. Gray bars highlight significant correlations (p < 0.05). Figure 5. rPCA model of the concentration of the molecules showing a signiﬁcant diﬀerence among the giraﬀes grouped by age. The scoreplot (A) shows the samples from the three groups with squares (Young), circles (Adult), and triangles (Old). The median of each sample group is represented by wide circles. The boxplot (B) summarizes the positions of the samples along PC1 and compares them by two-way ANOVA, followed by Tukey post-hoc test. The loading plot (C) reports the correlation between the importance of each substance over PC 1 and its concentration. Gray bars highlight signiﬁcant correlations (p < 0.05). Figure 5. rPCA model of the concentration of the molecules showing a significant difference among the giraffes grouped by age. The scoreplot (A) shows the samples from the three groups with squares (Young), circles (Adult), and triangles (Old). The median of each sample group is represented by wide circles. The boxplot (B) summarizes the positions of the samples along PC1 and compares them by two-way ANOVA, followed by Tukey post-hoc test. The loading plot (C) reports the correlation between the importance of each substance over PC 1 and its concentration. Gray bars highlight significant correlations (p < 0 05) Figure 5. rPCA model of the concentration of the molecules showing a significant difference among the giraffes grouped by age. The scoreplot (A) shows the samples from the three groups with squares (Young), circles (Adult), and triangles (Old). The median of each sample group is represented by wide circles. The boxplot (B) summarizes the positions of the samples along PC1 and compares them by two-way ANOVA, followed by Tukey post-hoc test. The loading plot (C) reports the correlation between the importance of each substance over PC 1 and its concentration. Gray bars highlight significant correlations (p < 0.05). Figure 5. individuals, while valerate showed an opp 2.5. Pregnancy Related Urinary Metabolome 2.5. Pregnancy Related Urinary Metabolome 2.5. Pregnancy Related Urinary Metabolome Urine samples were obtained from two female giraffes during and after pregnancy (Table S2). Despite the limited number of samples, it was possible to observe a variation of five metabolites during the pregnancy These molecules showed consistent trends in the samples from both giraffes Urine samples were obtained from two female giraffes during and after pregnancy (Table S2). Despite the limited number of samples, it was possible to observe a variation of five metabolites during the pregnancy. These molecules showed consistent trends in the samples from both giraffes. Urine samples were obtained from two female giraﬀes during and after pregnancy (Table S2). Despite the limited number of samples, it was possible to observe a variation of ﬁve metabolites during the pregnancy. These molecules showed consistent trends in the samples from both giraﬀes. 6 of 13 Metabolites 2020, 10, 157 All these molecules showed a relevant increase in concentration during the pregnancy, except for phenylacetylglycine, as shown in Table 2. All these molecules showed a relevant increase in concentration during the pregnancy, except for phenylacetylglycine, as shown in Table 2. Table 2. Urinary metabolites (mmol/L) affected by pregnancy consistently across the two giraffes observed. Table 2. Urinary metabolites (mmol/L) affected by pregnancy consistently across the two giraffes observed. Giulietta Nicole Not Pregnant Pregnant 1 Not Pregnant Pregnant Phenylacetylglycine 10.20 3.52 ↓ 10.40 5.02 ↓ Benzoate 2.14 3.88 ↑ 2.46 12.22 ↑ Glycine 1.06 3.04 ↑ 1.79 11.65 ↑ Taurine 1.75 × 10−1 2.93 × 10−1 ↑ 7.98 × 10−2 1.33 × 10−1 ↑ p-Cresol sulfate 1.46 × 10−2 2.15 × 10−2 ↑ 6.37 × 10−2 3.50 × 10−1 ↑ 1 For readability, only molecules changing by more than 40% for both giraﬀes are shown. 1 For readability, only molecules changing by more than 40% for both giraﬀes are shown. 3. Discussion The present paper describes one of the ﬁrst studies ever devoted to the urinary metabolome of nonfarmed animals, and the very ﬁrst focusing on the giraﬀe metabolome. Due to such paucity of studies on the topic, a key point that needs to be addressed before giraﬀe urine can be used for metabolomics studies is the possibility of relying on samples collected from the ground. Several aspects, in fact, make the collection of urine directly from the individual during urination highly impractical. To obtain a ﬁrst insight on this point, we managed to collect the same urine sample either at the start of a spontaneous voiding or from the ground with a syringe at the end the voiding. The corresponding 1H-NMR spectra were highly superimposable, except for four molecules, namely benzoate, citrate, p-cresol sulfate, and glycine. The fact that the non-volatile glycine showed the greatest diﬀerences gave hints that the discrepancies could be mainly connected to dynamic variations in composition during urination, in agreement with Sink and Weinstein [16]. Modiﬁcations induced by the collection method could therefore be considered a confounding factor of lower entities than inhomogeneity in the composition of urine during voiding. The 39 molecules identiﬁed give information about protein digestion, diet, gut-microbial co-metabolism, and energy production. Their quantitative observation therefore oﬀers a handy perspective of the health status of giraﬀes, through a quintessentially non-invasive sampling method. Comparisons with the urinary metabolome of other animals are also possible, giving indirect information about the diﬀerences in metabolism. An example of this possibility is oﬀered by allantoin. This molecule is the fourth most concentrated in giraﬀe urine (Table S1), identically to yak (Bos grunniens) [17] and horse [18]. Diﬀerently from these strictly herbivorous animals, this molecule is the most concentrated in the urine of the giant panda [19], even if the giant panda consumes an amount of vegetables in relation to body weight (as much as 30%) much higher than ruminants or horses, which should lead to the lowest concentration of urinary allantoin [20]. This apparent contradiction leads to speculate that the main mechanism determining the concentration of allantoin in the urine of the above-mentioned animals is likely to be its renal reabsorption, which is very eﬀective in strictly herbivorous animals [20]. 3.1. Sex Aﬀects the Giraﬀe Urine Molecular Proﬁle In the current study acetate, succinate, and lactate concentrations appeared to be signiﬁcantly higher in female giraﬀe urine, while hippurate, phenylacetylglycine, and thymine were more concentrated in male urine. For acetate, two of the authors of the present paper identiﬁed a similar situation in horse urine [18]. For the other molecules, indirect connections with published ﬁndings can be devised. There is an abundance of references, focusing on humans, showing that exercise leads to higher concentrations of acetate, succinate, and lactate in urine, and lower concentrations of thymine and hippurate [21–23]. Ginnett et al. showed that female giraﬀes spend more time walking, foraging, 7 of 13 Metabolites 2020, 10, 157 feeding, and traveling than males [24]. The two observations seem to suggest that the sex-related diﬀerences observed in the urine of males and females may be partly due to the diﬀerent daily activities. Contrary to the previously reported molecules, phenylacetylglycine is mainly a co-metabolite of gut microorganisms, derived from valine, leucine, phenylalanine, lysine, or ornithine [25]. Its diﬀerent concentration in relation to sex may therefore reﬂect peculiarities in gut microbiota proﬁles or diﬀerent foraging behaviors, similarly to what was recently observed in the giant panda [19]. Ginnett et al., in fact, demonstrated that males prefer larger bites than females, with potential consequences on the food, and in turn urine, metabolome proﬁle [24]. It is tantalizing to speculate that the length of the neck, which is higher in males [5], may play a role too. In fact, Schüßler and Greven [26] found an allometric direct relationship between rumen-to-mouth distance and the duration of rumination intercycles, inﬂuencing in turn the digestive action of ruminal microorganisms. 3.2. Eﬀect of Age By removing the gender eﬀect by two-way ANOVA, it was possible to focus on the eﬀect of age. In parallel with previous studies in rats and humans [27,28], formate and alanine were negatively related to age. The trend observed for formate is very likely related to the gut microbiome. In fact, in the gut microbiota of the juvenile giraﬀes there is a prevalence of Bacteroides and Acinetobacter genera, responsible for the degradation of starch and cellulose to formate [29], while in the gut of adult giraﬀes other genera tend to prevail, such as Treponema [30]. The concentration of amino acids in urine has been consistently linked to the turnover of muscle amino acids [18,31], with urinary concentration of alanine speciﬁcally related to exercise [32]. Therefore, the diﬀerence in the concentration of alanine could be ascribed to a variation of daily activity intensity along age. 4.1. Compliance with Ethical Requirements 4.1. Compliance with Ethical Requirements All the procedures related to animals respected the Directive 2010/63/EU of the European Parliament and of the Council of September 22, 2010 on the protection of animals used for scientiﬁc purposes (Article 1, Paragraph 1, Letter b) and the Italian legislation (D. Lgs. n. 26/2014, Article 2, Paragraph 1, Letter b). 3.3. Eﬀect of Pregnancy here, these observations support the compelling possibility to use the urine metabolome to gain speciﬁc information about giraﬀe inﬂammatory status during pregnancy, as modulated by the gut microbiota. 3.3. Eﬀect of Pregnancy Early identiﬁcation of pregnant giraﬀes with maximum accuracy is an important issue for optimizing their management. Although some diagnostic methods (e.g., ultrasonography) have been described in domestic animals [33], their application to wild or captive animals is hindered by practical reasons. Metabolomics approaches seem in principle promising for setting up diagnostic methods that might be more convenient in speciﬁc contexts, due to the possibility to quantify a high number of molecules at the same time. However, previous studies performed in domestic animals were focused on serum [34,35], a sub-optimal sample from the point of view of non-invasivity. Therefore, despite the restricted number of samples analyzed in the present study, the obtained data can provide a preliminary urinary ﬁngerprint of pregnancy in giraﬀes. Taurine is an important amino acid during pregnancy and lactation, because it satisﬁes the needs of both the fetus and suckling infant. In our research, taurine excretion through urine increased during early pregnancy, consistent with human studies [36]. Taurine is rarely found in plants [37], so that herbivores cannot obtain a suﬃcient amount taurine from the diet. Remarkably, in ruminants the urinary taurine concentration is strongly diet-dependent, as can be inferred from the works of Bristow et al. on cows fed with maize silage compared to free grazing cows [38]. Diet is therefore likely to trigger biosynthetic pathways, such as the one leading to taurine from methionine [39]. Moreover, a speciﬁc pathway, converting homocysteine to taurine and glycine through cysteine, is known to become eﬀective in early pregnancy [39]. This latter mechanism is a likely reason for the increasing trend of taurine excretion we found in the present work. A further contribution to urine metabolome proﬁle modiﬁcations may be due to changes in the gut microbiota. In fact, among the molecules showing the greatest changes we found p-cresol sulfate and phenylacetylglycine, mainly described as gut microorganism co-metabolites [11,25], absorbed at the intestinal level and then expelled through urine. Interestingly, the change in the concentration of both has been related, in humans, with alterations in the microbiota proﬁle linked to inﬂammatory states [13,40], in which pregnancy is known to play a role [41]. Despite the very limited number of cases 8 of 13 Metabolites 2020, 10, 157 here, these observations support the compelling possibility to use the urine metabolome to gain speciﬁc information about giraﬀe inﬂammatory status during pregnancy, as modulated by the gut microbiota. 4.2. Sample Collection A total of 35 captive giraﬀes (Giraﬀa camelopardalis) were involved in the current study. Based on physical examinations, giraﬀes did not show symptoms of diseases both before and during the urine sampling period. The giraﬀes were housed in ﬁve Italian zoos: Zoosafari Fasanolandia (FA) (N = 11), Safari Ravenna (RA) (N = 4), Giardino Zoologico di Pistoia (PT) (N = 1), Parco Natura Viva (VR) (N = 4), and Parco Faunistico Le Cornelle (BG) (N = 15). The details for each giraﬀe are reported in Table 3. Their age ranged from a minimum of 6 months to a maximum of 20 years. The giraﬀes were categorized in 3 age classes: Young (from 6 months to 6 years old, N = 14), Adult (from 6 to 15 years old, N = 16), and Old (older than 15, N = 9), according to the following information. In female giraﬀes the ﬁrst birth is at about 6.4 years old, even if sexual maturity is reached at 3–4 years [42,43]. Giraﬀe males are considered as adults when older than 6 years old, according to Lee et al. [44]. Table 3. Animal information. Sample ID Name Sex Age (years) Zoo N.01 Ronny Male 14 FA N.02 Nicole Female 14 FA N.03 Giulietta Female 17 FA N.04 Marcello Male 9 FA N.05 Italia Female 8 FA N.06 Carlos Male 2 RA N.07 Daniele Male 11 RA N.08 Cleopatra Female 20 PT N.09 Alto Male 2 FA N.10 Congo Male 0.3 FA N.11 Roberto Male 0.6 RA N.12 Martina Female 0.6 RA N.13 Linda Female 16 BG N.14 Sandy Female 16 BG N.15 Raﬀa Female 7 BG N.16 Telete Female 2 BG N.17 Rusman Male 16 BG N.18 Akuna Female 10 BG N.19 Ciokwe Male 5 BG N.20 Miro Male 9 BG N.21 Lucia Female 16 BG N.22 Nuvola Female 7 BG N.23 Sahel Female 2 BG N.24 Russel Male 16 BG N.25 Ramiro Male 3 BG N.26 Madiba Male 6 BG N.27 Nasanta Female 2 BG Table 3. Animal information. 9 of 13 Metabolites 2020, 10, 157 Table 3. Cont. N.28 Macchia Male 5 VR N.29 Secondo Male 11 VR N.30 Akasha Male 7 VR N.31 Quarto Male 9 VR N.32 Luna Female 15 FA N.33 Kenya Female 20 FA N.34 Alessia Female 4 FA N.35 Mina Female 14 FA The samples were collected between 10:00 a.m. 4.3. Metabolomic Analysis We prepared urine samples for NMR by thawing and centrifuging them for 15 min at 18,630× g at 4 ◦C. We added the supernatant (350 µL) to bi-distilled water (350 µL) and to a D2O solution (200 µL) of TSP (3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid) 10 mM and of NaN3 2 mM. A 1M phosphate buﬀer had been used to set the D2O solution to pH of 7.00 ± 0.02. After a further centrifugation, we recorded 1H-NMR spectra at 298 K with an AVANCE III spectrometer (Bruker, Milan, Italy), at a frequency of 600.13 MHz, equipped with Topspin software (Ver. 3.5). According to Zhu et al. [17], we suppressed the signals from broad resonances using a CPMG-(Carr-Purcell-Meiboom-Gill) ﬁlter composed of 400 echoes with a of 400 s and a 180◦pulse of 24 s, for a total ﬁlter of 330 ms. We also applied pre-saturation, to reduce the signal from water. We employed Topspin software to apply a line broadening of 0.3 Hz and to adjust the phase of each spectrum. We set the recycle delay to 5 s, by considering the relaxation time of the protons under investigation. We employed R computational language [45] for any further processing of spectra, quantiﬁcation of molecules, and data mining, with custom scripts. We aligned the spectra by using the TSP signal as a reference (−0.017 ppm). We adjusted the baseline of each spectrum by distinguishing irregularities of the baseline from genuine signals, according to the “rolling ball” idea [46], implemented in the R package “baseline” [47]. We performed the assignment of the signals by comparing chemical shift and multiplicity with the libraries (Ver. 10) of Chenomx software (Chenomx Inc., Canada, v. 8.3). According to Dieterle et al. [48], water intake behavior can change the dilution of urine as much as ﬁve times, obscuring any trend in metabolite concentrations. We removed this confounding factor by calculating, for each sample, the ratio between the area of TSP peak and the intensity of the spectrum. This allowed us to estimate the dilution of each sample and to select the one with the mostly representative dilution. We used this sample as a reference by quantifying the molecules from the added TSP. We then normalized the other samples towards the reference by probabilistic quotient normalization (PQN) [48]. 4.2. Sample Collection and 2:00 p.m., in connection to the daily activities of the keepers. Urine samples were collected with a syringe from the ground. To limit the soil contaminants, only the upper part of the urine was collected, immediately after the spontaneous voiding, before it was absorbed by the soil. A sample from one male was also collected directly into a sterile beaker, preventing the sample from touching the ground. Four urine samples were collected from two females during and after pregnancy. After collection, the urine samples were centrifuged at 1500× g for 10 min, to further remove potential ground contaminants, and the supernatants were frozen at −80 ◦C. 4.4. Statistical Analysis We conducted the statistical analysis in R computational language [45] and we reﬁned the artwork by GIMP (version 2.10, www.gimp.org). Prior to univariate analysis, we transformed the data to normality by BoxCox transformation [49]. To investigate the eﬀects of sex on urinary metabolites, 10 of 13 Metabolites 2020, 10, 157 we considered only adult, non-pregnant giraﬀes. This allowed us to reduce potential interferences due to diﬀerent age classes. We then highlighted any diﬀerence by t-test. To investigate age related eﬀects, by removing sex eﬀect, we applied a two-way ANOVA test followed by Tukey-HSD, by taking advantage of the “aov” function of the R package “stats” [50]. For the above statistical tests, we accepted a cut-oﬀp-value of 0.05. p In agreement with Bazzano et al. [51], we highlighted any trend characterizing the samples with robust principal component analysis (rPCA) models [52], using the molecules accepted by univariate analysis as a base. We took advantage of the PcaHubert algorithm implemented in the “rrcov” package. The algorithm grants robustness with a two-steps approach. In the ﬁrst step outlying samples are detected according to their distance from the others along and orthogonally to the PCA plane. A second step determines the optimal number of principal components (PCs). The main features of each rPCA model are summarized by a scoreplot and by a Pearson correlation plot. The former is the projection of the samples in the PC space and highlights the underlying structure of the data. The latter relates the concentration of each variable to the components of the model. 5. Conclusions This work represents a primer in giving quantitative information about the urinary metabolome of captive giraﬀes, as detected by untargeted 1H-NMR. Foraging behaviors and daily activity could be considered as one of the main reasons for the diﬀerences we highlighted that are linked to sex and age. A preliminary observation conducted on two female giraﬀes suggests that 1H-NMR based metabolomics could be conveniently applied to monitor modiﬁcations occurring during pregnancy, some of which are potentially related to inﬂammatory status triggered by modiﬁcation of the microbiota proﬁle. Supplementary Materials: The following are available online at http://www.mdpi.com/2218-1989/10/4/157/s1, Table S1: Concentration (mmol/L, median (IQR)) of the molecules quantiﬁed by 1H-NMR in all the samples studied in the present investigation, sorted by abundance. Table S2. Concentration (mmol/L) of the molecules quantiﬁed by 1H-NMR in the samples collected during and after pregnancy. Author Contributions: C.Z., L.L., S.F., and G.I. conceived and designed the research. C.Z. and L.L. performed metabolomics analysis. S.F. collected the samples. C.Z., L.L., S.F., and G.I. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: Chenglin Zhu gratefully acknowledges ﬁnancial support from Chinese Scholarship Council (grant n◦201606910076). Part of the samples used in this study was collected with the ﬁnancial Grant won by Sabrina Fasoli and donated by the Zebra Foundation for Veterinary Zoological Education in 2019. The authors would like to thank Bandoli Francesca, Cordon Rossana, Cotignoli Chiara, Laguardia Daniele, Laricchiuta Pietro, Sandri Camillo, Schneider Rainer, and Spiezio Caterina for their support. The authors also would like to thank all staﬀof the zoos involved in this study for their precious help during the sampling and Vito Barnaba, Pietro Ciaccia, Giacomo Melani, and Fulvio Pendezza for their fundamental assistance. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Normando, S.; Pollastri, I.; Florio, D.; Ferrante, L.; Macchi, E.; Isaja, V.; de Mori, B. Assessing animal welfare in animal-visitor interactions in zoos and other facilities. A pilot study involving giraﬀes. Animals 2018, 8, 153. [CrossRef] [PubMed] 3. Hosey, G.; Melﬁ, V.; Pankhurst, S. Animal Welfare. In Zoo Animals: Behaviour, Management, and Welfare, 2nd ed.; Oxford University Press: Oxford, UK, 2013; pp. 212–250. ISBN 0199693528. 2. Paul-Murphy, J.; Molter, C. Overview of Animal Welfare in Zoos. In Fowler’s Zoo and Wild Animal Medicine Current Therapy; Miller, R.E., Lamberski, N., Calle, P.P., Eds.; Elsevier: St. Louis, MO, USA, 2019; Volume 9, pp. 64–72. ISBN 978-0-323-55228-8. References 1. Muller, Z.; Bercovitch, F.; Brand, R.; Brown, D.; Brown, M.; Bolger, D.; Carter, K.; Deacon, F.; Doherty, J.B.; Fennessy, J.; et al. Giraﬀa Camelopardalis. The IUCN Red List of Threatened Species 2016: e.T9194A51140239. Available online: https://www.iucnredlist.org/species/9194/109326950 (accessed on 17 April 2020). 2. Paul-Murphy, J.; Molter, C. Overview of Animal Welfare in Zoos. In Fowler’s Zoo and Wild Animal Medicine Current Therapy; Miller, R.E., Lamberski, N., Calle, P.P., Eds.; Elsevier: St. Louis, MO, USA, 2019; Volume 9, pp. 64–72. ISBN 978-0-323-55228-8. 3. Hosey, G.; Melﬁ, V.; Pankhurst, S. Animal Welfare. In Zoo Animals: Behaviour, Management, and Welfare, 2nd ed.; Oxford University Press: Oxford, UK, 2013; pp. 212–250. ISBN 0199693528. 4. Normando, S.; Pollastri, I.; Florio, D.; Ferrante, L.; Macchi, E.; Isaja, V.; de Mori, B. Assessing animal welfare in animal-visitor interactions in zoos and other facilities. A pilot study involving giraﬀes. Animals 2018, 8, 153. [CrossRef] [PubMed] 11 of 13 Metabolites 2020, 10, 157 5. Dagg, A.I. Physiology. In Giraﬀe: Biology, Behaviour and Conservation; Dagg, A.I., Ed.; Cambridge University Press: New York, NY, USA, 2014; pp. 117–134. ISBN 9781139542302. 6. Gage, L.J. Giraﬀe Husbandry and Welfare. In Fowler’s Zoo and Wild Animal Medicine Current Therapy; Miller, R.E., Lamberski, N., Calle, P.P., Eds.; Elsevier: St. Louis, MO, USA, 2019; Volume 9, pp. 619–622. ISBN 978-0-323-55228-8. 7. Glatston, A.R.; Smit, S. Analysis of the urine of the okapi (Okapia johnstoni). Acta Zool. Pathol. Antverp. 1980, 75, 49–58. 8. Zhang, Q. Hypertension and Counter-Hypertension Mechanisms in Giraﬀes. Cardiovasc. Hematol. Disord. Targets 2012, 6, 63–67. [CrossRef] [PubMed] 9. Hargens, A.R.; Millard, R.W.; Pettersson, K.; Johansen, K. Gravitational haemodynamics and oedema prevention in the giraﬀe. Nature 1987, 329, 59–60. [CrossRef] 10. Agaba, M.; Ishengoma, E.; Miller, W.C.; McGrath, B.C.; Hudson, C.N.; Bedoya Reina, O.C.; Ratan, A.; Burhans, R.; Chikhi, R.; Medvedev, P.; et al. Giraﬀe genome sequence reveals clues to its unique morphology and physiology. Nat. Commun. 2016, 7, 1–8. [CrossRef] 11. Patel, K.P.; Luo, F.J.G.; Plummer, N.S.; Hostetter, T.H.; Meyer, T.W. The production of p-Cresol sulfate and indoxyl sulfate in vegetarians versus omnivores. Clin. J. Am. Soc. Nephrol. 2012, 7, 982–988. [CrossRef] 11. Patel, K.P.; Luo, F.J.G.; Plummer, N.S.; Hostetter, T.H.; Meyer, T.W. The production of p-Cresol sulfate and indoxyl sulfate in vegetarians versus omnivores. Clin. J. Am. Soc. Nephrol. 2012, 7, 982–988. [CrossRef] 12. Laghi, L.; Zhu, C.; Campagna, G.; Rossi, G.; Bazzano, M.; Laus, F. Probiotic supplementation in trained trotter 12. References Laghi, L.; Zhu, C.; Campagna, G.; Rossi, G.; Bazzano, M.; Laus, F. Probiotic supplementation in trained trotter horses: Eﬀect on blood clinical pathology data and urine metabolomic assessed in ﬁeld. J. Appl. Physiol. 2018, 125, 654–660. [CrossRef] 13. Barbara, G.; Scaioli, E.; Barbaro, M.R.; Biagi, E.; Laghi, L.; Cremon, C.; Marasco, G.; Colecchia, A.; Picone, G.; Salﬁ, N.; et al. Gut microbiota, metabolome and immune signatures in patients with uncomplicated diverticular disease. Gut 2017, 66, 1252–1261. [CrossRef] 14. Kok, D.E.G.; Rusli, F.; van der Lugt, B.; Lute, C.; Laghi, L.; Salvioli, S.; Picone, G.; Franceschi, C.; Smidt, H.; Vervoort, J.; et al. Lifelong calorie restriction aﬀects indicators of colonic health in aging C57Bl/6J mice. J. Nutr. Biochem. 2018, 56, 152–164. [CrossRef] 15. Liao, Y.; Hu, R.; Wang, Z.; Peng, Q.; Dong, X.; Zhang, X.; Zou, H.; Pu, Q.; Xue, B.; Wang, L. Metabolomics Proﬁling of Serum and Urine in Three Beef Cattle Breeds Revealed Diﬀerent Levels of Tolerance to Heat Stress. J. Agric. Food Chem. 2018, 66, 6926–6935. [CrossRef] [PubMed] g 16. Sink, C.A.; Weinstein, N.M. Specimen procurement. In Practical Veterinary Urinalysis; John Wiley & Sons: Chichester, West Sussex, UK, 2012; pp. 9–18. 17. Zhu, C.; Li, C.; Wang, Y.; Laghi, L. Characterization of yak common bioﬂuids metabolome by means of proton nuclear magnetic resonance spectroscopy. Metabolites 2019, 9, 41. [CrossRef] [PubMed] 18. Zhu, C.; Faillace, V.; Laus, F.; Bazzano, M.; Laghi, L. Characterization of trotter horses urine metabolome by means of proton nuclear magnetic resonance spectroscopy. Metabolomics 2018, 14, 106. [CrossRef] [PubMed] 19. Zhu, C.; Laghi, L.; Zhang, Z.; He, Y.; Wu, D.; Zhang, H.; Huang, Y.; Li, C.; Zou, L. First Steps toward the Giant Panda Metabolome Database: Untargeted Metabolomics of Feces, Urine, Serum, and Saliva by 1 H NMR. J. Proteome Res. 2020, 19, 1052–1059. [CrossRef] 20. Chen, X.; Kyle, D.; Orskov, E.; Hovell, F. Renal clearance of plasma allantoin in sheep. Exp. Physiol. 1991, 76, 59–65. [CrossRef] 21. Sheedy, J.R.; Gooley, P.R.; Nahid, A.; Tull, D.L.; McConville, M.J.; Kukuljan, S.; Nowson, C.A.; Daly, R.M.; Ebeling, P.R. 1H-NMR analysis of the human urinary metabolome in response to an 18-month multi-component exercise program and calcium–vitamin-D3 supplementation in older men. Appl. Physiol. Nutr. Metab. 2014, 39, 1294–1304. [CrossRef] 22. Mukherjee, K.; Edgett, B.A.; Burrows, H.W.; Castro, C.; Griﬃn, J.L.; Schwertani, A.G.; Gurd, B.J.; Funk, C.D. References Whole blood transcriptomics and urinary metabolomics to deﬁne adaptive biochemical pathways of high- intensity exercise in 50-60 year old masters athletes. PLoS ONE 2014, 9, e92031. [CrossRef] 23. Enea, C.; Seguin, F.; Petitpas-Mulliez, J.; Boildieu, N.; Boisseau, N.; Delpech, N.; Diaz, V.; Eugène, M.; Dugué, B. 1H NMR-based metabolomics approach for exploring urinary metabolome modiﬁcations after acute and chronic physical exercise. Anal. Bioanal. Chem. 2010, 396, 1167–1176. [CrossRef] 24. Ginnett, T.F.; Demment, M.W. Sex diﬀerences in giraﬀe foraging behavior at two spatial scales. Oecologia 1997, 110, 291–300. [CrossRef] 12 of 13 Metabolites 2020, 10, 157 25. Mayneris-Perxachs, J.; Bolick, D.T.; Leng, J.; Medlock, G.L.; Kolling, G.L.; Papin, J.A.; Swann, J.R.; Guerrant, R.L. Protein-and zinc-deﬁcient diets modulate the murine microbiome and metabolic phenotype. Am. J. Clin. Nutr. 2016, 104, 1253–1262. [CrossRef] 6. Schüßler, D.; Greven, H. Quantitative aspects of the ruminating process in giraﬀes (Giraﬀa camelopard fed with diﬀerent diets. Zoo Biol. 2017, 36, 407–412. [CrossRef] [PubMed] 27. Schnackenberg, L.K.; Sun, J.; Espandiari, P.; Holland, R.D.; Hanig, J.; Beger, R.D. Metabonomics evaluations of age-related changes in the urinary compositions of male Sprague Dawley rats and eﬀects of data normalization methods on statistical and quantitative analysis. In Proceedings of the BMC Bioinformatics, Hong Kong, China, 27–30 August 2007. 28. Slupsky, C.M.; Rankin, K.N.; Wagner, J.; Fu, H.; Chang, D.; Weljie, A.M.; Saude, E.J.; Lix, B.; Adamko, D.J.; Shah, S.; et al. Investigations of the eﬀects of gender, diurnal variation, and age in human urinary metabolomic proﬁles. Anal. Chem. 2007, 79, 6995–7004. [CrossRef] [PubMed] 29. Theodorou, M.K.; France, J. Rumen microorganisms and their interactions. In Quantitative Aspects of Ruminant Digestion and Metabolism; Dijkstra, J., Forbes, J., France, J., Eds.; CAB International: Wallingford, Oxfordshire, UK, 2009; Volume 2, pp. 207–228. 30. Schmidt, J.M.; Henken, S.; Dowd, S.E.; McLaughlin, R.W. Analysis of the Microbial Diversity in the Fecal Material of Giraﬀes. Curr. Microbiol. 2018, 75, 323–327. [CrossRef] [PubMed] 31. Soupart, P. Urinary excretion of free amino acids in normal adult men and women. Clin. Chim. Acta 1959, 4, 265–271. [CrossRef] 32. Pechlivanis, A.; Kostidis, S.; Saraslanidis, P.; Petridou, A.; Tsalis, G.; Mougios, V.; Gika, H.G.; Mikros, E.; Theodoridis, G.A. 1H NMR-based metabonomic investigation of the eﬀect of two diﬀerent exercise sessions on the metabolic ﬁngerprint of human urine. J. Proteome Res. 2010, 9, 6405–6416. [CrossRef] [PubMed] 33. Karen, A.; Szabados, K.; Reiczigel, J.; Beckers, J.F.; Szenci, O. References Accuracy of transrectal ultrasonography for determination of pregnancy in sheep: Eﬀect of fasting and handling of the animals. Theriogenology 2004, 61, 1291–1298. [CrossRef] 34. Sun, L.; Guo, Y.; Fan, Y.; Nie, H.; Wang, R.; Wang, F. Metabolic proﬁling of stages of healthy pregnancy in Hu sheep using nuclear magnetic resonance (NMR). Theriogenology 2017, 92, 121–128. [CrossRef] 35. Kenéz, Á.; Dänicke, S.; Rolle-Kampczyk, U.; von Bergen, M.; Huber, K. A metabolomics approach to characterize phenotypes of metabolic transition from late pregnancy to early lactation in dairy cows. Metabolomics 2016, 12, 165. [CrossRef] 36. Diaz, S.O.; Barros, A.S.; Goodfellow, B.J.; Duarte, I.F.; Carreira, I.M.; Galhano, E.; Pita, C.; Almeida, M.D.C.; Gil, A.M. Following healthy pregnancy by nuclear magnetic resonance (NMR) metabolic proﬁling of human urine. J. Proteome Res. 2013, 12, 969–979. [CrossRef] 37. Bouckenooghe, T.; Remacle, C.; Reusens, B. Is taurine a functional nutrient? Curr. Opin. Clin. Nutr. Metab. Care 2006, 9, 728–733. [CrossRef] 38. Bristow, A.W.; Whitehead, D.C.; Cockburn, J.E. Nitrogenous constituents in the urine of cattle, sheep and goats. J. Sci. Food Agric. 1992, 59, 387–394. [CrossRef] 39. Dasarathy, J.; Gruca, L.L.; Bennett, C.; Parimi, P.S.; Duenas, C.; Marczewski, S.; Fierro, J.L.; Kalhan, S.C. Methionine metabolism in human pregnancy. Am. J. Clin. Nutr. 2010, 91, 357–365. [CrossRef] [PubMed] 40. Sarosiek, I.; Schicho, R.; Blandon, P.; Bashashati, M. Urinary metabolites as noninvasive biomarkers of gastrointestinal diseases: A clinical review. World J. Gastrointest. Oncol. 2016, 8, 459. [CrossRef] [PubMed] 41. Edwards, S.M.; Cunningham, S.A.; Dunlop, A.L.; Corwin, E.J. The Maternal Gut Microbiome during Pregnancy. MCN Am. J. Matern. Nurs. 2017, 42, 310–316. [CrossRef] [PubMed] 42. Bercovitch, F.B.; Berry, P.S.M. Reproductive life history of Thornicroft’s giraﬀe in Zambia. Afr. J. Ecol. 2010, 48, 535–538. [CrossRef] 43. Bercovitch, F.B.; Berry, P.S.M. Giraﬀe birth locations in the South Luangwa National Park, Zambia: Site ﬁdelity or microhabitat selection? Afr. J. Ecol. 2015, 53, 206–213. [CrossRef] 44. Lee, D.E.; Bond, M.L.; Bolger, D.T. Season of birth aﬀects juvenile survival of giraﬀe. Popul. Ecol. 2017, 59, 45–54. [CrossRef] 45. R Development Core Team. R: A Language and Environment for Statistical Computing; Vienna, Austria, 2011; Volume 1. Available online: http://www.r-project.org (accessed on 24 March 2020). 46. Kneen, M.A.; Annegarn, H.J. Algorithm for ﬁtting XRF, SEM and PIXE X-ray spectra backgrounds. Nucl. Instrum. Methods Phys. Res. Sect. B Beam Interact. Mater. Atoms 1996, 109–110, 209–213. [CrossRef] 13 of 13 13 of 13 Metabolites 2020, 10, 157 47. References Liland, K.H.; Almøy, T.; Mevik, B.H. Optimal choice of baseline correction for multivariate calibration of spectra. Appl. Spectrosc. 2010, 64, 1007–1016. [CrossRef] 48. Dieterle, F.; Ross, A.; Schlotterbeck, G.; Senn, H. Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabonomics. Anal. Chem. 2006, 78, 4281–4290. [CrossRef] 49. Box, G.E.P.; Cox, D.R. An Analysis of Transformations. J. R. Stat. Soc. Ser. B 2018, 26, 211–243. [CrossRef] 49. Box, G.E.P.; Cox, D.R. An Analysis of Transformations. J. R. Stat. Soc. Ser. B 2018, 26, 211–243. [CrossRef] 50. Chambers, J.M.; Freeny, A.; Heiberger, R.M. Analysis of variance; designed experiments. In Statistical Models in S; Wadsworth and Brooks/Cole Advanced Books and Software; Springer: Paciﬁc Grove, CA, USA, 1992; pp. 145–193. 51. Bazzano, M.; Laghi, L.; Zhu, C.; Magi, G.E.; Serri, E.; Spaterna, A.; Tesei, B.; Laus, F. Metabolomics of tracheal wash samples and exhaled breath condensates in healthy horses and horses aﬀected by equine asthma. J. Breath Res. 2018, 12, 46015. [CrossRef] [PubMed] 52. Hubert, M.; Rousseeuw, P.J.; Vanden Branden, K. ROBPCA: A new approach to robust principal component analysis. Technometrics 2005, 47, 64–79. [CrossRef] © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W2118480036	https://europepmc.org/articles/pmc4522058?pdf=render	English	null	A systematic review including meta-analysis of work environment and depressive symptoms	BMC public health	2,015	cc-by	11,947	* Correspondence: tores.theorell@stressforskning.su.se 1Stress Research Institute, Stockholm University, SE-106 91 Stockholm, Sweden 2Department of Neuroscience, Karolinska Institutet, SE- 171 77 Stockholm, Sweden Full list of author information is available at the end of the article Abstract Background: Depressive symptoms are potential outcomes of poorly functioning work environments. Such symptoms are frequent and cause considerable suffering for the employees as well as financial loss for the employers. Accordingly good prospective studies of psychosocial working conditions and depressive symptoms are valuable. Scientific reviews of such studies have pointed at methodological difficulties but still established a few job risk factors. Those reviews were published some years ago. There is need for an updated systematic review using the GRADE system. In addition, gender related questions have been insufficiently reviewed. Method: Inclusion criteria for the studies published 1990 to June 2013: 1. European and English speaking countries. 2. Quantified results describing the relationship between exposure (psychosocial or physical/chemical) and outcome (standardized questionnaire assessment of depressive symptoms or interview-based clinical depression). 3. Prospective or comparable case-control design with at least 100 participants. 4. Assessments of exposure (working conditions) and outcome at baseline and outcome (depressive symptoms) once again after follow-up 1-5 years later. 5. Adjustment for age and adjustment or stratification for gender. g j g Studies filling inclusion criteria were subjected to assessment of 1.) relevance and 2.) quality using predefined criteria. Systematic review of the evidence was made using the GRADE system. When applicable, meta-analysis of the magnitude of associations was made. Consistency of findings was examined for a number of possible confounders and publication bias was discussed. Results: Fifty-nine articles of high or medium high scientific quality were included. Moderately strong evidence (grade three out of four) was found for job strain (high psychological demands and low decision latitude), low decision latitude and bullying having significant impact on development of depressive symptoms. Limited evidence (grade two) was shown for psychological demands, effort reward imbalance, low support, unfavorable social climate, lack of work justice, conflicts, limited skill discretion, job insecurity and long working hours. There was no differential gender effect of adverse job conditions on depressive symptoms Conclusion: There is substantial empirical evidence that employees, both men and women, who report lack of decision latitude, job strain and bullying, will experience increasing depressive symptoms over time. These conditions are amenable to organizational interventions. Keywords: Depression, Work stress, Prevention, Ergonomic, Toxicology Full list of author information is available at the end of the article © 2015 Theorell et al. A systematic review including meta-analysis of work environment and depressive symptoms Töres Theorell1,2, Anne Hammarström3, Gunnar Aronsson4, Lil Träskman Bendz5, Tom Grape6, Christer Hogstedt7, Ina Marteinsdottir8, Ingmar Skoog9 and Charlotte Hall10 Theorell et al. BMC Public Health (2015) 15:738 DOI 10.1186/s12889-015-1954-4 Open Access Background depressive conditions/symptoms as outcome. Most of the reviews have used multiple kinds of mental health outcomes. However, depression is the most widely re- ported outcome in the field of mental health in epi- demiological research. Depressive symptoms are well understood in psychiatry which has resulted in a large number of studies. Accordingly this outcome should provide a good basis for a focused systematic review. As far as the authors know there is no published study that has used the international GRADE system [9] for evalu- ating the evidence in this field. In addition there is a need for a systematic review utilizing the most recent developments in search technology. Depressive symptoms are potential outcomes of poorly functioning work environments. Such symptoms are fre- quent and may cause considerable suffering for the em- ployees themselves as well as financial loss for the employers. Accordingly good prospective studies of psy- chosocial working conditions and depressive symptoms are valuable. Several reviews including prospective studies of psychosocial factors at work in relation to depression have been published. Bonde [1] concluded that there were consistent findings that perception of adverse psy- chosocial factors in the workplace is related to an ele- vated risk of subsequent depressive symptoms or major depressive episode but also that methodological limita- tions preclude causal inference. Netterström et al. [2] made a similar conclusion but pointed out that studies are needed that assess in more detail the duration and intensity of exposure necessary for developing depres- sion. The conclusions in a review by Siegrist from the same year [3] were similar. Also, Michie and Williams [4] concluded that” many of the work related variables associated with high levels of psychological ill health, are potentially amenable to change which has been shown in intervention studies that have successfully improved psychological health and reduced sickness absence”. A review of psychosocial and health effects of workplace reorganization by Egan et al. [5] concluded that” some organizational-level participation interventions may benefit employee health, as predicted by the demand- control model”. However, several other psychosocial exposures should be examined more in detail. An important aspect of the systematic review process is to systematically and transparently assess the scientific evidence. We have chosen to use the internationally rec- ognized GRADE- system for scientific evaluation. The GRADE system uses four levels of evidence, namely High, Moderate, Limited and Very Limited. Aim of the study The aim of this study was to provide systematically graded evidence for possible associations between work environment factors and near-future development of de- pressive symptoms Background We are well aware that the system has been developed primarily for assessing interventions in a health care context, but the system has been adapted to epidemiological evaluation. Beside the transparency, an advantage is that the GRADE system [9] - a system often applied in reviews conducted within the Cochrane Collaboration - is in- creasingly used internationally e.g., by the World Health Organization. Hence results from systematic reviews can be more easily compared. Time has elapsed since most of the previous reviews were published and new studies are published continu- ously. The most relevant reviews were published in 2008. They pointed at several methodological shortcomings, and it is not known whether researchers more recently have tried to address the identified scientific problems. In particular, the reviews have pointed at the paucity of stud- ies on physical/chemical/ergonomic exposures. Most of the work environment reviews published so far have not been confined to depression only - they have included for instance stress related disorders, psychologic- ally related sick leave and suicide or combinations [4, 6–8] as outcomes, and it has sometimes been difficult to disen- tangle them. Studied work environment factors have mostly been limited to psychosocial factors although two reviews have included physical/chemical/ergonomic expo- sures as well. The conclusion from them [4, 7] was that the evidence for physical/chemical/ergonomic exposures is limited and inconclusive. Nieuwenhuijsen et al. [8] pub- lished a review of the effects of the psychosocial environ- ment on risk of stress-related disorders (SRDs) and concluded that there is” strong evidence that high job de- mands, low job control, low co-worker support, low supervisor support, low procedural justice and a high ef- fort- reward imbalance predicted the incidence of SRDs”. A topic that has not been addressed sufficiently in pre- vious reviews is gender in the relationship between working conditions and the development of depressive symptoms. Are the associations different for men and women? Abstract 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. Theorell et al. BMC Public Health (2015) 15:738 Page 2 of 14 Inclusion criteria p y After that, the scientific experts started their examin- ation. Pre-set evaluation forms were used. The experts judged relevance and quality of the studies on the basis of the relevance/quality criteria, their experience as re- searchers and their knowledge of the field. Accordingly they were recruited among Swedish academic high ranking specialists in fields of relevance for the process, namely psychiatry (three), epidemiology and stress re- search (three), work psychology (one) and family prac- tice (one). This group was divided into pairs with as widely differing specialty in the pair as possible. In the following process, the articles remaining in the process were randomly assigned to the four pairs (with avoid- ance of author bias). Concordance in judgments of rele- vance and quality was trained. After the training session, each member of the pair did the assessments separately, and then discordances were discussed within the pair. If disagreement remained another pair was asked to make an independent judgment. If that deci- sion was in disagreement with the first group, we made the decision in the whole group. The inclusion criteria for studies were: 1. The study should have examined the importance of the work environment for depressive symptoms. Our review was not confined to any specific kind of work environment factors. Physical/chemical/ ergonomic exposures as well as psychosocial factors were screened. 2. The study should be relevant for Swedish conditions and focused on people at work. Work environments in Europe, North America, Australia and New Zealand were included. 3. In the study symptoms of depression should have been analyzed. These should have have been certified through diagnostic investigation or with established scales. We argued that not only diagnosed major depression, but also milder states with depressive symptoms are relevant since depressive feelings give rise to suffering, increase the risk of long term sick leave and cause productivity decline and quality loss in work places [11]. Thus, our review included both studies with standardized clinical interviews regarding diagnosed depression and studies based upon rating scales on depressive symptoms. As diagnosed depression is also to a large extent based on symptoms we decided that the most accurate naming of the outcome of our review was depressive symptoms. A few studies were based upon either sick leave data or registered anti- depression medication as outcome but these studies are not included in this review. 3. Methods In summary, the evidence about the negative impact of certain work environments for depressive symptoms is accumulating but so far there has been no review taking the entire spectrum of adverse working condi- tions into account and at the same time focusing on The present review was based upon studies with a pro- spective design and is focused on the relationship between working conditions and development of symp- toms of depression among the employees.. We con- ducted and funded this systematic review within the Theorell et al. BMC Public Health (2015) 15:738 Page 3 of 14 5. The study should have been published between the years 1990 and (June) 2013 and written in English. 5. The study should have been published between the years 1990 and (June) 2013 and written in English. 5. The study should have been published between the years 1990 and (June) 2013 and written in English. framework for the Swedish Council on Health and Tech- nology Assessment, a public agency with the charge of providing impartial and scientifically reliable information to decision makers and health care providers [10]. 6. Prospective or comparable case-control design. Only prospective cohort, case control (with design equivalent to prospective) and randomized intervention studies with at least 100 participants were included. By case control studies with “design equivalent to prospective” we are referring to studies with strict definition of cases recruited in a representative way in the same population as the control group. Search strategy Systematic literature search was performed in the following data bases: PubMed, Embase, Psycinfo, Arbline (Swedish database), Cochrane library and NIOSHTIC-2. A combin- ation of controlled search words (e.g., MeSH) and free- text words was used. The search strategy for the outcome was performed for mesh terms (‘Depression’ and ‘Depressive Disorders’) and as free search in title and abstract (depress and dysthym*). The whole search strategy is available at http://www.sbu.se/upload/Publikationer/Content0/1/223E/ Inclusion%20criteria_occupational%20exposure_depression _burnout.pdf. We only accepted as articles in scientific journals with independent reviews. Assessments of exposure should have been made be- fore disease onset. Doublets were systematically identified and only the most relevant publication in a doublet was included. Analyses of relevance and quality Abstract screening and full-text assessment were con- ducted by a specialist in occupational medicine and a psychiatrist. GRADE procedure A i An important aspect of the systematic review process was to systematically and transparently assess the scien- tific evidence. According to the GRADE instructions explicit consideration should be given to each of the GRADE criteria for assessing the quality of evidence (risk of bias/study limitations, directness, consistency of results, precision, publication bias, magnitude of the ef- fect, dose-response gradient, influence of residual plaus- ible confounding and bias “antagonistic bias”) although different terminology may be used. For level 4 (=High), randomized trials are required and there were no such published relevant studies in our search. For observa- tional studies of the kind included in the present review, the highest possible grade is Moderate = 3 if there is sufficient reason for an upgrading from the normal level for such studies of 2 (=Limited). Level 1 (=Very limited) corresponds to evidence based on case reports and case series or on reports downgraded evidence from observa- tional studies. 2.) Confounding. Age and at least some aspect of socioeconomic conditions should have been considered. Gender specific analyses were preferred but when such analyses were not available, adjustment for gender was required. Life habits such as smoking habits and alcohol consumption were not taken into account as confounders in our review. 2.) Confounding. Age and at least some aspect of socioeconomic conditions should have been considered. Gender specific analyses were preferred but when such analyses were not available, adjustment for gender was required. Life habits such as smoking habits and alcohol consumption were not taken into account as confounders in our review. 3.) Prospective data collection. All results of the studies included in this review (apart from case-control studies) are based upon assessments of exposure and depressive symptoms in the beginning and of the depressive symptoms again at least one year later. In the calculations of associations a design with either exclusion of subjects with depressive symptoms at baseline or adjustment for baseline level of depressive symptoms was required. Qualified statistics and thorough discussion of longitudinal data rendered higher quality ratings. We allowed for upgrading the scientific evidence when there was strong coherence of results between studies - according to the most recent guidelines [12]. Inclusion criteria In the study symptoms of depression should have been analyzed. These should have have been certified through diagnostic investigation or with established scales. We argued that not only diagnosed major depression, but also milder states with depressive symptoms are relevant since depressive feelings give rise to suffering, increase the risk of long term sick leave and cause productivity decline and quality loss in work places [11]. Thus, our review included both studies with standardized clinical interviews regarding diagnosed depression and studies based upon rating scales on depressive symptoms. As diagnosed depression is also to a large extent based on symptoms we decided that the most accurate naming of the outcome of our review was depressive symptoms. A few studies were based upon either sick leave data or registered anti- depression medication as outcome but these studies are not included in this review. In the first expert phase, the group judged relevance. Relevance criteria are presented in http://www.sbu.se/up- load/Publikationer/Content0/1/223E/Inclusion%20criteria _occupational%20exposure_depression_burnout.pdf. Secondly, we performed a quality assessment. Three levels of quality rating were used, (low, medium high and high quality) and in the final grading process only those with medium high and high quality were accepted. Accordingly the important dividing line was between poor and medium high quality whereas the distinction between medium high and high was less crucial. Studies on the borderline between low and medium high quality were accordingly re-examined by the whole group. A list 4. A minimum of 100 persons should have been included in the exposed group and the results were controlled for at least age and gender. Theorell et al. BMC Public Health (2015) 15:738 Page 4 of 14 of relevant articles meeting the inclusion criteria judged to be of low quality is available at http://www.sbu.se/up- load/Publikationer/Content0/1/223E/Inclusion%20cri- teria_occupational%20exposure_depression_burnout.pdf high effort and poor reward). Since the overall aim of the present study was to grade total evidence, not to as- sess magnitude of associations, and since it was impos- sible to re-construct operationalizations in such a way that they would match one another we decided to use the definitions presented by the authors themselves and to mostly abstain from assessment of overall magnitude of the different relationships. The following aspects of quality were considered: 1.) Representativeness of study sample. Representativeness and ways of defining and recruiting the sample as well as attrition in different steps were considered in the quality rating. Inclusion criteria Statistical considerations and an insightful discussion of possible consequences of a possible systematic drop-out for findings were required in case of marked drop-out problems. The final list of studies judged to be of high or medium high quality is listed in Appendix. GRADE procedure A i Accordingly when there were many published observa- tional studies of medium high or high quality with homogenous results (almost all pointing in the same direction although all findings may not have been statis- tically significant) the evidence was graded on level 3 (two exposures, high decision latitude as protective and job strain as negative exposure, see below). Level 3 can also be used according to the GRADE system even when there are relatively few studies if there are unanimous findings with high odds ratios (above 2.0). This occurred for one exposure – bullying (see below). 4.) For both exposure and outcome assessment, psychometrically standardized and validated methods were required. Well established methods enable comparison across studies and therefore contributed to higher quality rating. 5.) Designs that enable the analysis of a dose response relationship contributed to a high quality rating. For instance, in a few studies the work environment was assessed in two or three subsequent waves and the development of depressive symptoms followed up after the last assessment. Exposure to given work environment factor on one, two or three occasions could be regarded as a progressive duration of exposure and was regarded as equivalent of a dose- response analysis. 5.) Designs that enable the analysis of a dose response relationship contributed to a high quality rating. For instance, in a few studies the work environment was assessed in two or three subsequent waves and the development of depressive symptoms followed up after the last assessment. Exposure to given work environment factor on one, two or three occasions could be regarded as a progressive duration of exposure and was regarded as equivalent of a dose- response analysis. Ethics In these tests, we conducted sub-analyses of the presented find- ings and compared results between the sub-categories, e.g., if the association between job exposure and de- pressive symptoms differed according to the instru- ment used for assessing the symptoms. All studies perused in this review have been approved by the scientific ethical committees in their universities. They have all been published in international scientific journals with peer review. Accordingly, no additional ethical approval has been required. Meta-analyses/Forest plots y p In the studies results were reported as calculations of association, e.g., expressed as odds ratios, from mul- tiple logistic regression, multivariate correlations or multiple linear regression coefficients. Whenever pos- sible, the results were transformed into multiple logis- tic regression odds ratios. Forest plots were used for visual interpretation. To assist in illustrating the Even between studies of specific work environment factors there were differences with regard to operationa- lization of exposure. Examples are job strain (combin- ation of high psychological demands and low decision latitude) and effort reward imbalance (combination of Theorell et al. BMC Public Health (2015) 15:738 Page 5 of 14 Ethics results, and as a contribution to the overall assess- ment, these forest plots (meta-analyses) were con- ducted when in at least two studies the same risk factor was analysed and mathematically comparable data was provided using the Comprehensive Meta- Analysis software package (www.meta-analysis.com/ index.php). Since the participants in the various stud- ies might be construed as coming from the same popu- lation (workers) or from different populations (i.e., according to each study’s inclusion criteria) we chose to use a fixed effects model. The strength of the scien- tific evidence, using data from all of the included stud- ies (not just those illustrated in the meta-analyses), was determined by pairs of the authors of this paper and then discussed and confirmed by all authors. In- formal homogeneity tests were performed in order to compare results from studies using standardized de- pression interviews versus self-reported question- naires, high quality versus medium high quality studies, general population studies versus specific oc- cupational cohorts and men versus women. In these tests, we conducted sub-analyses of the presented find- ings and compared results between the sub-categories, e.g., if the association between job exposure and de- pressive symptoms differed according to the instru- ment used for assessing the symptoms. results, and as a contribution to the overall assess- ment, these forest plots (meta-analyses) were con- ducted when in at least two studies the same risk factor was analysed and mathematically comparable data was provided using the Comprehensive Meta- Analysis software package (www.meta-analysis.com/ index.php). Since the participants in the various stud- ies might be construed as coming from the same popu- lation (workers) or from different populations (i.e., according to each study’s inclusion criteria) we chose to use a fixed effects model. The strength of the scien- tific evidence, using data from all of the included stud- ies (not just those illustrated in the meta-analyses), was determined by pairs of the authors of this paper and then discussed and confirmed by all authors. In- formal homogeneity tests were performed in order to compare results from studies using standardized de- pression interviews versus self-reported question- naires, high quality versus medium high quality studies, general population studies versus specific oc- cupational cohorts and men versus women. Results Figure 1 shows the number of articles that were perused in the different steps. The process also included burnout as outcome. The results of the burnout review will be reported elsewhere. Altogether 20 828 articles were screened in the initial search process, and 488 of those were eligible in the review of depressive symptoms (and 202 for the review of burnout). 324 full text articles with depression as outcome were found not to fill inclu- sion criteria. Hence, 164 studies remained for relevance assessment. 84 of those were judged as not relevant and hence 80 studies were assessed with regard to quality. 19 were judged to be of high, 40 of moderately high and 21 of low quality. The grading of evidence has been based upon the 59 relevant studies with high/medium high quality. A detailed table showing the full results of the data extraction is available at http://www.sbu.se/upload/ Publikationer/Content0/1/223E/Inclusion%20criteria_ occupational%20exposure_depression_burnout.pdf. Fig. 1 Flow chart of the literature search, screening, review- and quality assessment Fig. 1 Flow chart of the literature search, screening, review- and quality assessment Fig. 1 Flow chart of the literature search, screening, review- and quality assessment Theorell et al. BMC Public Health (2015) 15:738 Theorell et al. BMC Public Health (2015) 15:738 Page 6 of 14 Studies- Depression Scale, CES-D, and Hospital Anx- iety and Depression Scale, HAD, and Hamilton Depres- sion Scale, HRSD) for depressive symptoms were used. Most studies were based on population samples al- though studies of samples from companies and occupa- tional groups were also present. Few studies that were judged to be relevant were based upon objective assess- ments of exposure. Subjective assessments based upon standardized and validated questionnaires (for instance demand/control/support, effort/reward, procedural just- ice and bullying) were used in most studies. The most widely used established questionnaires rendered high quality ratings. With regard to depression outcome, both standardized interviews (mostly Composite Inter- national Diagnostic Interview, CIDI) performed by trained interviewers and different versions of standard- ized questionnaires (such as Center for Epidemiological Studies- Depression Scale, CES-D, and Hospital Anx- iety and Depression Scale, HAD, and Hamilton Depres- sion Scale, HRSD) for depressive symptoms were used. Table 1 shows the results of the evidence grading process. Three exposures, two harmful (job strain and bullying) and one protective (control/decision latitude) were judged to have moderate evidence (grade 3) while 18 exposures were judged to have limited (grade 2) evidence. - There is scientific evidence for an association between exposure and outcome. The result is based on studies of high or moderate quality - It is not possible to determine if there is any association between exposure and outcome. The motivation is that one or several conditions apply: 1) no study fulfilled the inclusion criteria, 2) none of the studies fulfilling the inclusion criteria were relevant to the hypothesis tested in the present review, 3) all relevant studies were of low quality or 4) studies were of high or moderate quality - but one or several limitations applied, e.g. inconsistency of data between studies - There is scientific evidence for an association between exposure and outcome. The result is based on studies of high or moderate quality. The quality of evidence has been upgraded due to consistency of the data (control and job strain) or large magnitude of effect (bullying) evidence has been upgraded due to consistency of the data (control and job strain) or large magnitude of effect (bullying) - There is scientific evidence for an association between exposure and outcome. The result is based on studies of high or modera is scientific evidence for an association between exposure and outcome. The result is based on studies of high or moderate quality. Th ce has been upgraded due to consistency of the data (control and job strain) or large magnitude of effect (bullying) Results Ten exposures were judged to have very lim- ited evidence (grade 1). Three of the exposures judged to have very limited evidence were related to heavy metals and other chemical exposures. The most exten- sively studied factors were decision latitude (158 251 subjects in 19 studies) and job strain - the combination Table 1 A summary of the scientific evidence for variables with sufficient data to draw a conclusion on the association between work environment factors and future depressive symptoms Work-related factor Participants Studies Scientific evidence Relationship between occupational environment and less depressive symptoms Control 158 251 19 Relationship between occupational environment and more depressive symptoms Demands - psychological job demands 53 985 10 Job strain 197 682 14 Passive job (low decision latitude, low job demands) 11 419 2 High pressure job 34 554 5 Effort reward imbalance 27 136 3 Low support at the work place 82 772 17 - Low supervisor support 50 935 8 - Low co-worker support 27 170 6 Poor social climate at the work place 9 242 2 Poor social capital at the work place 59 340 2 Low work place justice 33 589 5 - Procedural injustice 33 589 5 - Relational injustice 30 761 3 Work place conflicts 13 732 3 - Conflicts with superiors 9 692 2 - Conflicts with co-workers 9 692 2 Bullying 15 173 3 Low job development 15 173 4 Job insecurity 24 833 7 Long working week 13 107 6 The scientific evidence is in-sufficient ( ) to determine if there is a relationship between the following occupational factors and depressive symptoms/ Demands (several types of demands), Demands (emotional), Distributive justice, Threats, Violence, Irregular, Irregular work hours, Physically demanding work, Pesticides, Solvents, Heavy metals - There is scientific evidence for an association between exposure and outcome. The result is based on studies of high or moderate quality. The quality of of the scientific evidence for variables with sufficient data to draw a conclusion on the association between ctors and future depressive symptoms The scientific evidence is in-sufficient ( ) to determine if there is a relationship between the following occupational fac - There is scientific evidence for an association between exposure and outcome. The result is based on studies of high or moderate quality. Discussion of high psychological demands and low decision latitude (197 682 subjects in 14 studies). It was possible to com- pute a weighted odds ratio 1.74 (95 % CI 1.54 to 1.96 for studies with odds ratio calculations). A high decision latitude protected statistically against worsening depres- sive symptoms – with a weighted odds ratio of 0.73 (95 % CI 0.68 to 0.77). Bullying had been studied in 15 173 subjects in three studies. One of these studies showed results for men and women separately. Despite the relatively small number of studies, bullying was judged to be related to worsening depressive symptoms with an evidence grade of 3 as the findings were very consistent and the odds ratios were high (the weighted odds ratio being 2.82; 95 % CI 2.21 to 3.59). of high psychological demands and low decision latitude (197 682 subjects in 14 studies). It was possible to com- pute a weighted odds ratio 1.74 (95 % CI 1.54 to 1.96 for studies with odds ratio calculations). A high decision latitude protected statistically against worsening depres- sive symptoms – with a weighted odds ratio of 0.73 (95 % CI 0.68 to 0.77). Bullying had been studied in 15 173 subjects in three studies. One of these studies showed results for men and women separately. Despite the relatively small number of studies, bullying was judged to be related to worsening depressive symptoms with an evidence grade of 3 as the findings were very consistent and the odds ratios were high (the weighted odds ratio being 2.82; 95 % CI 2.21 to 3.59). Main findings and recent developments in the field The aim of the study was to provide systematically graded evidence for possible associations between work environment factors and near-future development of de- pressive symptoms. A total of fifty-nine relevant articles with high or medium high scientific quality fulfilling our criteria were found. The results provide evidence for several work conditions being linked to depressive symp- toms among the employees in both positive and negative directions. Scientific evidence of grade three out of four (in other words moderately strong) was shown for job strain (high psychological demands and low decision latitude), low decision latitude and bullying. Discussion Further- more, scientific evidence of grade two was found for psychological demands, effort reward imbalance, low support, unfavorable social climate, lack of procedural and relational justice, conflicts with superiors and col- leagues, limited skill discretion, job insecurity and long working week. Figure 2 shows forest plots for the three factors with evidence grade 3 - decision latitude (a), job strain (b) and bullying (c). For high decision latitude, 17/18 point estimates were lower than 1.0 (separate point estimates for men and women in five studies). The upper 95 % confidence limit was above 1.0 in five studies. For job strain, 14/15 point estimates were above 1.0. Three lower confidence limits reached below 1.0. The forest plots were based upon studies from which odds ratios could be extracted or calculated. It should be pointed out, however, that the total evidence grading also included a few additional studies. Bullying, finally, had four point estimates in the diagram. All of those were higher than 2.0 and all the lower confidence limits were above 1.0. An important finding is that there were few prospect- ive studies with sufficient quality of the relationship be- tween adverse chemical (pesticides and heavy metals for instance) and physical (heavy loads, awkward positions, irradiation, cold and hot temperature) and depressive symptoms. This field needs more research. The results should primarily be interpreted in the con- text of the Western world. We deliberately limited our inclusion of studies to these countries. The rationale be- hind this was that we wanted to secure similar cultural framework around work in order to simplify our inter- pretation of the findings. The exposures with a limited level of evidence were psychological demands (quantitative psychological demands defined according to the widely used Job Content Questionnaire or alternative psychometrically tested versions), the combination of low psychological demands and low decision latitude (“passive work”), “pressing work” (mainly important life events at work), effort reward imbalance, low social support (from management and coworkers), poor social climate, poor social capital, low procedural and relational justice, conflicts with superiors and colleagues, poor skill discretion, job insecurity and long working weeks (the latter for women only). The review differs from earlier studies in the field due to its comprehensive and thorough approach. Our re- view is based on an extremely thorough literature search as well as on a well-described and systematic evaluation of a large number of publications. Results The quality of evidence has been upgraded due to consistency of the data (control and job strain) or large magnitude of effect (bullying) p g q y - It is not possible to determine if there is any association between exposure and outcome. The motivation is that one or several conditions apply: 1) no study fulfilled the inclusion criteria, 2) none of the studies fulfilling the inclusion criteria were relevant to the hypothesis tested in the present review, 3) all relevant studies were of low quality or 4) studies were of high or moderate quality - but one or several limitations applied, e.g. inconsistency of data between studies Theorell et al. BMC Public Health (2015) 15:738 Theorell et al. BMC Public Health (2015) 15:738 Page 7 of 14 Page 7 of 14 Page 7 of 14 Discussion Data have been re-calculated to show the association between high level of control and development of depressive symptoms (data in these studies are presented as association between low level of control and depressive symptoms). b. Job strain, The graph is based on data from the least adjusted model in studies expressing the strength of the association either as odds ratios or as correlations (the latter have been transformed into odds ratios). Please note that data from three more studies (Ibrahim et al. 2009, Wieclaw et al. 2008 and Mantyniemi et al. 2012) are included in the evidence-rated result; however data from these studies could not be illustrated in the graph due to the data format. c. Bullying, The graph is based on data from the least adjusted model in studies expressing the strength of the association either as odds ratios or as correlations (the latter have been transformed into odds ratios) Fig. 2 Association between work environment factors and development of depressive symptoms when evidence was judged as moderate (grade 3), a. Decision latitude, The graph is based on data from the least adjusted model in studies expressing the strength of the association either as odds ratios or as correlations (the latter have been transformed into odds ratios). Ylipaavalniemi et al.: “Healthy at baseline” refers to a doctor diagnosis/non-diagnosis of depression. Please note that data from six more studies (Dagher et al. 2011, Magnusson Hansson et al. 2009, Paterniti et al. 2002, Plaisier et al. 2007, Rugulies et al. 2006 and Wieclaw et al. 2008) are included in the evidence-rated result; however data from these studies could not be illustrated in the graph due to the data format. Data have been re-calculated to show the association between high level of control and development of depressive symptoms (data in these studies are presented as association between low level of control and depressive symptoms). b. Job strain, The graph is based on data from the least adjusted model in studies expressing the strength of the association either as odds ratios or as correlations (the latter have been transformed into odds ratios). Please note that data from three more Fig. 2 Association between work environment factors and development of depressive symptoms when evidence was judged as moderate (grade 3), a. Discussion Thus, it includes all kinds of environmental exposures, physical as well as psychosocial and that it is based upon a systematic ap- proach. This is the first review in which the examination of evidence follows (a slight modification of) GRADE principles. Furthermore it is including more recently published research than previous reviews. The exposures with very limited (= level 1) evidence were other kinds of demands (not quantitative) including emotional demands, distributive justice, threats, vio- lence, irregular working hours, long working hours (men), physically demanding work, exposure to pesti- cides and insecticides, solvents and heavy metals. Our review shows that the psychosocial research field has made progress since the reviews published in 2008 and 2010. Bonde [1] and Netterström et al. [2] made critical remarks about possible publication bias, lack of more “objective” measures of exposure and outcome and also about lack of time perspectives which would be needed for the understanding of time of exposure needed for the development of depression. With regard to object- ive measures, there are more published studies than previously with standardized interview based assessment of clinical depression. Comparison of the plots corresponding Homogeneity tests showed that results were compar- able for two groups of outcome measures (standardized interview versus standardized self-report questionnaire), for men and women, for general population versus spe- cific occupation cohorts and for white collar versus blue collar groups. Theorell et al. BMC Public Health (2015) 15:738 Page 8 of 14 Fig. 2 Association between work environment factors and development of depressive symptoms when evidence was judged as moderate (grade 3), a. Decision latitude, The graph is based on data from the least adjusted model in studies expressing the strength of the association either as odds ratios or as correlations (the latter have been transformed into odds ratios). Ylipaavalniemi et al.: “Healthy at baseline” refers to a doctor diagnosis/non-diagnosis of depression. Please note that data from six more studies (Dagher et al. 2011, Magnusson Hansson et al. 2009, Paterniti et al. 2002, Plaisier et al. 2007, Rugulies et al. 2006 and Wieclaw et al. 2008) are included in the evidence-rated result; however data from these studies could not be illustrated in the graph due to the data format. Technical issues I hi i In this review we have not reviewed evidence whether there is interaction or not between high psychological demands and low decision latitude (as discussed for in- stance in Karasek and Theorell [24]). We have regarded the combination simply as a theoretical construction and evaluated its possible success or lack of success as a predictor of development of depressive symptoms. In forest plots, we chose to use data from the least ad- justed model from each study. The main rationale for this was that these models were more comparable between studies than other models, since the more adjusted ones were adjusted to widely different potential confounders. The most powerful prognostic factor for incident depres- sive symptoms was manifest symptoms at the study base- line; a parameter that had to be assessed in each of the included studies. Generally, adjusting for other con- founders had very little effect. For transparency, we have listed data in both least and most adjusted models, see ex- tensive tables at http://www.sbu.se/upload/Publikationer/ Content0/1/223E/Inclusion%20criteria_occupational%20 exposure_depression_burnout.pdf. The literature search included articles published up to June 2013. For practical reasons it has not been possible to do a full review of the articles published after that date. However, a more informal search in the scientific litera- ture (PubMed and PsycInfo until February 2015) showed that a few more recent prospective studies of work envir- onment and development of depressive feelings relevant to the present review have been published. None of those would have changed our conclusions. Four of them sup- port the use of standardized measures of job strain or high psychological demands and low decision latitude in pre- dicting either depressive symptoms or major depressive disorder [18–21] and one of them supports the use of ef- fort reward imbalance (or low reward) in the prediction of disability pension due to depression [21]. An important point is that if a study presented data in several statistical models, all data from all models were in- cluded in the expert group assessment of scientific evidence for all of the results presented in this systematic review. Assessments of odds ratios may be somewhat unreli- able due to differences in methodology across studies and also due to the fact that summary odds ratios could not be calculated for some of the occupational expo- sures. Discussion Decision latitude, The graph is based on data from the least adjusted model in studies expressing the strength of the association either as odds ratios or as correlations (the latter have been transformed into odds ratios). Ylipaavalniemi et al.: “Healthy at baseline” refers to a doctor diagnosis/non-diagnosis of depression. Please note that data from six more studies (Dagher et al. 2011, Magnusson Hansson et al. 2009, Paterniti et al. 2002, Plaisier et al. 2007, Rugulies et al. 2006 and Wieclaw et al. 2008) are included in the evidence-rated result; however data from these studies could not be illustrated in the graph due to the data format. Data have been re-calculated to show the association between high level of control and development of depressive symptoms (data in these studies are presented as association between low level of control and depressive symptoms). b. Job strain, The graph is based on data from the least adjusted model in studies expressing the strength of the association either as odds ratios or as correlations (the latter have been transformed into odds ratios). Please note that data from three more studies (Ibrahim et al. 2009, Wieclaw et al. 2008 and Mantyniemi et al. 2012) are included in the evidence-rated result; however data from these studies could not be illustrated in the graph due to the data format. c. Bullying, The graph is based on data from the least adjusted model in studies expressing the strength of the association either as odds ratios or as correlations (the latter have been transformed into odds ratios) Theorell et al. BMC Public Health (2015) 15:738 Theorell et al. BMC Public Health (2015) 15:738 Page 9 of 14 (MDD). For example, a Canadian study showed that men had elevated risk of MDD only if they were exposed to extremely high level of job strain while women had elevated risk of MDD even when exposed to moderate job strain [23]. The study points to the need of context- ualizing findings about mental health and it may also illustrate that gender could be more relevant for the relationship between working conditions and major depressive disorder than for the relationship between working conditions and depressive symptoms. to results from studies based upon standardized interviews did not differ from those from studies based upon inter- nationally accepted depression questionnaires. Objective exposures are still uncommon, however. One interesting approach was used by Virtanen et al. Technical issues I hi i It should however be pointed out that for most of the studied exposures the observed risks were of moder- ate size. Discussion [13] who could show that hospital staff who experienced excess occupancy of hospital beds had increased risk of developing sick leave be- cause of depression in a dose-response manner, with excess occupancy exceeding 10 % being associated with an odds ratio of sick leave for depression of 1.94 (1.14-3.28). During later years research designs on the association between work environment factors and depressive feel- ings have become increasingly sophisticated. For in- stance, Shields [14], Stansfeld et al. [15], De Lange et al. [16] and Wang et al. [17] have examined possible effects of exposure to job strain at least twice, or even three times in the follow-up survey waves. Their findings indi- cate that accumulated or increasing job strain has a stronger adverse statistical effect on risk of experiencing increased ratings of depressive symptoms during follow- up than decreasing job strain. As might be expected, these studies show that two or more assessments of the job situation provide more precise information regarding risk than only one measurement. Therefore stronger evi- dence regarding the influence of working conditions on mental health may be expected in future research with a growing body of studies with such methodology. Gender Our results showed that similar work conditions were related to a similar increase in depressive symptoms among men and women. However, although there is no gender difference in excess risk associated with adverse work conditions, studies have shown that women actu- ally have higher levels of job strain than men [22]. This may be one reason for women’s higher prevalence of de- pressive symptoms. Other studies indicate that work conditions can affect men and women differently in rela- tion to development of major depressive disorder The operationalization of job strain differed between the studies. The majority of the published studies used the median split definition (above median for the psy- chological demands score and below median for the de- cision latitude score). When exposure to job strain is defined in this way and the remaining participants in the study are defined as unexposed there is relatively little contrast between unexposed and exposed subjects. This may lead to underestimation of the true association. Theorell et al. BMC Public Health (2015) 15:738 Page 10 of 14 Page 10 of 14 As recommended in the epidemiological literature we produced funnel plots to investigate possible publication bias. When there is pronounced publication bias, studies reporting “confirming” odds ratios with wide confidence intervals are more common than studies reporting “rejecting” odds ratios with wide confidence intervals. Such an analysis cannot replace a real analysis of publi- cation bias – the best analysis would be to contact re- searchers asking for unpublished studies. But according to our exploration of the material, there was no such evidence of publication bias. were examined in many studies but did not achieve level 3 in the grading system such as psychological demands - which only yielded evidence level 2. This illustrates the need for more detailed studies of different aspects of demands, such as emotional demands. In addition, ef- fort reward imbalance was consistently associated with worsening depressive symptoms in three studies of medium high quality and was classified as second grade evidence. The summarized odds ratio was 1.78 which is comparable to the corresponding odds ratio for job strain which was 1.74. However, job strain had been examined in 14 studies and therefore achieved third grade evidence. Societal relevance h f Despite the often moderate sizes of our findings, some of the associations are of considerable societal import- ance. An illustration of this is that if a work environ- ment factor has a prevalence of 25 % and is associated with a relative risk of 1.8, the resulting population at- tributable risk is 11 %. Accordingly, when an exposure is common (as is the case with job strain, low level of control and effort reward imbalance with the oper- ational definitions that have been used) even a moder- ately elevated risk associated with it becomes important in a societal context. Due to the fact that the researchers in the included studies had chosen a wide range of different statistical measures to express associations between occupational exposure and depressive symptoms, it was not possible to conduct formal mathematical homogeneity analyses including the entire data material. Instead, the expert group conducted a combination of mathematical and narrative sub-group analyses to explore whether the re- sults were homogenous when subgroups of studies were compared. Accordingly results were compared for men versus women, for self-reported versus clinically rated depression/depressive symptoms, for general population studies versus specific occupational cohorts and for high/medium high quality studies. Inspection of the con- fidence interval distributions, as well as sub-populations' summary odds ratios and their confidence intervals showed homogenous results across those dichotomies. The work environment factors for which we found scientific evidence for an association to depressive symptom development are possible to influence by means of work organization changes. For instance, it has been shown that decision latitude for employees can be improved by analysis of the work organization with subsequent goal-directed organization interven- tion [27, 28] or by a year-long education of managers about psychosocial factors [29]. A review of natural ex- periments designed to reduce psychosocial risks in the work environment for bus drivers showed that such in- terventions may result in reduced biological stress in that group [30]. The present results suggest that in as- sessment and treatment plans of depression, work en- vironment should be taken into account. Limitations Another limitation is that we have not included out- side work factors that may be of importance. As pointed out for instance by Wang and Schmitz [26] job strain may interact with psychosocial factors outside of the workplace in relation to the risk of major depression, and such interactions may in addition differ between men and women. Most studies were based upon self-reports of both work- ing conditions and depressive symptoms. Few of the studies were based upon in situ investigation of the work environment and standardized clinical interviews of em- ployees. Such interviews are more objective and may more often identify depression than standardized self- rating questionnaires which primarily have screening or follow-up indications. The risk of inflated associations may arise, when there are subjective descriptions both of explanatory and dependent factors [25]. This is particu- larly the case in cross-sectional studies while in pro- spective studies this risk is less pronounced. The risk of inflated association decreases as adjustments are per- formed for initial symptoms of mental disease and when the assessments of working conditions and mental symptoms are standardized. Accordingly, in this study we only included prospective studies (and comparable case-control studies) with data on initial symptoms and standardized measures of exposure and outcome. Appendix d d Studies judged to be of high or medium high quality Ahola K, Hakanen J. Job strain, burnout, and depres- sive symptoms: a prospective study among dentists. J Affect Disord 2007;104:103-10. Godin I, Kittel F, Coppieters Y, Siegrist J. A prospective study of cumulative job stress in relation to mental health. BMC Public Health 2005;5:67. Andrea H, Bultmann U, van Amelsvoort LG, Kant Y. The incidence of anxiety and depression among em- ployees – the role of psychosocial work characteristics. Depress Anxiety 2009;26:1040-8. Andrea H, Bultmann U, van Amelsvoort LG, Kant Y. The incidence of anxiety and depression among em- ployees – the role of psychosocial work characteristics. Depress Anxiety 2009;26:1040-8. Goodman WB, Crouter AC. Longitudinal associations between maternal work stress, negative work-family spillover, and depressive symptoms. Fam Relat 2009; 58:245-58. Beseler CL, Stallones L, Hoppin JA, Alavanja MC, Blair A, Keefe T, et al. Depression and pesticide expo- sures among private pesticide applicators enrolled in the Agricultural Health Study. Environ Health Per- spect 2008;116:1713-9. Beseler CL, Stallones L, Hoppin JA, Alavanja MC, Blair A, Keefe T, et al. Depression and pesticide expo- sures among private pesticide applicators enrolled in the Agricultural Health Study. Environ Health Per- spect 2008;116:1713-9. Griffin JM, Fuhrer R, Stansfeld SA, Marmot M. The importance of low control at work and home on depres- sion and anxiety: do these effects vary by gender and so- cial class? Soc Sci Med 2002;54:783-98. Grynderup MB, Mors O, Hansen AM, Andersen JH, Bonde JP, Kaergaard A, et al. Work-unit measures of or- ganisational justice and risk of depression –a 2-year co- hort study. Occup Environ Med 2013;70:380-5. Bonde JP, Munch-Hansen T, WieclawJ, Westergaard- Nielsen N, Agerbo E. Psychosocial work environment and antidepressant medication: a prospective cohort study. BMC Public Health 2009;9:262. Grynderup MB, Mors O, Hansen AM, Andersen JH, Bonde JP, Kaergaard A, et al. A two-year follow-up study of risk of depression according to work-unit measures of psychological demands and decision latitude. Scand J Work Environ Health 2012;38:527-36. Burgard SA, Brand JE, House JS. Perceived job inse- curity and worker health in the United States. Soc Sci Med 2009;69:777-85. Clays E, De Bacquer D, Leynen F, Kornitzer M, Kittel F, De Backer G. Job stress and depression symptoms in middle-aged workers – prospective results from the Bels- tress study. Scand J Work Environ Health 2007;33:252-9. Grzywacz JG, Quandt SA, Chen H, Isom S, Kiang L, Vallejos Q, et al. Conclusions A limitation of our review could be that we may have underestimated the importance of work environment factors that have not been subjected to many empirical studies. There were examples of exposure factors that There is substantial empirical evidence that employees, both men and women, who report lack of decision lati- tude, job strain and bullying, will experience increasing depressive symptoms over time. Page 11 of 14 Theorell et al. BMC Public Health (2015) 15:738 on a sample of Italian workers. Int Arch OccupEnviron Health 2011;84:413-24. on a sample of Italian workers. Int Arch OccupEnviron Health 2011;84:413-24. Many of the work environmental factors can be favor- ably influenced by effective organizational interventions. An important step in this research field would be the launching of good evaluations of psychosocial interven- tions. For some kinds of working conditions that are de- veloping in the modern working world, new research on depressive symptoms will be needed. Many of the work environmental factors can be favor- ably influenced by effective organizational interventions. De Santo Iennaco J, Cullen MR, Cantley L, Slade MD, Fiellin M, Kasl SV. Effects of externally rated job de- mand and control on depression diagnosis claims in an industrial cohort. Am J Epidemiol 2010;171:303-11. An important step in this research field would be the launching of good evaluations of psychosocial interven- tions. For some kinds of working conditions that are de- veloping in the modern working world, new research on depressive symptoms will be needed. Fandino-Losada A, Forsell Y, Lundberg I. Demands, skill discretion, decision authority and social climate at work as determinants of major depression in a 3-year follow-up study. Int Arch Occup Environ Health 2013;86:591-605. Epub 2012 Jul 4. Appendix d d Lang J, Bliese PD, Lang JW, Adler AB. Work gets un- fair for the depressed: cross-lagged relations between organizational justice perceptions and depressive symp- toms. J Appl Psychol 2011;96:602-18. Schonfeld IS. Stress in 1st-year women teachers: the context of social support and coping. Genet Soc Gen Psychol Monogr 2001;127:133-68. Levin A, Besser A, Albert L, Smith D, Neria Y. The effect of attorneys’ work with trauma- exposed clients on PTSD symptoms, depression, and functional impairment: a cross- lagged longitudinal study. Law Hum Behav 2012;36:538-47. Shields M. Stress and depression in the employed population. Health Rep 2006;17:11-29. Magnusson Hanson LL, Theorell T, Bech P, Rugulies R, Burr H, Hyde M, et al. Psychosocial working condi- tions and depressive symptoms among Swedish em- ployees. Int Arch Occup Environ Health 2009;82:951-60. Shields M. Long working hours and health. Health Rep 1999;11:33-48 (Eng); 37-55 (Fre). Sinokki M, Hinkka K, Ahola K, Koskinen S, Kivimäki M, Honkonen T, et al. The association of social support at work and in private life with mental health and anti- depressant use: the Health 2000 Study. J Affect Disord 2009;115:36-45. Mantyniemi A, Oksanen T, Salo P, Virtanen M, Sjosten N, Pentti J, et al. Job strain and the risk of disability pen- sion due to musculoskeletal disorders, depression or cor- onary heart disease: a prospective cohort study of 69,842 employees. Occup Environ Med 2012;69:574-81. Stansfeld SA, Shipley MJ, Head J, Fuhrer R. Repeated job strain and the risk of depression: longitudinal ana- lyses from the Whitehall II study. Am J Public Health 2012;102:2360-6. Niedhammer I, Goldberg M, Leclerc A, Bugel I, David S. Psychosocial factors at work and subsequent depres- sive symptoms in the Gazel cohort. Scand J Work En- viron Health 1998;24:197-205. Stoetzer U, Ahlberg G, Johansson G, Bergman P, Hallsten L, Forsell Y, et al. Problematic interpersonal re- lationships at work and depression: a Swedish prospect- ive cohort study. J Occup Health 2009;51:144-51. Oksanen T, Kouvonen A, Vahtera J, Virtanen M, Kivimäki M. Prospective study of workplace social capital and depression: are vertical and horizontal com- ponents equally important? J Epidemiol Community Health 2010;64:684-9. Theorell T, Nyberg A, Leineweber C, Magnusson Hanson LL, Oxenstierna G, Westerlund H. Non- listening and self centered leadership – relationships to socioeconomic conditions and employee mental health. PLoS One 2012;7:e44119. Parker SK. Longitudinal effects of lean production on employee outcomes and the mediating role of work characteristics. J Appl Psychol 2003;88:620-34. Appendix d d Depressive symptoms among Latino farm workers across the agricultural season: structural and situational influences. Cultur Divers Ethnic Minor Psychol 2010;16:335-43. Clumeck N, Kempenaers C, Godin I, Dramaix M, Kornitzer M, Linkowski P, et al. Working conditions pre- dict incidence of long-term spells of sick leave due to de- pression: results from the Belstress I prospective study. J Epidemiol Community Health 2009;63:286-9 Ibrahim S, Smith P, Muntaner C. A multi-group cross- lagged analysis of work stressors and health using a Canadian National sample. Soc Sci Med 2009;68:49-59. Dagher RK, McGovern PM, Dowd BE, Lundberg U. Postpartum depressive symptoms and the combined load of paid and unpaid work: a longitudinal analysis. Int Arch Occup Environ Health 2011;84:735-43. Jensen HK, Wieclaw J, Munch-Hansen T, Thulstrup AM, Bonde JP. Does dissatisfaction with psychosocial work climate predict depressive, anxiety and substance abuse disorders? A prospective study of Danish public service employees. J Epidemiol Community Health 2010;64:796-801. De Lange AH, Taris TW, Kompier MAJ, Houtman ILD, Bongers PM. The relationships between work char- acteristics and mental health: examining normal, re- versed and reciprocal relationships in a 4-wavestudy. Work & Stress 2004;18:149-66. Kivimaki M, Vahtera J, Elovainio M, Virtanen M, Siegrist J. Effort-reward imbalance, procedural injustice and rela- tional injustice as psychosocial predictors of health: com- plementary or redundant models? Occup Environ Med 2007;64:659-65. de Lange AH, Taris TW, Kompier MA, Houtman IL, Bongers PM. Effects of stable and changing demand- control histories on worker health. Scand J Work En- viron Health 2002;28:94-108. Kivimäki M, Virtanen M, Vartia M, Elovainio M, Vahtera J, Keltikangas-Jarvinen L. Workplace bullying and the risk of cardiovascular disease and depression. Occup Environ Med 2003;60:779- 83. d’Errico A, Cardano M, LandriscinaT, Marinacci C, Pasian S, Petrelli A, et al. Workplace stress and prescrip- tion of antidepressant medications: a prospective study Theorell et al. BMC Public Health (2015) 15:738 Page 12 of 14 prolonged unemployment: a 3.5-year follow-up study. J Epidemiol Community Health 2010;64:75-81. prolonged unemployment: a 3.5-year follow-up study. J Epidemiol Community Health 2010;64:75-81. Kouvonen A, Oksanen T, Vahtera J, Stafford M, Wilkinson R, Schneider J, et al. Low workplace social capital as a predictor of depression: the Finnish Public Sector Study. Am J Epidemiol 2008;167:1143-51. Rugulies R, Bultmann U, Aust B, Burr H. Psychosocial work environment and incidence of severe depressive symptoms: prospective findings from a 5-yearfollow-up of the Danish work environment cohort study. Am J Epidemiol 2006;163:877-87. Appendix d d 5Division of Psychiatry, Lund University, SE-221 00 Lund, Sweden. 6South Health Care Centre, SE-761 46 Norrtälje, Sweden. 7Division of Occupational medicine, Institute for Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 8Division of Psychiatry, Linköping University, SE-581 83 Linköping, Sweden. 9Department of Psychiatry and Neurochemistry, Göteborg University, SE-411 24 Stockholm, Sweden. Wieclaw J, Agerbo E, Mortensen PB, Burr H, Tuchsen F, Bonde JP. Work related violence and threats and the risk of depression and stress disorders. J Epidemiol Community Health 2006;60:771-5. 10Swedish Council of Health Technology Assessment, SE-103 59 Stockholm, Sweden. 10Swedish Council of Health Technology Assessment, SE-103 59 Stockholm, Sweden. Received: 7 March 2015 Accepted: 19 June 2015 Received: 7 March 2015 Accepted: 19 June 2015 Virtanen M, Stansfeld SA, Fuhrer R, Ferrie JE, Kivimäki M. Overtime work as a predictor of major depressive epi- sode: a 5-year follow-up of the Whitehall II study. PLoS One 2012;7:e30719. Authors’ information The authors deliberately represent a diversity of academic fields, namely psychiatry (LTB, IS, IM), work psychology (GA), occupational epidemiology (AH, CH), stress research (TT) and general practice (TG). 16. de Lange AH, Taris TW, Kompier MA, Houtman IL, Bongers PM. Effects of stable and changing demand-control histories on worker health. Scand J Work Environ Health. 2002;28:94–108. 16. de Lange AH, Taris TW, Kompier MA, Houtman IL, Bongers PM. Effects of stable and changing demand-control histories on worker health. Scand J Work Environ Health. 2002;28:94–108. References 1. Bonde JP. Psychosocial factors at work and risk of depression: a systematic review of the epidemiological evidence. Occup Environ Med. 2008;65:438–45. 1. Bonde JP. Psychosocial factors at work and risk of depression: a systematic review of the epidemiological evidence. Occup Environ Med. 2008;65:438–45. Virtanen M, Ferrie JE, Singh-Manoux A, Shipley MJ, Stansfeld SA, Marmot MG, et al. Long working hours and symptoms of anxiety and depression: a 5-year follow-up of the Whitehall II study. Psychol Med 2011;41:2485-94. 2. Netterström B, ConradM BP, Fink P, Olsen O, Rugulies R, Stansfeld S. The relation between work-related psychosocial factors and the development of depression. Epidemiol Rev. 2008;30:118–32. 3. Siegrist J. Chronic psychosocial stress at work and risk of depression: evidence from prospective studies. Eur Arch Psychiatry Clin Neurosci. 2008;258(suppl):115–9. Virtanen M, Batty GD, Pentti J, Vahtera J, Oksanen T, Tuisku K, et al. Patient overcrowding in hospital wards as a predictor of diagnosis-specific mental disorders among staff: a 2-year prospective cohort study. J Clin- Psychiatry 2010;71:1308-12. 4. Michie S, Williams S. Reducing work related psychological ill health and sickness absence: a systematic literature review. Occup Environ Med. 2003;60:3–9. 5. Egan M, Bambra C, Thomas S, Petticrew M, Whitehead M, Thomson H. The psychosocial and health effects of workplace reorganisation. A systematic review of organisational-level interventions that aim to increase employee control. J Epidemiol Community Health. 2007;61:945–54. Virtanen M, Honkonen T, KivimäkiM, Ahola K, Vahtera J, Aromaa A, et al. Work stress, mental health and antidepressant medication findings from the Health 2000 Study. J Affect Disord 2007;98:189-97. Virtanen M, Honkonen T, KivimäkiM, Ahola K, Vahtera J, Aromaa A, et al. Work stress, mental health and antidepressant medication findings from the Health 2000 Study. J Affect Disord 2007;98:189-97. control. J Epidemiol Community Health. 2007;61:945–54. 6. Woo J-M, Postolache TT. The impact of work environment on mood disorders and suicide: Evidence and implications. Int J Disabil Hum Dev. 2008;7:185–200. Ybema JF, van den Bos K. Effects of organizational justice on depressive symptoms and sickness absence: a longitudinal perspective. Soc Sci Med 2010;70:1609-17. Ybema JF, van den Bos K. Effects of organizational justice on depressive symptoms and sickness absence: a longitudinal perspective. Soc Sci Med 2010;70:1609-17. 7. Freire C, Koifman S. Pesticides, depression and suicide: A systematic review of the epidemiological evidence. Int J Hygiene and Environ Health. 2013;216:445–60. 8. Niuwenhuijsen N, Bruinvelds D, Frings-Dresen M. Psychosocial work environment and stress-related disorders, a systematic review. References Occup Med. 2010;60:277–86. Ylipaavalniemi J, Kivimaki M, Elovainio M, Virtanen M, Keltikangas-Jarvinen L, Vahtera J. Psychosocial work characteristics and incidence of newly diagnosed depres- sion: a prospective cohort study of three different models. Soc Sci Med 2005;61:111-22. 9. Guyatt G, Oxman AD, Akl EA, Kunz R, Vist G, Brozek J, et al. GRADE guidelines: 1. Introduction—GRADE evidence profiles and summary of findings tables. J Clin Epidemiol. 2011;64(4):383–94. 11. Jauregui M, Schnall PL. Work, psychosocial stressors and the bottom line. In Schnall PL, Dobson M and Rosskam E (eds) Unhealthy Work. Amityville, NY: Baywood Publ; 2009. 11. Jauregui M, Schnall PL. Work, psychosocial stressors and the bottom line. In Schnall PL, Dobson M and Rosskam E (eds) Unhealthy Work. Amityville, NY: Baywood Publ; 2009. Sweden. 4Department of Psychology, Stockholm University, SE-106 91 Stockholm, Sweden. 5Division of Psychiatry, Lund University, SE-221 00 Lund, Sweden. 6South Health Care Centre, SE-761 46 Norrtälje, Sweden. 7Division of Occupational medicine, Institute for Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm, Sweden. 8Division of Psychiatry, Linköping University, SE-581 83 Linköping, Sweden. 9Department of Psychiatry and Neurochemistry, Göteborg University, SE-411 24 Stockholm, Sweden. 10 Acknowledgements 17. Wang J, Schmitz N, Dewa C, Stansfeld S. Changes in perceived job strain and the risk of major depression: results from a population-based longitudinal study. Am J Epidemiol. 2009;169:1085–91. 17. Wang J, Schmitz N, Dewa C, Stansfeld S. Changes in perceived job strain and the risk of major depression: results from a population-based longitudinal study. Am J Epidemiol. 2009;169:1085–91. This study has been funded by the Swedish Council of Health Technology Assessment. The Swedish Government has had no role in the work, neither in the scientific work itself nor in the writing of the article. The review has been funded by the Swedish Council of Health Technology Assessment. 18. Bailey TS, Dollard MF, Richards PA. A national standard for psychosocial safety climate (PSC): PSC 41 as the benchmark for low risk of job strain and depressive symptoms. J Occup Health Psychol. 2015;20(1):15–26. doi:10.1037/a0038166. Epub 2014 Oct 27. 18. Bailey TS, Dollard MF, Richards PA. A national standard for psychosocial safety climate (PSC): PSC 41 as the benchmark for low risk of job strain and depressive symptoms. J Occup Health Psychol. 2015;20(1):15–26. doi:10.1037/a0038166. Epub 2014 Oct 27. Authors’ contributions 13. Virtanen M, Batty GD, Pentti J, Vahtera J, Oksanen T, Tuisku K, et al. Patient overcrowding in hospital wards as a predictor of diagnosis-specific mental disorders among staff: a 2-year prospective cohort study. J Clin Psychiatry. 2010;71:1308–12. 13. Virtanen M, Batty GD, Pentti J, Vahtera J, Oksanen T, Tuisku K, et al. Patient overcrowding in hospital wards as a predictor of diagnosis-specific mental disorders among staff: a 2-year prospective cohort study. J Clin Psychiatry. 2010;71:1308–12. TT was chairman of the expert committee and has conceptualized and written large parts of the manuscript. GA, LTB, TG, AH, CH (1), IM and IS have all contributed substantially to the expert panel work and have made important comments to the manuscript. CH (2) has performed most of the statistical work and contributed both to the conceptualizing and the writing of the manuscript. All authors read and approved the final manuscript. 14. Shields M. Stress and depression in the employed population. Health Rep. 2006;17:11–29. 14. Shields M. Stress and depression in the employed population. Health Rep. 2006;17:11–29. 15. Stansfeld SA, Shipley MJ, Head J, Fuhrer R. Repeated job strain and the risk of depression: longitudinal analyses from the Whitehall II study. Am J Public Health. 2012;102:2360–6. 15. Stansfeld SA, Shipley MJ, Head J, Fuhrer R. Repeated job strain and the risk of depression: longitudinal analyses from the Whitehall II study. Am J Public Health. 2012;102:2360–6. Competing interests p g The authors declare that they have no competing interests. g The authors declare that they have no competing interests. 12. The Grade Approach. http://handbook.cochrane.org/chapter_12/12_2_1_the _grade_approach.htm 12. The Grade Approach. http://handbook.cochrane.org/chapter_12/12_2_1_the _grade_approach.htm Appendix d d Wang J, Schmitz N. Does job strain interact with psy- chosocial factors outside of the workplace in relation to the risk of major depression? The CanadianNational Population Health Survey. Soc Psychiatry Psychiatr Epi- demiol 2011;46:577-84. Paterniti S, Niedhammer I, Lang T, Consoli SM. Psy- chosocial factors at work, personality traits and depres- sive symptoms. Longitudinal results from the GAZEL Study. Br J Psychiatry 2002;181:111-7. Plaisier I, de Bruijn JG, de Graaf R, ten Have M, Beekman AT, Penninx BW. The contribution of working conditions and social support to the onset of depressive and anxiety disorders among male and female em- ployees. Soc Sci Med 2007;64:401-10. Wang J, Schmitz N, Dewa C, Stansfeld S. Changes in perceived job strain and the risk of major depression: re- sults from a population-based longitudinal study. Am J Epidemiol 2009;169:1085- 91. Wang J. Work stress as a risk factor for major depres- sive episode(s). Psychol Med 2005;35:865- 71. Rugulies R, Aust B, Madsen IE, Burr H, Siegrist J, Bultmann U. Adverse psychosocial working conditions and risk of severe depressive symptoms. Do effects differ by occupational grade? Eur J Public Health 2013;23:415- 20. Epub 2012 Jun 8. Wang J. Perceived work stress and major depressive episodes in a population of employed Canadians over 18 years old. J Nerv Ment Dis 2004;192:160-3. Varma A, Marott JL, Stoltenberg CD, Wieclaw J, Kolstad HA, Bonde JP. With long hours of work, might depression then lurk? A nationwide prospective follow- up study among Danish senior medical consultants. Scand J Work Environ Health 2012;38:418-26. Rugulies R, Madsen IE, Hjarsbech PU, Hogh A, Borg V, Carneiro IG, et al. Bullying at work and onset of a major depressive episode among Danish female eldercare workers. Scand J Work Environ Health 2012;38:218-27. Rugulies R, Thielen K, Nygaard E, Diderichsen F. Job insecurity and the use of antidepressant medication among Danish employees with and without a history of Weisskopf MG, Moisan F, TzourioC, Rathouz PJ, Elbaz A. Pesticide exposure and depression among agricultural workers in France. Am J Epidemiol 2013;178:1051-8. Theorell et al. BMC Public Health (2015) 15:738 Page 13 of 14 Page 13 of 14 Wieclaw J, Agerbo E, Mortensen PB, Burr H, Tuchsen F, Bonde JP. Psychosocial working conditions and the risk of depression and anxiety disorders in the Danish workforce. BMC Public Health 2008;8:280. Sweden. 4Department of Psychology, Stockholm University, SE-106 91 Stockholm, Sweden. Theorell et al. BMC Public Health (2015) 15:738 Theorell et al. BMC Public Health (2015) 15:738 20. Niedhammer I, Malard L, Chastang J-F. Occupational factors and subsequent depressive and generalized anxiety disorders in the prospective French SIP study. BMC Public Health. http://www.biomedcentral.com/1471-2458/15/200 21. Juvani A, Oksanen T, Salo P, Virtanen M, Kivimäki M, Pentti J, et al. Effort- reward imbalance as a risk factor for disability pension: the Finnish Public Sector Study. Scand J Work Environ Health. 2014;40(3):266–77. doi:10.5271/ sjweh.3402. Epub 2013 Nov 18. 22. Theorell T, Hammarström A, Gustafsson PE, Magnusson Hanson L, Janlert U, Westerlund H. Job strain and depressive symptoms in men and women: a prospective study of the working population in Sweden. J Epidemiol Community Health. 2014;68:78–82. 23. Wang J, Patten SB, Currie S, Sareen J, Schmitz N. A population-based longitudinal study on work environmental factors and the risk of major depressive disorder. Am J Epidemiol. 2012;176(1):52–9. 24. Karasek RA, Theorell T. Healthy Work: stress, productivity and the reconstruction of working life. New York: Basic Books; 1990. 25. Theorell T, Hasselhorn HM. On cross-sectional questionnaire studies of relationships between psychosocial conditions at work and health – are they reliable? Int Arch Occup Environ Health. 2005;78:517–22. 26. Wang J, Schmitz N. Does job strain interact with psychosocial factors outside of the workplace in relation to the risk of major depression? The Canadian National Population Health Survey. Soc Psychiatry Epidemiol. 2011;46:577–84. 27. Bond FW, Bunce D. Job control mediates change in a work reorganization intervention for stress reduction. J Occup Health Psychol. 2001;6:290–302. 28. Bourbonnais R, Brisson C, Vézina M. Long-term effects of an intervention on psychosocial work factors among healthcare professionals in a hospital setting. Occup Environ Med. 2011;68(7):479–86. 29. Theorell T, Emdad R, Arnetz B, Weingarten AM. Employee effects of an educational program for managers at an insurance company. Psychosom Med. 2001;63(5):724–33. 30. Kompier MA, Aust B, von den Berg AM, Siegrist J. Stress prevention in bus drivers: evaluation of 13 natural experiments. Occup Health Psychol. 2000;5(1):11–31. Author details 1 1Stress Research Institute, Stockholm University, SE-106 91 Stockholm, Sweden. 2Department of Neuroscience, Karolinska Institutet, SE- 171 77 Stockholm, Sweden. 3Department of Public Health and Clinical Medicine, Department of social medicine, Umeå University Hospital, SE-901 85 Umeå, 19. Grzywacz JG, Alterman T, Gabbard S, Shen R, Nakamoto J, Carroll DJ, et al. Job control, psychological demand, and farmworker health: evidence from the national agricultural workers survey. J Occup Environ Med. 2014;56(1):66–71. doi:10.1097/JOM.0000000000000025. Page 14 of 14 Theorell et al. 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https://openalex.org/W2741661958	https://www.scielo.br/j/cerne/a/7SbjGWzXDBTfTSVjwz5X96f/?lang=en&format=pdf	English	null	MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. Amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE	Cerne	2,017	cc-by	5,465	ISSN 0104-7760 Kaléo Dias Pereira1+, Rafael Gomes Viana2, Jonilson Ribeiro Trindade2, Rafael Amaral Cardoso2 MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. Amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE ABSTRACT: The objective of this study was to evaluate the morphological and physiological changes in paricá plants (Schizolobium parahyba var. amazonicum) intoxicated by glyphosate. The experiment was conducted in a protected environment using paricá plants during their planting stage, which were intoxicated with increasing doses of glyphosate: 0 (control); 43.2; 86.2; 129.6 and 172.8 g.ha-1. At 7 and 21 days after the application of the herbicide, the photosynthesis, transpiration, stomatal conductance and leaf temperature were measured. The visual intoxication degree and the growth of the shoot and the root of the plants were evaluated 21 days after the application. Paricá shows symptoms of visual intoxication characterized by chlorosis/winding, evolving to necrosis/abscission of the youngest leaflets. The growth of the stem and the roots of the intoxicated plants is preserved; however, an expressive leaf loss occurs, and paricá may have adaptation mechanisms to tolerate the action of the herbicide molecule. The photosynthesis decrease promoted by an indirect action of glyphosate represents the main reduction on the growth of plants. The decrease on the stomatal conductance, which was the most sensitive physiological variable to glyphosate, resulted in lower transpiration rates, which, consequently, caused increases on the leaf temperature. Histórico: Recebido 24/02/2017 Aceito 05/06/2017 Histórico: Recebido 24/02/2017 Aceito 05/06/2017 Keywords: Chemical control Drift Paricá Weeds +Correspondência: kaleoef@gmail.com Palavras chave: Controle químico Deriva Paricá Plantas daninhas Histórico: Recebido 24/02/2017 Aceito 05/06/2017 MATERIAL AND METHODS The study was conducted in a protected environment with 50% shading and lateral openings for air circulation. The three-month old paricá plantings were planted on pots with capacity for 7 L, containing a substrate constituted by soil and nutshells at a 2:1 proportion (v/v). After planting, the seedlings went through an acclimatization period of 20 days, during which two fertilizations were conducted with 10 g of NPK per pot at a 10-28-20 formulation (CARVALHO, 2005; LOPES et al., 2015). Although some silvicultural aspects related to the culture of paricá have already been relatively widely studied, there are still gaps of information, such as in relation to the management of weeds. Rosa (2006b) mentions the procedure used by the company Pampa, Vigia – PA, as an example of weed management in paricá plantations in the Amazon, which uses mechanical hoeing and chemical control of weeds with glyphosate. According to the monitoring data of the company, the elimination of weeds resulted in a reduction from 25 to 6% of paricá mortality during the first year in the field. At the end of the acclimatization period, a drift simulation was conducted, applying five treatments related to reduced doses of glyphosate: 0 (control); 43.2; 86.2; 129.6 and 172.8 g.ha-1 of active ingredient on acid equivalent, according to the sub-doses used by Machado et al. (2010) on a study with eucalyptus. The herbicide was applied by a CO2 pressurized sprayer, constituted by a 1m-wide bar equipped with two TT11002 spraying nozzles, previously calibrated for a spray volume of 200 L.ha-1. The sprayed solution was directly applied on the plant leaves. Therefore, weed control is necessary with the purpose of minimizing the potential interferences on the culture of paricá that may, somehow, reduce the productivity of the species. The use of herbicides is an attractive option, since it reduces costs and increases the operational yield (SILVA et al., 2007a). On the day of the application, the plants were distributed so that the treatments had equal height means, which was equivalent to a general mean of 41.34 cm. The experiment was established on random blocks, with five replications, and an experimental unit was constituted by one pot with one plant. INTRODUCTION on the plant development and physiological changes (TUFFI SANTOS et al., 2007b; MACHADO et al., 2010). In practice, according to Tuffi Santos et al. (2007a), the damages caused by the glyphosate drift, or by some other herbicide, distributed at different levels on the plantation, would increase the heterogeneity of the plot, since the intraspecific competition is favorable to the less affected individuals. The commercial plantations of paricá are still constituted by seminal plantings, therefore, the drift effect would be added to the genetic variability of the plants, making the heterogeneity of the population more accentuated. In 2012, the Brazilian government presented the final version of the Low Carbon Agricultural Plan – ABC Plan, which, among other goals, established the commitment of expanding the planted forest areas to 9 million hectares until 2020 (MAPA/MDA, 2011). According to IBGE (2014), in 2014, the area with forest plantations had exceeded 9.3 million hectares, mostly occupied by eucalyptus and pine plantations. Among the native species of the Amazon, Schizolobium parahyba var. amazonicum (Huber ex Ducke) Barneby (LEWIS, 2016), locally known as paricá, is currently one of the main species used for reforestation purposes, with over 89 thousand planted hectares (IBÁ, 2015). Considering the implications of the drift on the development of the forest plantation, our objective was to evaluate the morphological and physiological changes on young paricá plants (Schizolobium parahyba var. amazonicum) intoxicated by glyphosate. The growing interest for paricá is due to its quick growth. It may reach a productivity of 38 m3.ha-1.year-1 (CARVALHO, 2007) even with no improvement degree; therefore, it is one of the few native species that may catch up with the productivity of eucalyptus. Another determining factor, according to Marques et al. (2006), is the increase on the demand for wood to produce laminates and plywood, a context in which paricá is inserted (ROSA, 2006a). MODIFICAÇÕES MORFOLÓGICAS E FISIOLÓGICAS EM PLANTAS DE Schizolobium parahyba VAR. amazonicum (HUBER EX DUCKE) BARNEBY INTOXICADAS POR GLYPHOSATE RESUMO: Objetivou-se avaliar as variações morfofisiológicas em plantas de paricá (Schizolobium parahyba var. amazonicum) intoxicadas por glyphosate. O experimento foi realizado em ambiente protegido utilizando plantas de paricá em idade de plantio, as quais foram intoxicadas com doses crescentes de glyphosate: 0 (testemunha); 43,2; 86,2; 129,6 e 172,8 g.ha-1. Aos 7 e 21 dias após a aplicação do herbicida, foram realizadas medições da fotossíntese líquida, transpiração, condutância estomática e temperatura da folha. O grau de intoxicação visual e crescimento da parte aérea e da raiz das plantas foram avaliados aos 21 dias após a aplicação. O paricá manifesta sintomas de intoxicação visual caracterizados por clorose/enrolamento e evolução para necrose/abscisão dos foliolulos mais novos. O crescimento do caule e da raiz das plantas intoxicadas é conservado, contudo, há expressiva perda foliar, podendo o paricá possuir mecanismos de adaptação para tolerar a ação da molécula do herbicida. O decréscimo promovido na fotossíntese por ação indireta do glyphosate representa a principal causa da redução no crescimento das plantas. O decréscimo na condutância estomática, a qual demonstrou ser a variável fisiológica mais sensível ao glyphosate, resultou em menores taxas de transpiração, que, consequentemente, ocasionou elevações na temperatura da folha. DOI: 10.1590/01047760201723022316 1 Federal University of Viçosa - Viçosa, Minas Gerais, Brazil 2 Federal Rural University of Amazon - Belém, Pará, Brazil 1 Federal University of Viçosa - Viçosa, Minas Gerais, Brazil 2 Federal Rural University of Amazon - Belém, Pará, Brazil DOI: 10.1590/01047760201723022316 CERNE \| v. 23 n. 2 \| p. 267-274 \| 2017 MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE MATERIAL AND METHODS It is important to point out that there are no herbicides to be used on paricá plantations registered at the Ministry of Agriculture, Livestock and Supply, nor there are studies that evaluate the effects of herbicides on the species. It is necessary to determine the levels and types of damages that may be caused to paricá in case of herbicide intoxication, or even whether selectivity to the applied product occurs. The physiological evaluations were conducted 7 and 21 days after the application of glyphosate, between 9a.m. and 12p.m., every day, using an infrared gas analyzer (model LI-6400XT from LI-COR). The physiological Studies conducted with eucalyptus under the effect of glyphosate drift found significant reductions CERNE \| v. 23 n. 2 \| p. 267-274 \| 2017 268 PEREIRA et al. plant (Table 1). Although paricá has shown relatively reasonable resistance when intoxication symptoms are considered, it was observed that a reduction occurred on the leaf area from the dose of 43.2 g.ha-1 of glyphosate in relation to the control. The mean leaf area of the plants sprayed with 172.8 g.ha-1 was 507.57 cm2 (Table 1), which is equivalent to approximately 20% of the mean on the control (Figure 1B). parameters analyzed were photosynthesis (A), stomatal conductance (gs), transpiration (E) and leaf temperature (Tf), at constant light (1500 µmol.m-2.s-1) and CO2 (400 µmol) levels. For the readings, were selected leaves located on the intermediate region of the plants. At 21 days after the application, the visual toxicity degree was verifi ed according to grade scales from 0 to 100% of intoxication (FRANS, 1972), the stem base diameter and height were measured, and the root, stem and leaves of all plants were collected. After collecting the material, the leaf area was measured using a leaf area analyzer (model LAI 3100C). Finally, all of the collected material was subjected to the drying process on a force- air-circulation oven at 50º C for 72 hours, posteriorly weighting the dry mass of the root, stem and leaves. Evaluating the effect of glyphosate on different eucalyptus clones, Machado et al. (2010) verifi ed, 21 days after the application, that the 172.8 g.ha-1 dose of a.i. on acid equivalent caused from 70 to 86% of leaf damages. The same dose was tested on fi ve eucalyptus species by Tuffi Santos et al. MATERIAL AND METHODS (2006), and a mean of 47.5% of intoxication 15 days after the application was observed; only Eucalyptus resinifera and E. grandis showed damages below 50%. The morphology and physiology data were subjected to the normality analysis by the Kolmogorov- Smirnov test at 5% of signifi cance, homogeneity analysis through the Levene test at 5%, and homogenization of the treatments based on the standard deviation. A regression analysis was conducted with the use of models that showed an adequate adjustment. The comparisons with the control plants were conducted through the Dunnett test at 1 and 5% of signifi cance. TABLE 1 Morphological variables of S. parahyba var. amazonicum 21 days after the application of glyphosate. TABLE 1 Morphological variables of S. parahyba var. amazonicum 21 days after the application of glyphosate. Variables Glyphosate dose (g.ha-1 on acid equivalent) 0 43,2 86,2 129,6 172,8 Visual intoxication 0.001 1.33ns 28.71* 30.50 43.00 Leaf area 2,527.82 1,725.27* 806.64 548.32 507.57** Stem Diameter 9.25 9.14ns 8.73ns 7.57* 8.22ns Height 57.08 51.90ns 52.08ns 46.28** 48.58* Leaf dry mass 10.82 8.32ns 3.94 2.63 2.33** Stem dry mass 9.35 8.23ns 6.89ns 5.51* 6.39ns Root dry mass 2.24 2.48ns 1.65ns 1.42ns 1.57ns Total dry mass 22.42 19.03ns 12.48 9.56 10.29** 1Comparison with dose 0 (control) on the row: ns – non-signifi cant; * and ** signifi cant difference according to the Dunnett test at 5 and 1%, respectively. FIGURE 2 Height and stem diameter of S. parahyba var. amazonicum 21 days after the application of glyphosate. On a study conducted by Wagner Júnior et al. (2008) with passion fruit (Passiﬂ ora edulis), the intoxication was below 10% up to 28 days after the application of 172.8 g.ha-1 of glyphosate, similarly to what Yamashita et al. (2006) observed on Parkia multijuga plants. The drift effect on Jatropha curcas evaluated on the study by Costa et al. (2009) reached 38.7% of intoxication 21 days after the application of 180 g.ha-1. Costa et al. (2012) observed a more mild leaf loss tendency on Eucalyptus grandis, reaching a reduction of 57.6% with 120 g.ha-1 of glyphosate applied only on the leaves. Magalhães et al. (2001a) and Magalhães et al. (2001b) did not observe a signifi cant variation on the leaf area of maize and sorghum, respectively, using doses of up to 172.8 g.ha-1 of glyphosate. FIGURE 2 Height and stem diameter of S. parahyba var. amazonicum 21 days after the application of glyphosate. The leaf abscission observed must have occurred probably due to the increase on the ethylene production through an indirect action of glyphosate (FUCHS et al., 2002; GRAVENA, 2006). According to Yamada and Castro (2007), ethylene increases the cellulase activity, which makes the leaves more susceptible to abscission, in addition to promoting the loss of chlorophyll; therefore, it is one of the factors responsible for the chlorosis. Ethylene derives from the amino acid methionine (SCHALLER; KIEBER, 2002), which is, in turn, produced at a greater amount when the molecule of the herbicide inhibits the route that originates the aromatic amino acids phenylalanine, tryptophan and tyrosine (YAMADA; CASTRO, 2007). The total dry mass was reduced in approximately 55% with the application of 172.8 g.ha-1 (Figure 3A), observing a signifi cant variation from the 86.2 g.ha-1 dose in relation to the control mean (Table 1). Both the stem dry mass and the root dry mass suffered a reduction of approximately 30% on the most intoxicated plants when compared to the control plants. Although the decrease on the total dry mass was expressive, this is mostly due to the loss of leaf dry mass (Figure 3A and B), with a reduction of almost 80% upon the application of the highest dose. Only the mean of the lowest dose was equal to the control (Table 1). RESULTS AND DISCUSSION The visual toxicity symptoms on paricá were clearly manifested on plants that received 172.8 g.ha-1 of glyphosate. They were initially characterized by chlorosis and winding of the leafl ets before evolving to necrosis and leaf abscission. The symptoms started to manifest between the fi fth and the seventh day after application, always from the youngest leafl ets to the oldest ones, from the top of the leaf blade toward the basis. 1Comparison with dose 0 (control) on the row: ns – non-signifi cant; * and ** signifi cant difference according to the Dunnett test at 5 and 1%, respectively. FIGURE 1 Visual intoxication degree and leaf area of S. parahyba var. amazonicum 21 days after the application of glyphosate. Since glyphosate is a molecule that is translocated through the phloem, a higher concentration of it and an effect on parts of the plant with greater carbohydrate discharge is common, such as on growth points and strong drains (HETHERINGTON et al., 1999; MONQUERO et al., 2004). Although no records of the effect of glyphosate on paricá were found, the symptoms observed on this study are similar to the ones observed on eucalyptus by Tuffi Santos et al. (2009), with the difference that the symptoms on paricá were more evident for the dose of 172.8 g.ha-1 of glyphosate. The visual intoxication observed 21 days after the application of glyphosate reached its maximum degree at a dose of 172.8 g.ha-1 of a.i. on acid equivalent, causing 43% of leaf damages (Figure 1A). From the dose of 86.2 g.ha-1, signifi cant damages occurred in relation to the control FIGURE 1 Visual intoxication degree and leaf area of S. parahyba var. amazonicum 21 days after the application of glyphosate. 269 MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE PEREIRA et al. In order to reduce the dry mass of the root, leaves and stem of paricá in 50%, doses of 219.86; 90.10 and 216.35 g.ha-1, respectively, are necessary, while the total dry mass is reduced with 131.73 g.ha-1. route of shikimate, thus, reducing the carbon fi xation capacity of the plant (YAMADA; CASTRO, 2007). Such phenomenon was shown by Servaites et al. (1987) on a study with common beat (Beta vulgaris), in which they observed a 50% reduction on the ribulose bisphosphate activity 12 hours after the application of 17 mmol.plant-1. In opposition to the reduction on the leaf area and the leaf dry mass, the growth of the diameter and the root was relatively not much affect; this could be the effect of some self-preservation mechanism, such as the change on the translocation of carbohydrates in order to limit the action of the herbicide molecule (SILVA et al., 2007b). Air temperature: 32.39° C (7 days) and 33.87° C (21 days). According to Salgado et al. (2011), the dry mass of the leaves and stem of hybrids of Eucalyptus grandis x E. urophylla is reduced in half with the application of 143.3 to 277.4 g.ha-1 of glyphosate. In the case of the study conducted by Silva et al. (2015), the mass of the shoot components of Eucalyptus “urograndis” was reduced in 50% with doses of 72 g.ha-1 (stem) and 122 g.ha-1 (leaf) of glyphosate, while, for Pinus taeda, the same effect was promoted by doses of 120 g.ha-1 (stem) and 44 g.ha-1 (leaf) of glyphosate. The growth inhibition due to the action of glyphosate, both on the shoot and on the root, occurs partially due to the reduction on the synthesis and the concentration of indoleacetic acid (IAA) (LEE; DUMAS, 1983; LEE; DUMAS, 1985), as well as due to the reduction on the translocation speed of the hormone (BAUR, 1979). Yamada and Castro (2007) explain that, by inhibiting the synthesis of phenolic compounds, glyphosate reduces the concentration of the IAA precursor (tryptophan) and increases the IAA-oxidase activity, which would be normally regulated by the phenolic compounds. With the increase on the activity of the IAA-oxidase enzyme, the metabolism of free IAA increases, especially on plants that are sensitive to the herbicide, as observed by Lee and Dumas (1985). Air temperature: 32.39° C (7 days) and 33.87° C (21 days). FIGURE 3 Dry mass of the root, leaf, stem, total dry mass (A) and participation of assimilates on the total dry mass (B) of S. parahyba var. amazonicum 21 days after the application of glyphosate. The height of the plants varied signifi cantly from the dose of 129.6 g.ha-1 (Table 1), and a reduction occurred on approximately 15% with the dose of 172.8 g ha-1 in relation to the control (Figure 2A). There was a smaller variation on the diameter, and a decrease slightly above 11% occurred at the highest dose in comparison to the control (Figure 2B). Using clones of hybrids of Eucalyptus grandis x E. urophylla, Santos Junior et al. (2005) evaluated the total height and diameter (measured at 1.3 m) 30 days after the application of 1,080 g.ha-1 of glyphosate on 100% of the basal branches and observed reductions of 33.19 and 31.44%, respectively. Costa et al. (2012) verifi ed a reduction of 37.5% on the stem diameter 28 days after the application of 120 g.ha-1 of glyphosate. Tuffi Santos et al. (2006) did not observe variations on the height and diameter of E. urophylla 45 days after the application of up to 172.8 g.ha-1 of glyphosate, although it has also been compared together with other molecules and combinations. FIGURE 3 Dry mass of the root, leaf, stem, total dry mass (A) and participation of assimilates on the total dry mass (B) of S. parahyba var. amazonicum 21 days after the application of glyphosate. CERNE \| v. 23 n. 2 \| p. 267-274 \| 2017 CERNE \| v. 23 n. 2 \| p. 267-274 \| 2017 270 PEREIRA et al. REFERENCES BAUR, J. R. Effect of glyphosate on auxin transport in corn and cotton tissues. Plant Physiology, v. 63, p. 882-886, 1979. CARVALHO, C. J. R. de. Respostas de plantas de Schizolobium amazonicum [S. parahyba var. amazonicum] e Schizolobium parahyba [Schizolobium parahybum] à deficiência hídrica. Revista Árvore, v.29, n.6, p.907-914, 2005. When working with common beat, Geiger et al. (1986) observed a considerable decrease on gs approximately four hours after the application of 17 mmol.plant-1 of glyphosate with and without surfactant. Machado et al. (2010) reported an accentuated reduction on gs of eucalyptus 21 days after the application of up to 172.8 g.ha-1 of glyphosate. CARVALHO, F. P. de; FRANÇA, A. C.; LEMOS, V. T.; FERREIRA, E. A.; SANTOS, J. B. dos; SILVA, A. A. da. Photosynthetic activity of coffee after application of glyphosate subdoses. Acta Scientiarum. Agronomy, v. 35, n. 1, p. 109-115, 2013. CARVALHO, P. E. R. Paricá - Schizolobium amazonicum. Colombo: Embrapa Florestas, 2007. 8 p. (Circular Técnica, 142). It is known that the stomatal opening is promoted by the blue light through a specific stimulation and through the activation of the photosynthesis process on the chloroplasts or guard cells (TAIZ; ZEIGER, 2002). Therefore, if glyphosate has a negative effect on the photosynthesis, then, the opening and gs are partially reduced. CATANEO, A. C.; DÉSTRO, G. F. G.; FERREIRA, L. C.; CHAMMA, K. L.; SOUSA, D. C. F. Atividade de glutationa S-transferase na degradação do herbicida glyphosate em plantas de milho (Zea mays). Planta Daninha, v. 21, n. 2, p. 307-312, 2003. As a consequence of the reduction on gs, there was a decrease on E for paricá as the glyphosate doses increased. On both evaluations, the 129.6 and 172.8 g .ha-1 doses differed from the control (Table 2), with a more expressive variation 21 days after the application (Figure 4C). As a response to the lower transpiration rate, an increase was observed on the Tf (Figure 4D) with a significant variation, in comparison to the control, at the dose of 172.8 g.ha-1 at 7 days, and from the 129.6 g.ha-1 at 21 days after the application (Table 2). COSTA, A. C. P. R. da; COSTA, N. V. da; PEREIRA, M. R. R.; MARTINS, D. Efeito da deriva simulada de glyphosate em diferentes partes da planta de Eucalyptus grandis. Semina: Ciências Agrárias, v. 33, n. 5, p. 1663-1672, 2012. COSTA, N. V.; ERASMO, E. A. L.; QUEIROZ, P. The application of 196.27 g.ha-1 of active ingredient on acid equivalent causes 50% of intoxication on paricá. The application of 196.27 g.ha-1 of active ingredient on acid equivalent causes 50% of intoxication on paricá. The gs varied substantially from the 129.6 g.ha-1 dose of glyphosate (Table 2), both 7 and 21 days after the application, however, with a more accentuated difference between two higher doses and the other doses on the second evaluation. Among the analyzed physiological variables, the gs response was the most expressive one due to the intoxication, reaching a reduction of almost 85% on the 172.8 g.ha-1 dose on the evaluation after 21 days (Figure 4B). REFERENCES A.; DORNELAS, D. F.; DORNELAS, B. F. Efeito da deriva simulada de glyphosate no crescimento inicial de plantas de pinhão- manso. Planta Daninha, v. 27, p. 1105-1110, 2009. The rise on the Tf leads to an increase of the photorespiration and, concomitantly, to a decrease of the rubisco activity (ribulose-1,5-bisphosphate carboxylase/ oxygenase) and of the quantic yield of the photosynthesis on C3 plants, which becomes lower than on C4 plants from approximately 30º C on (TAIZ; ZEIGER, 2002). The increase on the photorespiration rate results on a linear decrease of the photosynthesis, that is, a lower production of photoassimilates. FUCHS, M. A.; GEIGER, D. R.; REYNOLDS, Tracey L.; BOURQUEB, J. E. Mechanisms of glyphosate toxicity in velvetleaf (Abutilon theophrasti medikus). Pesticide Biochemistry and Physiology, v. 74, p. 27-39, 2002. FRANS, R. E. Measuring plant response. In: WILKINSON, R. E. (Ed.). Research methods in weed science. Melbourne: Southern Weed Science Society, 1972. p. 28-41. GEIGER, D. R.; KAPITAN, S. W.; TUCCI, M. A. Glyphosate Inhibits Photosynthesis and Allocation of Carbon to Starch in Sugar Beet Leaves. Plant Physiology, v. 82, p. 468-472, 1986. PEREIRA et al. The effect of glyphosate on A for paricá led to a more accentuated reduction 21 days after the application (Figure 4A), and there was a signifi cant difference in relation to the control from the 129.6 g.ha-1 dose (Table 2). Considering the data collected 21 days after the application, the dose of 124.7 g.ha-1 of active ingredient on acid equivalent of glyphosate is enough to cause a 50% reduction of A for paricá. FIGURE 4 Photosynthesis (A), stomatal conductance (gs), transpiration (E) and leaf temperature (Tf) of S. parahyba var. amazonicum 7 and 21 days after the application of glyphosate. TABLE 2 Physiological variables of S. parahyba var. amazonicum 7 and 21 days after the application of glyphosate. TABLE 2 Physiological variables of S. parahyba var. amazonicum 7 and 21 days after the application of glyphosate. Variables Glyphosate dose (g.ha-1 on acid equivalent) 0 43,2 86,2 129,6 172,8 7 days after the application Photosynthesis 12.591 10.40ns 10.80ns 7.03ns 5.94* Stomatal conductance 0.21 0.15ns 0.14ns 0.09* 0.08* Transpiration 7.97 6.01ns 5.85ns 4.04* 3.68* Leaf temperature 31.63 32.87ns 33.32ns 33.24ns 34.52* 21 days after the application Photosynthesis 12.200 9.071ns 9.821ns 4.302 4.025 Stomatal conductance 0.246 0.157ns 0.186ns 0.037 0.039 Transpiration 9.668 7.140ns 7.846ns 2.175 2.124 Leaf temperature 33.546 34.947ns 34.434ns 36.145 37.206 1Comparison with dose 0 (control) on the row: ns – non-signifi cant; * and ** signifi cant difference according to the Dunnett test at 5 and 1%, respectively. On a study with Eucalyptus, Machado et al. (2010) only observed a change on A 21 days after the intoxication by glyphosate. Carvalho et al. (2013) observed a decrease on the photosynthesis rate of coffee 15 days after the application of 60.8 g.ha-1 of glyphosate. The reduction of A is related, among other factors, to an important secondary effect of glyphosate, which consists in deviating erythrose-4-phosphate, which would be used for the regeneration of ribulose bisphosphate on the Calvin cycle, for the deregulated 271 MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE CONCLUSIONS Morphological and physiological changes occur on paricá when it is intoxicated by glyphosate. Paricá is susceptible to glyphosate with intoxication symptoms from a dose of 172.8 g.ha-1 of active ingredient on acid equivalent. There are no relevant damages on the stem and root of intoxicated plants, however, severe leaf loss occurs. Increasing doses of glyphosate reduce the photosynthesis, stomatal conductance and transpiration. The leaf temperature rises as the glyphosate dose increases. GRAVENA, R. Respostas bioquímicas e fisiológicas de plantas de citros atingidas pelo glyphosate. 2006. 144f. Tese (Doutorado em Agronomia) – Escola Superior de Agricultura Luiz de Queiroz, Piracicaba, 2006. HETHERINGTON, P. R.; REYNOLDS, T. L.; MARSHALL, G.; KIRKWOOD, R. C. The absorption, translocation and distribution of the herbicide glyphosate in maize expressing the CP-4 transgene. Journal of Experimental Botany, v. 50, n. 339, p. 1567-1576, 1999. CERNE \| v. 23 n. 2 \| p. 267-274 \| 2017 272 PEREIRA et al. IBÁ. Relatório Ibá 2015. 2015. Disponível em:<http://iba.org/ images/shared/iba_2015.pdf> Acesso em: 22 de dez. de 2015. SALGADO, T. P.; ALVES, P. L. C. A.; KUVA, M. A.; TAKAHASHI, E. N.; DIAS, T. C. S.; LEMES, L. N. Sintomas da intoxicação inicial de Eucalyptus proporcionados por subdoses de glyphosate aplicadas no caule ou nas folhas. Planta Daninha, v. 29, n. 4, p. 913-922, 2011. IBGE. Produção da extração vegetal e da silvicultura. 2014. Disponível em: <http://www.sidra.ibge.gov.br/bda/ pesquisas/pevs/default.asp> Acesso em: 22 de dez. de 2015. SANTOS JUNIOR, A.; TUFFI SANTOS, L. D.; FERREIRA, F. A.; FERREIRA, L. R.; FELIX, R. C.; AMARAL, G. C.; CRUZ, L. R.; Glyphosate drift in eucalyptus plants. Planta Daninha, v. 33, n. 3, p. 615-621, 2015. LEE, T. T.; DUMAS, T. Effect of glyphosate on ethylene production in tobacco callus. Plant Physiology, v. 72, p. 855-857, 1983. LEE, T. T.; DUMAS, T. Effect of glyphosate on indole-3-acetic acid metabolism in tolerant and susceptible plats. Journal of Plant Growth Regulation, v. 4, p. 29-39, 1985. SCHALLER, G. E.; KIEBER, J. J. Ethylene. The Arabidopsis Book, v. 1, p.1-17, 2002. SERVAITES, J. C.; TUCCI, M. A.; GEIGER, D. R. Glyphosate Effects on Carbon Assimilation, Ribulose Bisphosphate Carboxylase Activity, and Metabolite Levels in Sugar Beet Leaves. Plant Physiology, v. 85, p. 370-374, 1987. LOPES, M. J. dos S.; DIAS-FILHO, M. B.; MENEZES NETO, M. A.; SANTOS, J. U. M. dos; CRUZ, E. D.; DIAS, H. do S. da S. CONCLUSIONS Morphological and physiological responses to shade in seedlings of Parkia gigantocarpa Ducke and Schizolobium parahyba var. amazonicum (Huber ex Ducke) Barneby (Leguminosae). Scientia Forestalis, v. 43, n. 107, p. 573-580, 2015. SILVA, A. A. da; FERREIRA, F. A.; FERREIRA, L. R.; SANTOS, J. B. dos. Métodos de controle de plantas daninhas. In: SILVA, A. A. da; SILVA, J. F. da (Ed.). Tópicos em manejo de plantas daninhas. Viçosa: Ed. UFV, 2007a. p 41-57. MACHADO, A. F. L.; FERREIRA, L. R.; SANTOS, L. D. T.; FERREIRA, F. A.; VIANA, R. G.; MACHADO, M. S.; FREITAS, F. C. L. Eficiência fotossintética e uso da água em plantas de eucalipto pulverizadas com glyphosate. Planta Daninha, v. 28, n. 2, p. 319-327, 2010. SILVA, A. A. da; VARGAS, L.; FERREIRA, A. E. Herbicidas: resistência de plantas. In: SILVA, A. A. da; SILVA, J. F. da (Ed.). Tópicos em manejo de plantas daninhas. Viçosa: Ed. UFV, 2007b. p 240-281. MAGALHÃES, P. C.; SILVA, J. B.; DURÃES, F. O. M.; KARAM, D.; RIBEIRO, L. S. Efeito de doses reduzidas de glyphosate e paraquat simulando deriva na cultura do milho. Planta Daninha, v.19, n.2, p.247-253, 2001a. SILVA, N. R. da; COSTA, F. R. da; CARVALHO, L. B. de. Acúmulo diferencial de massa seca em eucalipto e pinus expostos a glyphosate. Revista de Ciências Agroveterinárias, v.14, n.2, p.186-190, 2015. MAGALHÃES, P. C.; SILVA, J. B.; DURÃES, F. O. M.; KARAM, D.; RIBEIRO, L. S. Efeito de doses reduzidas de glyphosate e paraquat simulando deriva na cultura do sorgo. Planta Daninha, v.19, n.2, p.255-262, 2001b. TAIZ, L.; ZEIGER, E. Plant physiology. 3.ed. Sunderland: Sinauer Associates, 2002. 690 p. MAPA/MDA.Plano Setorial de Mitigação e de Adaptação às Mudanças Climáticas para a Consolidação de uma Economia de Baixa Emissão de Carbono na Agricultura. Brasília, 2011, 107p. TUFFI SANTOS, L. D.; FERREIRA, F. A.; FERREIRA, L. R.; DUARTE, W. M.; TIBURCIO, R. A. S.; SANTOS, M. V. Intoxicação de espécies de eucalipto submetidas à deriva do glyphosate. Planta Daninha, v. 24, n. 2, p. 359-364, 2006. TUFFI SANTOS, L. D;, MACHADO, A. F. L.; VIANA, R. G.; FERREIRA, L. R.; FERREIRA, F. A.; SOUZA, G. V. R. Crescimento do eucalipto sob efeito da deriva de glyphosate. Planta Daninha, v. 25, n. 1, p. 133-137, 2007a. MARQUES, L. C. T.; YARED, J. A. G.; SIVIERO, M. A. A evolução do conhecimento sobre o paricá para reflorestamento no Estado do Pará. MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE YAMADA, T.; CASTRO, P. R. de C. e. Efeitos do glifosato nas plantas: implicações fisiológicas e agronômicas. Informações Agronômicas, n. 119, p. 1-32, 2007. YAMASHITA, O. M.; VIEIRA, R. G.; SANTI, A.; RONDON NETO, R. M.; ALBERGUINI, S. E. Resposta de varjão (Parkia multijuga) a subdoses de glyphosate. Planta Daninha, v. 24, n. 3, p. 527-531, 2006. MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE CONCLUSIONS Belém: Embrapa Amazônia Oriental, 2006, 5 p. (Comunicado Técnico 158). MONQUEIRO, P. A.; CHRISTOFFOLETI, P. J.; OSUNA, M. D.; DE PRADO, R. A. Absorção, translocação e metabolismo do glyphosate por plantas tolerantes e susceptíveis a estes herbicidas. Planta Daninha, v. 22, n. 3, p. 123-132, 2004. TUFFI SANTOS, L. D.; MEIRA, R. M. S. A.; FERREIRA, F. A.; SANT’ANNA-SANTOS, B. F.; FERREIRA, L. R. Morphological responses of different eucalypt clones submitted to glyphosate drift. Environmental and Experimental Botany, v. 59, p. 11-20, 2007b. Lewis, G. P. Schizolobium in Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. Disponível em: <http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/ FB23144>. Acesso em: 06 de maio 2016. TUFFI SANTOS, L. D.; SANT’ANNA-SANTOS, B. F.; MEIRA, R. M. S. A.; FERREIRA, F. A.; TIBURCIO, R. A. S.; MACHADO, A. F. L. Leaf anatomy and morphometry in three eucalypt clones treated with glyphosate. Brazilian Journal of Biology, v. 69, n. 1, p. 129-136, 2009. ROSA, L. dos S. Características botânicas, anatômicas e tecnológicas do paricá (Schizolobium amazonicum Huberr ex Ducke). Revista de Ciências Agrárias, v. 46, p. 63-79, 2006a. WAGNER JÚNIOR, A.; TUFFI SANTOS, L. D.; SANTOS, C. E. M.; SILVA, J. O. C.; PIMENTEL, L. D.; BRUCKNER, C. H.; FERREIRA, F. A. Deriva simulada de formulações comerciais de glyphosate sobre maracujazeiro amarelo. Planta Daninha, v. 26, n. 3, p. 677-683, 2008. ROSA, L. S. Ecologia e silvicultura do paricá (Schizolobium amazonicum Huber ex Ducke) na Amazônia brasileira. Revista de Ciências Agrárias, v. 45, p. 135-174, 2006b. 273 MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES ON Schizolobium parahyba VAR. amazonicum (HUBER EX DUCKE) BARNEBY PLANTS INTOXICATED BY GLYPHOSATE YAMADA, T.; CASTRO, P. R. de C. e. Efeitos do glifosato nas plantas: implicações fisiológicas e agronômicas. Informações Agronômicas, n. 119, p. 1-32, 2007. YAMASHITA, O. M.; VIEIRA, R. G.; SANTI, A.; RONDON NETO, R. M.; ALBERGUINI, S. E. Resposta de varjão (Parkia multijuga) a subdoses de glyphosate. Planta Daninha, v. 24, n. 3, p. 527-531, 2006. CERNE \| v. 23 n. 2 \| p. 267-274 \| 2017 274
https://openalex.org/W3109514503	https://dergipark.org.tr/tr/download/article-file/1373982	Turkish	null	A Look at Teacher Training Schools In Turkish Education: Gönen Teacher Training School	Alınteri sosyal bilimler dergisi (Online)/Alınteri sosyal bilimler dergisi	2,020	cc-by	6,283	Ö Z Öğretmen okulu olarak 1954-1974 yılları arasında 2.381 kadar öğretmen yetiştiren, Gönen İlköğretmen Okulu, ülkede öğretmen yetiştirme konusunda önemli bir rol üstlenmiştir. Okul bölgenin en iyi okulu olup, Gönen İlköğretmen Okulu’na gitmenin ayrıcalık olduğu bir dönemi yaşatmıştır. Parasız yatılı olarak öğrenim veren Gönen İlköğretmen Okulu’nun öğrencileri aldıkları öğretmenlik derslerinin yanı sıra okulun atölyelerinde uygulamalı dersler de alarak zanaat da öğrenmişlerdir. Ayrıca bu okulun her bir öğrencisinin mutlaka bir enstrüman çalması istenmiştir. 6 yıl eğitim veren Gönen İlköğretmen Okulu’nun öğrencileri hem laboratuvar hem atölye hem de tarım çalışmalarında bulunarak pratik yapmış ve atölyelerde ders araçlarını kendileri üretmiştir. Üretilen bu ders araçları kullanılmak üzere staj okullarına ve bazı ilkokullara gönderilmiştir. Bu çalışmada, 1954-1974 yılları arasında Gönen İlköğretmen Okulu’nun öğrenci, öğretmen ve mezun sayıları, öğrencilerin ders bilgileri ve yaptıkları etkinliklerin yanı sıra öğrencilerin bu okuldaki günlük yaşamları, öğretmen-öğrenci ilişkileri de ele alınmıştır. Çalışmanın eğitim tarihindeki literatüre katkı sunması ve literatürdeki boşluğu doldurması düşünülmektedir. Anahtar Kelimeler: İlköğretmen Okulları, Gönen İlköğretmen Okulu, Eğitim, Isparta’da Eğitim, Gönen, Isparta. A Look at Teacher Training Schools in Turkish Education: Gönen Teacher Training School Esin YÜZBAŞI* Uncalı, Konyaaltı, Antalya, Türkiye. Esin YÜZBAŞI* Uncalı, Konyaaltı, Antalya, Türkiye. Uncalı, Konyaaltı, Antalya, Türkiye. Geliş Tarihi/Received Date: 01.11.2020 Kabul Tarihi/Accepted Date: 27.11.2020 Kabul Tarihi/Accepted Date: 27.11.2020 * Sorumlu yazar/Corresponding author E-mail: esinn-38@hotmail.com Alınteri Sosyal Bilimler Dergisi (2020) 4(2): 111-124 Alınteri Journal of Social Sciences (2020) 4(2): 111-124 Alınteri Sosyal Bilimler Dergisi (2020) 4(2): 111-124 Alınteri Journal of Social Sciences (2020) 4(2): 111-124 dergipark.org.tr/en/pub/alinterisosbil DOI: 10.30913/alinterisosbil.819370 ARAŞTIRMA MAKALESİ RESEARCH ARTICLE TÜRK EĞİTİMİNDE İLKÖĞRETMEN OKULLARINA BİR BAKIŞ: GÖNEN İLKÖĞRETMEN OKULU A Look at Teacher Training Schools in Turkish Education: Gönen Teacher Training School i Ü * Yüzbaşı, E. (2020). Türk Eğitiminde İlköğretmen Okullarına Bir Bakış: Gönen İlköğretmen Okulu. Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. TÜRK EĞİTİMİNDE İLKÖĞRETMEN OKULLARINA BİR BAKIŞ: GÖNEN İLKÖĞRETMEN OKULU A Look at Teacher Training Schools in Turkish Education: Gönen Teacher Training School GİRİŞ 19.yy’da Isparta’da ileri bir eğitim seviyesi olmamakla (Isparta Valiliği 1973 İl Yıllığı, 1973: 68) birlikte Isparta merkezde 17 sıbyan okulu bulunmuştur (Isparta, 1982: 3528-3529). Bugünkü anlamda öğretmen yetiştiren ilk öğretmen okulu olan Darulmuallimin, 16 Mart 1848 tarihinde İstanbul’da açılmıştır. Daha sonra Edirne, Bursa, Diyarbakır, Girit, Bosna, Kastamonu, Van, Erzurum, Halep, Kudüs, Sivas, Trabzon, Musul, Konya ve Bağdat’ta açılan öğretmen okullarıyla (Kamer, 2012: 2) rüştiyelere öğretmen yetiştirmek amaçlanmıştır (Kuru & Uzun, 2008: 210). Isparta’da ilk rüştiye 1860’da açılmıştır (Katırcıoğlu, 1958: 95). Rüştiye’ye öğrenci yetiştirmek amacıyla onun bir bölümüne de Sa’diye İlkokulu adıyla bir ilkokul açılmıştır (Süldür E., 1951: 41-43). Isparta’da ilk muallim mektebi ise, 1903 yılında açılan (Taşer, 2010: 670-672) Darulmuallimini İptidaiyye Mektebi olmuştur (Süldür E., 1951: 44). Darulmuallimini İptidaiyye Mektebi, 1901-1910 yılları arasında verdiği mezunlarla şehrin eğitimine önemli katkı sağlamıştır. (Süldür E., 1951: 44). Isparta’da İstiklâl Savaşı’ndan sonra da dini eğitimin verildiği Medrese-i İlmiye Okulu açılmış, 1922 yılına gelindiğinde bu okulun adı Darul-Hilafetül-Aliye olarak değiştirilmiştir. Bu okul 1924 yılında İmam Hatip Mektebi olmuştur. Bu İmam Hatip Mektebi’nden mezun olan öğrenciler, Konya’da 3 ay süren bir eğitimden sonra ilkokullarda öğretmenlik yapabilmişlerdir (Katırcıoğlu, 1958: 97). A B S T R A C T Gönen Teacher Training School which trained about 2.381 teachers between 1954 and 1974 as a teacher school, has played an important role in training teachers in the country. The school is the best school in the region and has experienced a period in which it was a privilege to go to Gönen Teacher Training School. The students of Gönen Teacher Training School, which provides free boarding education, learned the craft by taking applied lessons in the school's workshops in addition to the teaching lessons they took. Also, it was wanted that each student of this school play an instrument. The teacher candidates, who received 6 years of education in Gönen school, practiced both in laboratory, workshop and agriculture studies, and produced the teaching tools themselves in the workshops. These produced teaching tools were sent to internship schools and some primary schools to be used. In this study, in addition to the information about the number of students, teachers and graduates of Gönen Teacher Training School between 1954-1974, the course information of the students, their activities and the daily lives of the students in this school were also considered. Keywords: Teacher Training Schools, Gönen Teacher Training School, Education, Education in Isparta, Gönen, Ispar Please cite this paper as follows/Atıf için: Yüzbaşı, E. (2020). Türk Eğitiminde İlköğretmen Okullarına Bir Bakış: Gönen İlköğretmen Okulu. Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. * Sorumlu yazar/Corresponding author E-mail: esinn-38@hotmail.com Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. YÖNTEM Cumhuriyetin ilanından sonra eğitim alanında karşılaşılan sorunlar (Şimşek & Mercanoğlu, 2018: 262), halkın %80’inin okuma-yazma bilmemesi nedeniyle eğitim sorununun üzerinde ayrıca durulmuştur (Şimşir, 2006: 18-21). Darulmualliminler, il özel idaresi altında gelişim gösteremedikleri gerekçesiyle 22.03.1926 tarihinde 789 Sayılı kanunla Maarif Vekâletine bağlanmıştır (Kamer, 2012: 2). 1926’da Denizli ve Kayseri’de “Köy Muallim Mektepleri” açılarak köylerdeki öğretmen eksikliği giderilmeye çalışılmıştır. 6 yıllık bir eğitim veren ve (Yıldız & Akandere, 2017: 281) eğitimle üretimin iç içe olduğu Köy Muallim Mektepleri (Altunya, 1990: 84), 1933 yılında kapatılmıştır (Yıldız & Akandere, 2017: 281). Ülkede 1927’de okur-yazarlık oranına bakıldığında; kentlerde %32,4 iken köylerde bu oran %6’da kalmıştır (Gül, 2013: 25). Atatürk’ün emri doğrultusunda köylerdeki öğretmen sorununu gidermek adına, 1936-1937 eğitim-öğretim yılında eğitmen kursları faaliyete geçirilmiştir. Bu kurslar ilk önce Eskişehir Çiftelerde açılmış olup, daha sonra Kırklareli Kepirtepe, Kastamonu Gölköy ve İzmir Kızılçullu’da da açılmıştır (Özbek, 27 Nisan 1971: 1). Isparta Gönen Eğitmen Kursu ise 1939 yılında açılmış olup (Okan, 5 Haziran 1955: 2), 17 Nisan 1940 tarihindeki 3803 Sayılı Köy Enstitüleri Kanunu ile (Özbek, 27 Nisan 1971: 1) köy enstitüsüne dönüştürülmüştür (Okan, 5 Haziran 1955: 2). Köy Enstitülerinin açılmasında Milli Eğitim Bakanı Hasan Ali Yücel ve İlköğretim Genel Müdürü İsmail Hakkı Tonguç’un etkisi olmuştur (Yalçın ve diğerleri, 2005: 134-135). Köy Enstitülerinde gündüzlü eğitim olarak adlandırılan yatısız eğitimin yanında yatılı olarak ta eğitim verilmiştir. (Özbek, 27 Nisan 1971: 1) Köy Enstitülerinde, eğitim kelimesinin yanında ziraat ve sanat kelimelerinin yer alışı bu okullarla köy kalkınmasının da amaçlandığını göstermektedir. Köycülük politikası 112 112 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. doğrultusunda oluşturulan bu okullar, öğrencilerinden hem geleceğin yaratıcısı köy öğretmeni hem ziraatçısı hem de sanatçısı olması beklenilmiştir (Köy Enstitüleri Kanunu, 15 Eylül 1948: 2). 1947 yılında köy enstitülerinde teknik dersler azaltılarak kültür ve teknik derslerin isimlerinde değişik yapılmıştır. Kültür dersleri “genel bilgi’’, teknik dersler de “sanat ve atölye” dersi olarak adlandırılmıştır. 1950 yılında karma eğitimin sonlandırıldığı köy enstitülerinde, 1952-1953 eğitim-öğretim döneminde derslerin genel bilgi derslerinden oluşmasına karar verilmiştir. Köy enstitüleri Şubat 1954’te 6234 Sayılı kanunla ilköğretmen okullarıyla birleştirilip (Yalçın ve diğerleri, 2005: 134-135), öğretmen okulları ve köy enstitüleri ikiliğine son verilmiştir (Kamer, 2012: 2). GÖNEN İLKÖĞRETMEN OKULU 1954 yılında 6234 Sayılı kanunla öğretmen okulları ile köy enstitüleri birleştirilmiş ve Gönen Köy Enstitüsü, Gönen İlköğretmen Okulu’na dönüştürülmüştür. Okul binası Gönen Köy Enstitüsü’ne ait olup, Isparta’nın 28 km kuzey batısındaki Gönen köyünün Tınaztepe’nin eteğinde bulunmuştur (Okan, Gönen İlköğretmen Okulu, 5 Haziran 1955, s. 2). Gönen Köy Enstitüsü, 1954 yılına kadar ülkenin çeşitli yerlerinde görev yapan 42 kız, 883 erkek olmak üzere 925 öğretmen mezun ederek ülkenin öğretmen ihtiyacını karşılamada önemli eğitim kurumları arasında yer almıştır (Okan, 5 Haziran 1955: 2). 1954 yılında Gönen İlköğretmen Okulu’na girmek için başvuran 1.045 öğrenci olup, bunlardan 59’u sınavı kazanarak okula başlamaya hak kazanmıştır (Okan, 5 Haziran 1955: 2). Yine aynı yılda Gönen İlköğretmen Okulu’nda mesleki danışma bürosu kurularak ilkokul öğretmenlerine mesleki yaşamlarında yardımcı olmak amaçlanmıştır. Bu danışma bürosu aracılığıyla ilkokul öğretmenlerinin sorunlarına çözüm bulmak ve onların pratik, orijinal buluşlarından haberdar olunmak istenmiştir. Ayrıca danışma bürosundan 800 ilkokula mektup gönderilerek öğretmenlerin bürodan faydalanabileceği bildirilmiştir (Gönen İlköğretmen Okulunda Bir Mesleki Danışma Bürosu Kurulmuştur, 3 Kasım 1954: 4). 1955 yılında Gönen İlköğretmen Okulu’nda 23 öğretmen görev yapmış olup, mezun edilen öğretmen sayısı ise 120 olmuştur. Aynı yılda ülke genelinde 40 kadar öğretmen yetiştiren okul bulunmuştur. Bu okulların bir kısmı ortaokullardan sonra 3 yıllık okullar olurken bir kısmı ise ilkokuldan sonra 6 yıllık okullar olmuştur. Gönen İlköğretmen Okulu ilkokul mezunlarına 6 yıl eğitim veren İlköğretmen Okulları arasında yer almıştır. Isparta, Burdur, Denizli ve Afyon’dan öğrenci alan bu okula giriş için, ağustos ayında kaza merkezlerinde yazılı sınav yapılmıştır. Başarı derecelerine göre, o yıl okula alınacak öğrencilerin 1,5 katı kadar öğrenci 2. sınava girmeye hak kazanmıştır. 2. sınav genel bilgilerden oluşup sözlü olarak yapılmıştır. Bu sınavı da kazanan öğrenciler parasız yatılı veya gündüzlü olarak bu okulun öğrencisi olmaya hak kazanmıştır (Okan, 5 Haziran 1955: 2). Biçki, nakış, demircilik, bahçecilik vb. alanda da eğitilen Gönen İlköğretmen Okulu öğrencilerinin okullarında ihtiyaç duydukları malzemeler eğitim aldıkları alanla ilgili olmuştur. Okulun ihtiyaç malzemelerine örnek olarak; elbiselik, malina kumaş, oskardan iskarpin, kaput bezi, gömleklik poplin, kol astarı, astarlık saten vb. ihtiyaçlar okul müdürlüğünden okulun ihtiyaç listesi olarak ilin yerel gazetesinde yayınlanmıştır 113 113 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. (Isparta Gönen İlköğretmen Okulu Müdürlüğünden, 14 Temmuz 1954: 3). Isparta’da ilköğretim davasının önemli bir ayağını oluşturan Gönen İlköğretmen Okulu çevre okullarının ihtiyaçlarını karşılayarak ta eğitime katkı sağlamıştır. Gönen İlköğretmen Okulu, 1954 yılında 45 köydeki ilkokula ders araç gereç yardımı yapmıştır (Gönen İlköğretmen Okulundan Temin Edilen Ders Araçları, 13 Kasım 1954: 2). GÖNEN İLKÖĞRETMEN OKULU 1954-1955 eğitim-öğretim yılı sonunda ülkedeki bütün ilköğretmen okullarının katıldığı bir serginin açılması planlanmış ve sergide öğrencilerin yaptığı her türlü faaliyetlere yer verileceği belirtilmiştir. Gönen İlköğretmen Okulu’nun da katıldığı sergide, öğrencilerin yapmış olduğu resim, yazı, harita ve bütün derslere ait faaliyetler bulunmuştur (Öğretmen Okulları Sergisine Gönen Katılacak, 18 Kasım 1954: 2). Gönen İlköğretmen Okulu’nun 1955 yılı öğrenci sayısı 515 olup, bunların sadece 6’sı gündüzlü olarak öğrenim görmüştür. Okuldan mezun olan öğrenciler mecburi köy hizmetlerini yapmak ile yükümlü olmuşlar ve bu mecburi hizmet parasız yatılı olarak okudukları okul süresinin 1,5 katı kadar sürmüştür. Öğretmen okulunun birinci dönemi ortaokula denktir. Bu okulların ortaokullardan farkı ise dersleridir. Ortaokullardaki tarih, coğrafya ve yurt bilgisi dersleri, öğretmen okullarında sosyal bilgiler adı altında verilmiş olup; fizik, kimya ve tabiat bilgisi dersleri de verilmiştir. İkinci dönem ise, birleştirilmiş olan bu dersler ihtisas alanlarına ayrılmış ve öğrencilere öğretmenlik bilgisi olarak bazı meslek dersleri de verilmiştir. Son sınıfa gelen öğrenciler, öğretmenlerinden öğrendiklerini uygulayabilmeleri için Gönen köyündeki uygulama okulu kısmında öğrenimlerine devam etmişleridir. Ayrıca uygulamayı arttırmak amacıyla 6 köyde bulunan staj okullarında 2 ay boyunca staj yapmışlardır (Okan, 5 Haziran 1955: 2). Gönen İlköğretmen Okulu’nun son sınıf öğrencilerinin Isparta merkez ve Isparta’nın köylerinde 3 dönem süren staj dönemleri olmuştur (Öğretmen Adaylarının İlk Grubu Dün Staj Okullarında Görevlerine Başladı, 2 Kasım 1965: 1). Gönen İlköğretmen Okulu’nda 6 yıllık eğitim alan öğretmen adayı hem laboratuvar hem atölye hem de tarım çalışmaları yaparak, pratik kazanmış ve atölyelerde ders araçlarını kendisi üretmiştir. Üretilen bu ders araçları staj okullarına ve bazı ilkokullara gönderilmiştir (Okan, 5 Haziran 1955: 2). Gönen İlköğretmen Okulu’nda müziğe de önem verilmiş olup, okulun öğrencilerinden burada kaldıkları 6 yıl içinde mutlaka bir enstrüman çalmaları istenmiştir. Ayrıca ilgi gösterilen bir diğer ders ise beden eğitimi dersi olmuştur. Okulun geniş spor tesislerinde günün her saatinde öğrenci grupları görülebilmiştir. Spor dallarından en fazla basketbol ilgi görürmüş ve okulun basketbol takımı bölgedeki okullarla yapılan maçları genellikle kazanmıştır. Okul kapalı spor salonuna da sahip olup hem müsamere hem de sinema sunumu için kullanılmıştır. Ayrıca düzenlenen kurs programları ile öğrencilere sinemacılık, meteoroloji, enstrüman ve koro gibi çeşitli kurslar verilmiştir. Gönen İlköğretmen Okulu’nda bir öğrencinin bir günü şu şekilde geçmiştir: Öğrenciler sabah saat 06:00’da yeni bir güne kalkarlar. Saat 07:00’da nöbetçi öğretmen eşliğinde ders çalışma programına katılırlar. 07:45’te zil çalar çalmaz okul binasının önünde meydanda sabah jimnastiği yapılır, müzik öğretmeninin eşliğinde milli oyunlar oynanır. GÖNEN İLKÖĞRETMEN OKULU Dersler 08:30’da başlayıp, akşam 16:00’a kadar sürer ve saat 16:00’dan sonra öğrenciler serbest olmasına karşın bazı öğrenciler 16:15’te yine ders zili ile seçmeli kurslara katılmışlardır. Bu seçmeli kurslardan birine mutlaka katılmak 114 114 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. zorunda olmuştur. Akşam saat 18:00’a geldiğinde öğrenciler akşam çalışmalarına başlar ve öğretmenlerinin idaresinde serbest okuma yaparlar. Her sınıfın bir sınıf öğretmeni olmuştur. Serbest okuma saatinde ya sınıfça aynı kitap okunur ya da bir öğrenci okumuş olduğu bir kitabı arkadaşlarına aktarır. Bu okuma saatiyle amaç öğrenciye kitap sevgisini aşılamak olmuştur. Saat 19:00’da yemek zilinin çalması ile beraber öğrenciler 20:00’a kadar serbest kalırlar. Yatma saatleri 21:00 olan öğrenciler, 20:00 ile 21:00 arasında akşam çalışmalarını gerçekleştirirler (Okan, 5 Haziran 1955: 3). Gönen İlköğretmen Okulu’nda 13 öğrenci kolu faaliyette bulunmuş ve bunlar tamamen öğrenciler tarafından idare edilmiştir. Gönen İlköğretim Okulu’ndaki bu kolların ne tür faaliyetler yaptığına örnek verecek olursak; 1955 yılında gezi ve inceleme kolu; Ankara, Antalya, Keçiborlu, Senirkent ve Eğirdir’e geziler düzenlemiştir. Temsil kolu; sınıf geceleri düzenlemiş, yayın kolu; Gönen köyünde her 15 günde bir hoparlörle yayın yapmıştır. Yayın kolu; önemli Türk büyüklerimiz için anma törenleri ve şiir yarışmaları da düzenlemiştir. Müzik kolu ise, bütün öğrenci faaliyetlerinde üstüne düşen görevi yerine getirmiştir. Ayrıca okulda eğitsel faaliyetleri takip etmek amacıyla bir grup oluşturulmuş, grup üyelerinin seçimi ise tıpkı milletvekili seçimi gibi olmuştur. Okulun eğitsel faaliyetleri takip etmekle görevli grup üyesi seçimi yılda 2 kez olup, seçimden bir hafta önce seçim propagandaları yapılmıştır. Öğrencilere seçime 2 gün kala sonlandırmak koşulu ile okulun hoparlöründen seçim propagandası yapma izni de verilmiştir. Gizli oy açık tasnif esasına dayalı yapılan seçimin sonucunda seçilen üyelerden biri grup başkanı olurken, diğer üyeler arasında da görev dağılımı yapılmıştır. Her üye bir eğitsel kolun başkanı ve her eğitsel kolun da bir gözetici öğretmeni bulunmuştur. Gözetici öğretmenler, eğitsel kollarının bir yılık programını hazırlamaktan sorumlu tutulmuştur (Okan, 5 Haziran 1955: 3). İlköğretim davasını benimsemekle Türk ulusu kültürel gelişmişlik adına en büyük adımını atmıştır. Bu uğurda en büyük görev Türk öğretmenlerine verilmiştir (İdeal Bir Köy Öğretmeninin Vasıfları, 20 Ekim 1948: 2). 1961 yılında öğretmen okullarına Millî Eğitim Bakanlığı’ndan gelen emirle ortaokulların 1. ve 2. sınıflarını “iyi” ve “çok iyi” derece ile bitiren öğrenciler alınmıştır (Öğretmen Okullarına Ortaokullardan Öğrenci Alınacak, 6 Mayıs 1961: 1). Okullarındaki faaliyetleri halka taşıyan Gönen İlköğretmen Okulu öğrencileri, 21 Haziran 1961 tarihinde Ülkü İlkokulu’nda resim sergisi açmıştır. GÖNEN İLKÖĞRETMEN OKULU Sergideki resimlerin konuları; köy çocuğu olan Gönen İlköğretim Okulu öğrencisinin, köyü ve köy hayatı olmuştur (Çavuşoğlu, Resim Sergisi, 25 Haziran 1961: 1.). 1962’de ilköğretmen okullarının sayıları 19 olup, dönemin Milli Eğitim Bakanı Hasan Ali Yücel de onları korumuştur (Demokrat Eğirdir, 27 Ocak 1962: 2). Gönen İlköğretmen Okulu’ndan 1965 yılında mezun olan öğrenciler için okulda tören yapılmış olup İstiklal Marşı ile başlayan törene İlköğretmen Okulları Marşı ile devam edilmiştir. Sonrasında Okul Müdürü Mehmet Kahvecioğlu davetlilere hitap etmiş, derslerinde başarı gösteren öğrencilere ödüller vermiştir (Gönen İlköğretmen Okulu Mezunları İçin Dün Mesleğe Giriş Töreni Yapıldı, 2 Temmuz 1965: 1). Gönen İlköğretmen Okulu’ndan 1964-1965 eğitim öğretim yılında mezun olup, Isparta’ya ve diğer illere atanan öğretmenlerin isimleri ve atandıkları yerler de Isparta’nın yerel gazetesi olan “Yeni İnkılap” ta duyurulmuştur (Gönen Öğretmen Okulu Mezunlarından İlimize Tayin Edilen Öğretmenler, 21 Temmuz 1965:1). Hem öğrencilerini aydınlatan hem de köylüsüne ışık tutan Gönen İlköğretmen Okulu’nun meslek öğretmenlerinden 115 115 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. olan tarım öğretmeni, 1965’te Gönen halkının faydalanması adına bir traktör kullanma kursu açmıştır. Kursta traktörlerin kullanışı ve parçalarının tanıtılması vb. bilgileri Gönen halkı ile paylaşan tarım öğretmeninin yapmış olduğu bu davranış, Gönen halkı tarafından takdir ile karşılanmıştır (Öğretmen Okulu Tarım Öğretmeni Kurs Açtı, 22 Ekim 1965: 1). 30 Ekim 1965 tarihinde, kuruluş tarihi 1951 olan Göller Bölgesi Köy Öğretmenler Derneğinin 14. Genel Kurul Toplantısı yapılmıştır (Göller Bölgesi Köy Öğretmenler Derneğinin Yıllık Kongresi, 26 Ekim 1965: 1). Derneğin amacı; köy öğretmenleri arasındaki mesleki bağları güçlendirmek, elde bulunan araç gereçleri birbirine ulaştırmak olarak belirtilmiştir (Göller Bölgesi Köy Öğretmenleri Derneğinin Sütçüler Şubesi Açıldı, 30 Kasım 1954: 2). Göller Bölgesi Köy Öğretmenler Derneği “Demet” adında dergi çıkarmış olup, derginin yayın hayatı 19 sayı kadar sürmüştür. Dernek (Köy Öğretmenler Derneği Mümessilleri Maarif Vekillerimize Konuştular, 30 Aralık 1954: 1), okullardan her köyde bir kitaplık bulundurmalarını istemiş ancak bu pek mümkün olmamıştır. Bu nedenle Eğitim Bakanlığı Yayın Merkezi, Göller Bölgesi Öğretmenler Derneğinde kitaplık bulundurarak buradan köy öğretmenlerine kitap temin etmiştir (Köy Öğretmenler Derneği Mümessilleri Maarif Vekillerimize Konuştular, 30 Aralık 1954: 1). GÖNEN İLKÖĞRETMEN OKULU 1954-1974 yılları arasında Gönen Köy İlköğretmen Okulu öğrenci bilgileri ş şekildedir: 1954-1974 yılları arasında Gönen Köy İlköğretmen Okulu öğrenci bilgileri şu şekildedir: Eğitim Öğretim Dönemi Yeni Kayıt Olan Öğrenci Sayısı Diploma Alan Öğrenci Sayısı Toplam Öğrenci Sayısı E K T E K T E K T 1953-1954 2,3 40 - 40 84 - 84 558 - 558 1954-1955 4 51 - 51 132 - 132 514 - 514 1955-1956 5 132 - 132 126 - 126 512 1 513 1956-1957 6 138 - 138 92 - 92 515 - 515 1957-1958 7 122 - 122 57 - 57 532 - 532 1958-1959 8 52 - 52 34 - 34 544 - 544 1959-1960 9 54 - 54 43 - 43 558 - 558 1960-1961 10 81 - 81 117 - 117 601 4 605 1961-1962 11 171 4 175 99 5 99 668 9 677 1962-1963 12 94 - 94 119 1 120 665 7 672 1963-1964 13 112 - 112 93 2 93 695 2 697 116 2 (T.C.B.D.İ.E., 1963: 16). 3 (T.C.B.D.İ.E., 1963: 16). 4 (T.C.B.D.İ.E., 1963: 16). 5 (T.C.B.D.İ.E., 1963: 17). 6 (T.C.B.D.İ.E., 1963: 17). 7 (T.C.B.D.İ.E., 1963: 17). 8 (T.C.B.D.İ.E., 1963: 17). 9 (T.C.B.D.İ.E., 1963: 17). 10 (T.C.B.D.İ.E., 1965: 6). 11 (T.C.B.D.İ.E., 1968: 19). 12 (T.C.B.D.İ.E., 1968: 19). 13 (T.C.B.D.İ.E., 1968: 19). 116 2 (T.C.B.D.İ.E., 1963: 16). 3 (T.C.B.D.İ.E., 1963: 16). 4 (T.C.B.D.İ.E., 1963: 16). 5 (T.C.B.D.İ.E., 1963: 17). 6 (T.C.B.D.İ.E., 1963: 17). 7 (T.C.B.D.İ.E., 1963: 17). 8 (T.C.B.D.İ.E., 1963: 17). 9 (T.C.B.D.İ.E., 1963: 17). 10 (T.C.B.D.İ.E., 1965: 6). 11 (T.C.B.D.İ.E., 1968: 19). 12 (T.C.B.D.İ.E., 1968: 19). 13 (T.C.B.D.İ.E., 1968: 19). 116 116 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. GÖNEN İLKÖĞRETMEN OKULU Eğitim Öğretim Dönemi Yeni Kayıt Olan Öğrenci Sayısı Diploma Alan Öğrenci Sayısı Toplam Öğrenci Sayısı E K T E K T E K T 1964-1965 14 210 2 212 69 3 72 798 21 819 1965-1966 15 142 21 163 93 7 100 845 36 881 1966-1967 16 183 16 199 163 8 171 911 48 959 1967-1968 17 178 6 184 150 21 171 929 37 966 1968-1969 18 153 9 162 189 13 202 947 37 984 1969-1970 19 148 12 160 197 10 207 897 27 924 1970-1971 20 163 14 177 143 9 152 878 39 917 1971-1972 21 187 11 198 140 8 148 866 46 912 1972-1973 22 95 14 109 1 1 2 805 49 854 1973-1974 23 61 - 61 148 11 159 842 60 902 Bu tablodaki bilgilerden; 1954-1974 yılları arasında Gönen İlköğretmen Okulu’nun öğrenci sayısının en fazla olduğu dönemin 984 öğrenciyle 1968-1969 eğitim-öğretim dönemi olduğu görülmüştür. En az öğrencinin bulunduğu dönem ise 513 öğrenciyle 1955-1956 eğitim-öğretim dönemi olmuştur. Gönen İlköğretim Okulu’nun ilk kız öğrencisi 1955-1956 eğitim-öğretim döneminde olmuş ve bu dönemde sadece 1 kız öğrenci öğrenim görmüştür. Okulun 1956 yılından 1960 yılına kadar kız öğrencisi bulunmamıştır. 1960-1961 eğitim-öğretim döneminde ise 4 kız öğrenci öğrenim görmeye başlamış ve bu dönemden itibaren erkek öğrenci sayısı kadar olmasa da 1974 yılına kadar kız öğrenci bulunmuştur. 1954-1974 yılları arasında mezun olan öğrenci sayısı ise 2.381 kişi olup, bunların 2.289’u erkek, 92’si kızdır. Mezun sayısının en çok olduğu dönem ise 1969-1970 eğitim-öğretim dönemidir. Bu dönemde 197’si erkek, 10’u kız olmak üzere toplam 207 öğrenci mezun olmuştur. Mezun sayısının en az olduğu dönem ise 1972-1973 eğitim-öğretim dönemi olup, bu dönemde 1 kız, 1 erkek olmak üzere 2 öğrenci mezun olmuştur. Bu dönemde çok az öğrencinin mezun olmasının sebebi ise, 1971-1972 eğitim-öğretim döneminden itibaren 6 yıllık eğitim veren ilköğretmen okullarının eğitim süresinin 7 yıla çıkarılması olmuştur. Bu dönemde mezun olan bu 2 öğrenci de dışardan bu okula gelip mezun olanlardır24. Geçiş sürecindeki bu dönem dışında en az mezun veren dönem 1958-1959 eğitim-öğretim dönemi olup, bu dönemde 34 öğrenci mezun olmuştur ve öğrencilerin hepsi erkektir. 1954-1967 yılları arasında parasız yatılı okuyan öğrencilerin bilgileri ise şu şekildedir: 117 14 (T.C.B.D.İ.E., 1968: 19). 15 (D.İ.E., 1969: 12). 16 (D.İ.E., 1969: 12). 17 (D.İ.E., 1977: 5). 18 (D.İ.E., 1977: 5). 19 (D.İ.E., 1977: 5). 20 (D.İ.E., 1977: 5). 21 (D.İ.E., 1977: 5). 22 (D.İ.E., 1977: 5). 23 (B.D.İ.E., 1979: 3,101). GÖNEN İLKÖĞRETMEN OKULU 24 (D.İ.E., 1977: 4). 14 (T.C.B.D.İ.E., 1968: 19). 15 (D.İ.E., 1969: 12). 16 (D.İ.E., 1969: 12). 17 (D.İ.E., 1977: 5). 18 (D.İ.E., 1977: 5). 19 (D.İ.E., 1977: 5). 20 (D.İ.E., 1977: 5). 21 (D.İ.E., 1977: 5). 22 (D.İ.E., 1977: 5). 23 (B.D.İ.E., 1979: 3,101). 24 (D.İ.E., 1977: 4). 117 117 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. Eğitim-Öğretim Dönemi 1954-1967 Yılları Arasındaki Öğrenci Mevcutları 25 Gündüzlü Yatılı Toplam Paralı Parasız E K E K E K E K T 1954-1955 - - - - 508 - 508 - 508 1955-1956 - - - - 506 - 506 - 506 1956-1957 - - - - 510 - 510 - 510 1957-1958 - - - - 526 - 526 - 526 1958-1959 - - - - 534 - 534 - 534 1959-1960 - - - - 535 - 535 - 535 1960-1961 26 - - - - 555 - 555 - 555 1961-1962 27 - - - - 601 - 601 - 601 1962-1963 28 - - - - 602 - 602 - 602 1963-1964 29 - - - - 596 - 596 - 596 1964-1965 30 - - - - 627 - 627 - 627 1965-196631 - - - - 633 - 633 - 633 1966-196732 - - - - 623 - 623 - 623 1954-1967 yılları arasında parasız yatılı okuyan öğrencilerin hepsi erkek olup, en fazla öğrencinin okuduğu dönem 633 öğrenciyle 1965-1966 dönemi, en az öğrencinin öğrenim gördüğü dönem ise 506 öğrenciyle 1955-1956 eğitim-öğretim dönemi olmuştur. 1954-1974 yılları arasında Gönen İlköğretmen Okulu’nun öğretmen bilgileri ise şöyledir: Eğitim-Öğretim Dönemi Öğretmen Bilgileri Asil Öğretmenler Stajyerler Ücretli Öğretmenler Kadrosu Başka Okulda Olanlar Toplam E K E K E K E K E K T 1954-1955 33 18 4 - - - - - - 18 4 22 1955-1956 34 18 4 1 - - - - - 19 4 23 1956-1957 35 15 2 1 - - - - - 16 2 18 1957-1958 36 15 2 2 1 - - - - 17 3 20 1958-1959 37 12 2 3 2 1 - - - 16 4 20 1954-1974 yılları arasında Gönen İlköğretmen Okulu’nun öğretmen bilgileri ise şöyledir: 1954-1974 yılları arasında Gönen İlköğretmen Okulu’nun öğretmen bilgileri ise yledir: 118 25 (T.C.B.D.İ.E., 1963: 486). 26 (T.C.B.D.İ.E., 1965: 555). 27 (T.C.B.D.İ.E., 1968: 389). 28 (T.C.B.D.İ.E., 1968: 389). 29 (T.C.B.D.İ.E., 1968: 389). 30 (T.C.B.D.İ.E., 1968: 389). 31 (D.İ.E., 1969: 241). GÖNEN İLKÖĞRETMEN OKULU 32 (D.İ.E., 1969: 241). 33 (T.C.B.D.İ.E., 1963: 486). 34 (T.C.B.D.İ.E., 1963: 486). 35 (T.C.B.D.İ.E., 1963: 486). 36 (T.C.B.D.İ.E., 1963: 486). 37 (T.C.B.D.İ.E., 1963: 486). 118 118 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. Eğitim-Öğretim Dönemi Öğretmen Bilgileri Asil Öğretmenler Stajyerler Ücretli Öğretmenler Kadrosu Başka Okulda Olanlar Toplam E K E K E K E K E K T 1959-1960 38 17 4 1 - - - - - 18 4 22 1960-196139 16 4 1 - - - - - 17 4 21 1961-1962 40 15 4 - 2 - - - - 15 6 21 1962-1963 41 13 5 1 1 - - - - 14 6 20 1963-1964 42 19 - 1 1 - - - - 20 1 21 1964-1965 43 19 - 1 1 - - - - 20 1 21 1965-196644 21 2 2 2 - - - - 23 4 27 1966-196745 23 4 1 1 - - - - 24 5 29 Eğitim-Öğretim Dönemi Öğretmen Bilgileri 46 E K T 1967-1968 28 5 33 1968-1969 29 5 34 1969-1970 34 4 38 1970-1971 29 4 33 1971-1972 32 2 34 1972-1973 29 7 36 1973-1974 47 29 8 37 Bu tablodaki bilgilerden görülmektedir ki; 1954-1974 yılları arasında en çok öğretmenin bulunduğu dönem 38 öğretmenin görev yaptığı 1969-1970 eğitim-öğretim dönemidir. Bu dönemdeki 38 öğretmenin 34’ü erkek, 4’ü ise kadındır. En az öğretmenin görev yaptığı dönem ise, 18 öğretmenin bulunduğu 1956-1957 eğitim-öğretim dönemi olmuştur. Bu 18 öğretmenin 17’si asil, 1’i ise stajyer öğretmen olup, 17 asil öğretmenin 15’i erkek, 2’si kadındır. Ayrıca, bu dönemde 1 erkek stajyer öğretmen görev yapmıştır. Bunların dışında, 1958-1959 eğitim-öğretim döneminde ise ücretli olarak görev yapan 1 öğretmen bulunmuştur. Her eğitim-öğretim döneminde erkek öğretmenlerin sayısı kadın öğretmenlerin sayısından oldukça fazla olmuştur. 1954-1965 yılları arasında öğretmen sayısı 18-22 kişi arasında değişirken, 1965 yılında okulun öğrenmen sayısı bir önceki yıla göre 6 kişi artarak 27’ye yükselmiştir. Yıllara göre öğretmen sayısındaki artış en fazla 1965’de olmuştur. GÖNEN İLKÖĞRETMEN OKULU 1965 yılından sonra ise öğretmen sayıları 1970 yılına kadar artmış ancak 1970 yılında öğretmen sayısı bir önceki yıla göre 5 kişi azalarak 33’e Eğitim-Öğretim Dönemi Öğretmen Bilgileri Asil Öğretmenler Stajyerler Ücretli Öğretmenler Kadrosu Başka Okulda Olanlar Toplam E K E K E K E K E K T 1959-1960 38 17 4 1 - - - - - 18 4 22 1960-196139 16 4 1 - - - - - 17 4 21 1961-1962 40 15 4 - 2 - - - - 15 6 21 1962-1963 41 13 5 1 1 - - - - 14 6 20 1963-1964 42 19 - 1 1 - - - - 20 1 21 1964-1965 43 19 - 1 1 - - - - 20 1 21 1965-196644 21 2 2 2 - - - - 23 4 27 1966-196745 23 4 1 1 - - - - 24 5 29 Eğitim-Öğretim Dönemi Öğretmen Bilgileri 46 E K T 1967-1968 28 5 33 1968-1969 29 5 34 1969-1970 34 4 38 1970-1971 29 4 33 1971-1972 32 2 34 1972-1973 29 7 36 1973-1974 47 29 8 37 Bu tablodaki bilgilerden görülmektedir ki; 1954-1974 yılları arasında en çok öğretmenin bulunduğu dönem 38 öğretmenin görev yaptığı 1969-1970 eğitim-öğretim dönemidir. Bu dönemdeki 38 öğretmenin 34’ü erkek, 4’ü ise kadındır. En az öğretmenin görev yaptığı dönem ise, 18 öğretmenin bulunduğu 1956-1957 eğitim-öğretim dönemi olmuştur. Bu 18 öğretmenin 17’si asil, 1’i ise stajyer öğretmen olup, 17 asil öğretmenin 15’i erkek, 2’si kadındır. Ayrıca, bu dönemde 1 erkek stajyer öğretmen görev yapmıştır. Bunların dışında, 1958-1959 eğitim-öğretim döneminde ise ücretli olarak görev yapan 1 öğretmen bulunmuştur. Her eğitim-öğretim döneminde erkek öğretmenlerin sayısı kadın öğretmenlerin sayısından oldukça fazla olmuştur. 1954-1965 yılları arasında öğretmen sayısı 18-22 kişi arasında değişirken, 1965 yılında okulun öğrenmen sayısı bir önceki yıla göre 6 kişi artarak 27’ye yükselmiştir. Yıllara göre öğretmen sayısındaki artış en fazla 1965’de olmuştur. 1965 yılından sonra ise öğretmen sayıları 1970 yılına kadar artmış ancak 1970 yılında öğretmen sayısı bir önceki yıla göre 5 kişi azalarak 33’e 119 38 (T.C.B.D.İ.E., 1963: 486). 39 (T.C.B.D.İ.E., 1965: 302). 40 (T.C.B.D.İ.E., 1968: 401). 41 (T.C.B.D.İ.E., 1968: 401). 42 (T.C.B.D.İ.E., 1968: 401). 43 (T.C.B.D.İ.E., 1968: 401). 44 (D.İ.E., 1969: 249). 45 (D.İ.E., 1969: 249). 46 (D.İ.E., 1977: 5). 47 (B.D.İ.E., 1979: 72). 119 119 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. düşmüştür. GÖNEN İLKÖĞRETMEN OKULU Daha sonra ise öğretmen sayısı çok olmasa da her yıl 1-2 kişi artarak devam etmiş ve 1973 yılında okulun öğretmen sayısı 37’ye yükselmiştir. Öğretmen Okullarının 122. kuruluş yıl dönümü tüm öğretmen okullarında olduğu gibi Gönen’de de törenle kutlanmıştır. Törende konuşmalar yapılmış, şiirler okunmuş ve milli oyunlar oynanmıştır (Öğretmen Okullarının 122. Kuruluş Yıl Dönümü Dün Kutlandı, 17 Mart 1970: 1). 1966-1967 eğitim-öğretim yılında Gönen İlköğretmen Okulu’nda toplam 2.612 öğrenci öğrenim görmüştür. Bu öğrencilerin 2.165’i erkek, 447’si kız öğrenci olmuştur. Aynı dönemde bu okuldaki öğretmen sayısı 118 olup, öğretmenlerin 82’si erkek, 36’sı kadındır (Isparta Valiliği 1967 İl Yıllığı, 1967: 131).1971 yılında Millî Eğitim Bakanlığı tarafından Gönen İlköğretmen Okulu’ndan 60 öğrenci ilköğretmen okulları müfredat programı ve yönetmeliği hükümleri dahilinde Kuşadası’nda açılan yaz dönemi kursuna çağrılmıştır. Bu öğrencilerin kursa geliş-gidiş vb. ihtiyaçlarının ise Ortaklar İlköğretmen Okulu Müdürlüğünce karşılanacağı bildirilmiştir. Kursa Isparta’dan Gönen İlköğretmen Okulu çağrılırken Isparta Kız İlköğretmen Okulu ve Isparta Eğitim Enstitüsü öğrencileri çağrılmamıştır (Gönen İlköğretmen Okulundan 60 Öğrenci Kuşadası’ndaki Yaz Dönemi Kursuna Çağrıldı, 28 Nisan 1971: 1). 28 Nisan 1971 tarihinde Gönen İlköğretmen Okulunun 900 öğrencisi 5 gün olarak belirledikleri bir boykota başlamıştır. Ancak okulun 30 kız öğrencisi bu boykota katılmamıştır. Boykota katılan öğrencilerin istekleri: • Mesleki kitaplarının tekrar yazılması, • Öğrencilerin okul yönetiminde söz sahibi olmak istemeleri, • Öğrencilerin komşu köylerde yaptıkları 2 aylık staj masraflarının okulun karşılamasını istemeleri (Gönen İlköğretmen Okulunun 900 Öğrencisi Dün 5 Gün Sürece Boykota Başladı, 29 Nisan 1971: 1), • Amerikan gıda, eğitim ve düzen emperyalizmine son verilmesi, • Okul içindeki nurculuk politikasına göz yumulmaması, • Okul müdürü başta olmak üzere bazı öğretmenlerin değiştirilmesi… vb. şeklinde olmuştur (Vali Öztürk Gönen İlköğretmen Okulunu Kapattı, 10 Mayıs 1971: 2). Bu boykotu duyan Isparta Valisi Hilmi Öztürk, Okul Müdürü Mehmet Kahvecioğlu’ndan boykot hakkında bilgi almış ve bizzat olay yerine giderek 2 saat boyunca öğrencileri boykottan geri döndürmeye çalışmış ancak başarılı olamamıştır (Gönen İlköğretmen Okulunun 900 Öğrencisi Dün 5 Gün Sürece Boykota Başladı, 29 Nisan 1971: 1). Daha sonra öğrencilerin okul idaresini basmaları nedeniyle okul yetkilileri jandarma kuvvetlerini çağırmış ve okul 15 gün süre ile kapatılmıştır (Gönen İlköğretmen Okulu Çıkması Muhtemel Olaylara Karşı 15 Gün Süreyle Kapatıldı, 30 Nisan 1971: 1). Okul, 29 Nisan 1971 tarihinde de boykot yapan öğrencilerin taşkınlık çıkaracakları düşüncesiyle valilik tarafından süresiz olarak kapatılmıştır (Boykot Yüzünden 11 Gün Kapalı Kalan Gönen İlköğretmen Okulu Dün Açıldı, 11 Mayıs 1971: 1). Gazetecilerle görüşmeler yapan öğrenciler ise bu boykotta ideolojik bir tutumlarının 120 120 Yüzbaşı, E. (2020). GÖNEN İLKÖĞRETMEN OKULU Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. olmadığını, hakları olan istekleri talep ettiklerini ifade etmişlerdir (Kapatılan İlköğretmen Okulunu Jandarma Bekliyor, 1 Mayıs 1971: 1). Süresiz kapatıldıktan 11 gün sonra 10 Mayıs 1971 (Gönen Öğretmen Okulu Dün Törenle Öğrenime Açıldı, 17 Mayıs 1971: 2)48 tarihinde bu okul tekrar açılmış ve okul düzenlenen bir törenle normal öğretime (Boykot Yüzünden 11 Gün Kapalı Kalan Gönen İlköğretmen Okulu Dün Açıldı, 11 Mayıs 1971: 1) başlamıştır. Tören sonrasında Milli Eğitim Müdürü Sadık Güneş ve İl Jandarma Alay Komutanı Kd. Albay Mustafa Hacı Hanifioğlu öğrenci ve velileri uyarıcı konuşmalar yapmıştır. Daha sonra Okul Müdürü Mehmet Kahvecioğlu’nun da katılımıyla velilerle bir toplantı yapılarak ortak iş birliğine karar verilmiştir (Gönen Öğretmen Okulu Dün Törenle Öğrenime Açıldı, 17 Mayıs 1971: 2). 1972 yılında mevcudu 912 olan okulun hem köy hem de şehir kontenjanı olup, 8 öğrenci köy, 8 öğrenci de şehir kontenjanına girmeye hak kazanmıştır (İlköğretim Okulları Sınavını Kazanan Öğrenciler Belli Oldu, 2 Ağustos 1972: 1). Milliyet gazetesinin düzenlediği halk oyunları yarışmasında Türkiye 4.’sü olan Gönen İlköğretmen Okulu 25 Nisan 1974 tarihinde İstanbul’da yapılan final gecesinde halk oyunları kategorisinde adını duyurmayı başarmıştır (Gönen İlköğretmen Okulu Halk Oyunları Yarışmasında, 6 Nisan 1974: 1). 1940 yılında Köy Enstitüsü olarak kurulan okul, 1954’te Gönen İlköğretmen Okuluna dönüştürülmüştür (Okan, 5 Haziran 1955: 2). 1973-1974 eğitim-öğretim döneminden itibaren de öğretmen okullarının ismi öğretmen lisesi adını almış olup, öğretim süresi de 7 yıldan 6 yıla indirilmiştir. Ancak bu değişiklik yapıldığında Gönen İlköğretmen Okulu’nun son sınıf öğrencileri eski statüye göre 7 yıl olarak öğrenimlerine devam etmiştir. 6 yıllık eğitim-öğretime 1976 yılından itibaren başlanmıştır (B.D.İ.E., 1982: 1-2). 48 17 Mayıs 1971 tarihli Öz Yalvaç gazetesinde 1.000 mevcutlu olarak gösterilen Gönen İlköğretmen Okulu’nun boykot sonrası eğitim-öğretime başlama tarihi 16 Mayıs 1971 olarak gösterilmektedir. SONUÇ Cumhuriyet öncesinde bugünkü anlamda öğretmen yetiştiren ilk öğretmen okulu 16 Mart 1848 tarihinde İstanbul’da açılan Darulmuallimin okuludur. Isparta’da ilk muallim mektebi ise; 1903 yılında açılan Darulmuallimini İptidaiyye Mektebi olmuştur. Cumhuriyetin ilanından sonra halkın %80’inin okuma-yazma bilmemesi ve köylerde okur-yazarlık oranının daha da düşük olması nedeniyle eğitim sorununun üzerinde ayrıca durulmuştur. Atatürk’ün emri doğrultusunda köylerdeki öğretmen sorununu gidermek için 1936-1937 eğitim öğretim yılında eğitmen kurslarının ilki Eskişehir Çiftelerde faaliyete geçirilmiştir. Gönen Eğitmen Kursu ise 1939 yılında açılmış olup, 17 Nisan 1940 tarihindeki 3803 Sayılı Köy Enstitüleri Kanunuyla Gönen Köy Enstitüsü halini almıştır. Gönen Köy Enstitüsü, 1954’te 6234 Sayılı Köy Enstitüleri ile Öğretmen Okullarının birleştirilmesi kanunuyla Gönen İlköğretmen Okulu’na dönüştürülmüştür. Okul, 1955 yılına kadar 42 kız ve 883 erkek öğretmen mezun etmiştir. Gönen İlköğretmen Okulu’ndan mezun olan 925 öğretmen ülkenin çeşitli yerlerinde görev yapmıştır. Öğretmenlik bilgisinin yanında biçki, nakış, demircilik, bahçecilik vb. alanda 121 48 17 Mayıs 1971 tarihli Öz Yalvaç gazetesinde 1.000 mevcutlu olarak gösterilen Gönen İlköğretmen Okulu’nun boykot sonrası eğitim-öğretime başlama tarihi 16 Mayıs 1971 olarak gösterilmektedir. 48 17 Mayıs 1971 tarihli Öz Yalvaç gazetesinde 1.000 mevcutlu olarak gösterilen Gönen İlköğretmen Okulu’nun boykot sonrası eğitim-öğretime başlama tarihi 16 Mayıs 1971 olarak gösterilmektedir. 121 121 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. da eğitilen Gönen İlköğretmen Okulu öğrencilerinin ihtiyaç duydukları malzemeler de eğitim aldıkları alanla ilgili olmuştur. Öğrencilerin okullarında ürettikleri ürünler düzenlenen sergilerle halka sunulmuş olup, anma günleri, tiyatrolar, halk oyunları vb. etkinliklerle okulun ismi duyurulmuştur. Parasız yatılı olarak öğrenim veren Gönen Öğretmen Okulu 1971-1972 yılına kadar 6 yıllık eğitim-öğretim vermiştir. 1971-1972 döneminden itibaren de eğitim öğretim süresi 7 yıla çıkarılmıştır. Öğrenim süresinin 7 yıla çıkmasıyla 1972-1973 eğitim-öğretim döneminde sadece 2 öğrenci mezun olmuştur. Geçiş sürecindeki bu dönem dışında en az mezun veren dönem 1958-1959 eğitim-öğretim dönemi olup, bu dönemde mezun olan 34 öğrencinin hepsi erkektir. En çok mezun ise 1969-1970 eğitim- öğretim döneminde verilmiş, 197’si erkek, 10’u kız olmak üzere toplam 207 öğrenci mezun olmuştur. 1954-1974 yılları arasında Gönen Öğretmen Okulu’ndan 2.381 öğrenci mezun olmuş olup, mezun olan öğretmenlerin 2.289’u erkek, 92’si ise kızdır. Mezun olan bu öğretmenler yurdun çeşitli yerlerindeki okullarda göreve başlamışlardır. Gönen İlköğretmen Okulu’nun öğretmen sayısı 1954-1974 yılları arasında 18 ile 38 arasında değişmiştir. En çok öğretmenin bulunduğu dönem 1969- 1970 dönemi iken en az öğretmenin bulunduğu dönem ise 1956-1957 dönemi olmuştur. Gönen Köy Enstitüsü’nün devamı niteliğinde olan Gönen İlköğretmen Okulu, öğretmen yetiştirme konusunda önemli rol üstlenmiştir. SONUÇ Okul 1974’te Gönen öğretmen Lisesi, 1989’da Anadolu Öğretmen Lisesi ve 2014’te de Gönen Fen Lisesi olarak eğitim- öğretim faaliyetlerini sürdürmüş olup, halende Gönen Fen Lisesi olarak eğitim-öğretime devam etmektedir. KAYNAKÇA Akyüz, Y. (2011), Türk Eğitim Tarihi (M.Ö. 1000-M.S.2011). Ankara: Pegem Akademi Yayınları. Altunya, N. (1990), Köy Enstitülerinin Tarihçesi, Kuruluşunun 50. Yılında Köy Enstitüleri. Ankara: Eğit-der Yayınları. Başbakanlık Devlet İstatistik Enstitüsü [B.D.İ.E.]. (1979), Milli Eğitim İstatistikleri Mesleki ve Teknik Öğretim 1973-1975. Ankara. Başbakanlık Devlet İstatistik Enstitüsü [B.D.İ.E.]. (1982), Milli Eğitim İstatistikleri Mesleki ve Teknik Öğretim 1975-1978. Ankara. Boykot Yüzünden 11 Gün Kapalı Kalan Gönen İlköğretmen Okulu Dün Açıldı. (1971), Isparta. 11 Mayıs 1971. Çavuşoğlu, B. (1961), Resim Sergisi, Isparta Postası. 25 Haziran 1961. Devlet İstatistik Enstitüsü [D.İ.E]. (1969), Milli Eğitim İstatistikleri Mesleki ve Teknik Öğretim 1965-1967. Ankara. Devlet İstatistik Enstitüsü [D.İ.E.]. (1977), Milli Eğitim İstatistikleri Mesleki ve Teknik Öğretim 1967-1973. Ankara. Demokrat Eğirdir. (27 Ocak 1962). Göller Bölgesi Köy Öğretmenleri Derneğinin Sütçüler Şubesi Açıldı. (1954), Yalvaç. 30 Kasım 1954. 122 122 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. Göller Bölgesi Köy Öğretmenler Derneğinin Yıllık Kongresi. (1965), Yeni İnkılâp. 26 Ekim 1965 Göller Bölgesi Köy Öğretmenler Derneğinin Yıllık Kongresi. (1965), Yeni İnkılâp. 26 Ekim 1965 Gönen İlköğretmen Okulunda Bir Mesleki Danışma Bürosu Kurulmuştur. (1954), Yalvaç. 3 Kasım 1954. Gönen İlköğretmen Okulu Çıkması Muhtemel Olaylara Karşı 15 Gün Süreyle Kapatıldı. (1971), Isparta. 30 Nisan 1971. Gönen Öğretmen Okulu Dün Törenle Öğrenime Açıldı. (1971), Öz Yalvaç. 17 Mayıs 1971. Gönen İlköğretmen Okulu Halk Oyunları Yarışmasında. (1974), Isparta. 6 Nisan 1974 Gönen İlköğretmen Okulu Mezunları İçin Dün Mesleğe Giriş Töreni Yapıldı. (1965), Yeni İnkılâp. 2 Temmuz 1965. Gönen İlköğretmen Okulundan Temin Edilen Ders Araçları. (1954), Demokrat Isparta. 13 Kasım 1954. Gönen Öğretmen Okulu Mezunlarından İlimize Tayin Edilen Öğretmenler. (1965), Yeni İnkılâp. 21 Temmuz 1965. Gönen İlköğretmen Okulundan 60 Öğrenci Kuşadası’ndaki Yaz Dönemi Kursuna Çağrıldı. (1971), Isparta. 28 Nisan 1971 Gönen İlköğretmen Okulunun 900 Öğrencisi Dün 5 Gün Sürece Boykota Başladı. (1971), Isparta. 29 Nisan 1971. Gül, S. S. (2013), Gönen Köy Enstitüsü Işığı. İzmir: Yeni Kuşak Köy Enstitüsü Derneği Yayınları. Isparta. (1982), Yurt Ansiklopedisi Türkiye İl İl Dünü Bugünü Yarını (Cilt 5). İstanbul: Anadolu Yayıncılık. Isparta Gönen İlköğretmen Okulu Müdürlüğünden. (1954), Demokrat Isparta. 14 Temmuz 1954. Isparta Valiliği 1967 İl Yıllığı. (1967), Isparta: Isparta Valiliği. Isparta Valiliği 1967 İl Yıllığı. (1967), Isparta: Isparta Valiliği. Isparta Valiliği 1973 İl Yıllığı. (1973), Isparta: Isparta Valiliği. Isparta Valiliği 1973 İl Yıllığı. (1973), Isparta: Isparta Valiliği. İdeal Bir Köy Öğretmeninin Vasıfları. (1948), Isparta. 20 Ekim 1948. İlköğretim Okulları Sınavını Kazanan Öğrenciler Belli Oldu. (1972), Isparta. 2 Ağustos 1972. Kamer, S. T. KAYNAKÇA (2012), Öğretmen Yetiştirmede Kastamonu Kız İlköğretmen Okulu Uygulaması (1956-1975). Cumhuriyet Uluslararası Eğitim Dergisi, 1(2), 1-17. Kapatılan İlköğretmen Okulunu Jandarma Bekliyor. (1971), Isparta. 1 Mayıs 1971 Katırcıoğlu, N. (1958), Bütün Isparta. Ankara: Bereket Matbaası. Kayacan, İ. V. (5 Şubat 2019), Kişisel Görüşme. Köy Enstitüleri Kanunu. (1948), Isparta. 15 Eylül 1948 Köy Enstitüleri Kanunu. (1948), Isparta. 15 Eylül 1948 Köy Enstitüleri Kanunu. (1948), Isparta. 15 Eylül 1948 123 123 Yüzbaşı, E. (2020). Alınteri Sosyal Bilimler Dergisi, 4(2): 111-124. Köy Öğretmenler Derneği Mümessilleri Maarif Vekillerimize Konuştular. (1954), Yalvaç. 30 Aralık 1954. Köy Öğretmenler Derneği Mümessilleri Maarif Vekillerimize Konuştular. (1954), Yalvaç. 30 Aralık 1954. Kuru, M., ve Uzun, H. (2008), Türkiye’de Öğretmen Adaylarının Seçiminde 1954 Örneği. G.Ü. Gazi Eğitim Fakültesi Dergisi, 28(3), 207-232. Okan, K. (1955), Gönen İlköğretmen Okulu. Yalvaç, 5 Haziran 1955. Öğretmen Okulları Sergisine Gönen Katılacak. (1954), Yalvaç. 18 Kasım 1954. Öğretmen Adaylarının İlk Grubu Dün Staj Okullarında Görevlerine Başladı. (1965), Yeni İnkılâp. 2 Kasım 1965. Öğretmen Okullarına Ortaokullardan Öğrenci Alınacak. (1961), Demokrat Eğirdir. 6 Mayıs 1961. Öğretmen Okulu Tarım Öğretmeni Kurs Açtı. (1965), Yeni İnkılâp. 22 Ekim 1965 Öğretmen Okullarının 122. Kuruluş Yıl Dönümü Dün Kutlandı. (1970), Isparta. 17 Mart 1970. Vali Öztürk Gönen İlköğretmen Okulunu Kapattı. (1971), Öz Yalvaç. 10 Mayıs 1971. Özbek, H. (1971), Köy Enstitüleri. Isparta, 27 Nisan 1971. Özbek, H. (1971), Köy Enstitüleri. Isparta, 27 Nisan 1971. Süldür, E. (1951), İkinci Isparta Tarihi. İzmir: Üstün Sanat Matbaası. Şimşek, G., ve Mercanoğlu, C. (2018), Bir Planlanama Örneği Olarak Köy Enstitüleri Deneyimi. Planlama, 28(3), 261-281. Şimşir, B. N. (2006), Türk Harf Devrimi Üzerinde İncelemeler. Ankara: Atatürk Araştırma Merkezi. Taşer, S. (2010), Konya Vilayeti Sancak Merkezlerinde Eğitim Veren Darülmualliminler. Türkiyat Araştırmaları Dergisi, 27, 665-678. Türkiye Cumhuriyeti Başbakanlık Devlet İstatistik Enstitüsü [T.C.B.D.İ.E.]. (1963), Milli Eğitim Meslek, Teknik ve Yüksek Öğretim İstatistikleri 1953-1960. Ankara. Türkiye Cumhuriyeti Başbakanlık Devlet İstatistik Enstitüsü [T.C.B.D.İ.E.]. (1965), Milli Eğitim İstatistikleri Meslek, Teknik ve Yüksek Öğretim 1960-1961. Ankara. Türkiye Cumhuriyeti Başbakanlık Devlet İstatistik Enstitüsü [T.C.B.D.İ.E.]. (1968), Milli Eğitim İstatistikleri Meslek, Teknik ve Yüksek Öğretim 1961-1965. Ankara. Yalçın, D., Akbıyık, Y., Özkaya, Y., Bozkurt, G., Akbulut, D. A., Tokgöz, E., ve Eraslan, C. (2005), Türkiye Cumhuriyeti Tarihi II. Ankara: ATAM Yayınları. Yıldız, N., ve Akandere, O. (2017), Köy Enstitülerinin İdeolojik Yapısı, Çağdaş Türkiye Tarihi Araştırmaları, 27(35), 275-316. 124 124
https://openalex.org/W3109321398	https://hal.science/hal-03007482/document	English	null	Neural Networks approaches focused on French Spoken Language Understanding: application to the MEDIA Evaluation Task	null	2,020	cc-by	3,719	To cite this version: Sahar Ghannay, Christophe Servan, Sophie Rosset. Neural Networks approaches focused on French Spoken Language Understanding: application to the MEDIA Evaluation Task. In Proceedings of The 28th International Conference on Computational Linguistics (COLING’2020), 2020, Dec 2020, Barcelona (online), Spain. hal-03007482 Neural Networks approaches focused on French Spoken Language Understanding: application to the MEDIA Evaluation Task Sahar Ghannay, Christophe Servan, Sophie Rosset Sahar Ghannay, Christophe Servan, Sophie Rosset HAL Id: hal-03007482 https://hal.science/hal-03007482v1 Submitted on 16 Nov 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Abstract In this paper, we present a study on a French Spoken Language Understanding (SLU) task: the MEDIA task. Many works and studies have been proposed for many tasks, but most of them are focused on English language and tasks. The exploration of a richer language like French within the framework of a SLU task implies to recent approaches to handle this difﬁculty. Since the MEDIA task seems to be one of the most difﬁcult, according to several previous studies, we propose to explore Neural Networks approaches focusing of three aspects: ﬁrstly, the Neu- ral Network inputs and more speciﬁcally the word embeddings; secondly, we compared French version of BERT against the best setup through different ways; Finally, the comparison against State-of-the-Art approaches. Results show that the word embeddings trained on a small cor- pus need to be updated during SLU model training. Furthermore, the French BERT ﬁne-tuned approaches outperform the classical Neural Network Architectures and achieves state of the art results. However, the contextual embeddings extracted from one of the French BERT approaches achieve comparable results in comparison to word embedding, when integrated into the proposed neural architecture. This work is licensed under a Creative Commons Attribution 4.0 International Licence. http://creativecommons.org/licenses/by/4.0/ Neural Networks approaches focused on French Spoken Language Understanding: application to the MEDIA Evaluation Task Sahar Ghannay† Christophe Servan‡ Sophie Rosset† Sophie Rosset† Christophe Servan‡ ‡ QWANT 61 rue de Villier 92200 Neuilly-sur-Seine, France inital.lastname@qwant.com †Universit´e Paris-Saclay, CNRS, LIMSI, 91405 Orsay, France lastname@limsi.fr Abstract 1 Introduction Spoken language understanding (SLU) module is a key component for a spoken language dialogue sys- tem. It consists on semantically analyse user queries and identiﬁes text spans that mention semantic concepts. SLU task can fall into three sub-tasks: domain classiﬁcation, intent classiﬁcation, and slot- ﬁlling (Tur and Mori, 2011). The latter is the task that interests us in this study. Over the past ﬁve years, the studies developed for SLU task are based on neural network architec- tures (Yao et al., 2014; Mesnil et al., 2015; Guo et al., 2014; Zhang and Wang, 2016; Dinarelli et al., 2017; Simonnet et al., 2017; Korpusik et al., 2019; Ghannay et al., 2020). Recent approaches take ben- eﬁt from contextual or language model embeddings such as BERT (Devlin et al., 2019). Korpusik et al. (2019) investigated the transfer ability of a pre-trained BERT representation for English SLU tasks. But, as far as we know, there are no such studies on a French SLU task. Following Ghannay et al. (2020)’s study, many avenues can be explored. In their study, the word embeddings have been frozen during training (are not updated), since Lebret et al. (2013) show that ﬁne- tuned word embeddings show very similar performance and provide comparable results. However, the evaluation of whether updating the embeddings during SLU model training improves or not the results, for SLU task, is less studied. In addition, their SLU model is fed only with word embeddings, and they did not use any additional features, thus there are some rows for improvements. Finally, B´echet and Raymond (2019) benchmarked several SLU tasks and proposed a difﬁculty hierarchy in which the MEDIA evaluation (Bonneau-Maynard et al., 2006) seems to be the most difﬁcult SLU task. Contributions: This study focuses on a French SLU task: the MEDIA evaluation, in which we ﬁrstly propose the evaluation of whether updating the word embeddings during training can improve the results, according to several scenarios. Secondly, we propose to use a BiLSTM-CNN architecture (Ma, 2016) that integrates character embeddings as additional features, using a convolution layer. Finally, we propose to evaluate the performance of BERT approaches against the BiLSTM-CNN architecture and State-of-the- Art on the MEDIA task (Simonnet, 2019) through different ways: i) We propose to ﬁne-tune BERT on SLU task using two french models: CamemBERT (Martin et al., 2020) and FlauBERT (Le et al., 2020). 2the code an data needed to run the experiments are available here: https://github.com/saharghannay/MEDIA Eval 3MEDIA is publicly available for academic use: https://catalogue.elra.info/en-us/repository/browse/ELRA-S0272/ 3.1 Data Experiments are conducted on the French MEDIA3 corpus, composed of 1258 transcribed dialogues, which is about hotel reservation and information (Bonneau-Maynard et al., 2006). The corpus was manually annotated, following a BIO model, with semantic concepts characterized by a label and its value. The corpus is split into three parts: a training corpus composed of 13k sentences, a development corpus composed of 1.3k sentences, and a test corpus composed of 3.5k sentences. 1 Introduction ii) based on the results of i) we propose to integrate the extracted BERT contextual embeddings to the BiLSTM-CNN architecture and compare it to word embeddings. 3 Experiments In this section, we present the experiments we performed using our approaches and their setup2. For both BiLSTM and BiLSTM-CNN, we made some hyper-parameters tuning by varying the number of layers l ∈{1, 2, 3}, the size of the BiLSTM hidden layers n ∈{128, 256, 512} and the batch size b ∈{16, 32, 64}. For BiLSTM-CNN, in addition to the other parameters, the window size is set to 3 and the number of ﬁlters (dimension of character embeddings) is set to s ∈{30, 50, 100}. 2 SLU Model descriptions This section describe the SLU models used in this study. The ﬁrst two models are based on BiLSTM and its update: the BiLSTM-CNN. The NeuroNLP2 implementation1 was used for both BiLSTM implemen- tations. The third model is based on the BERT models. BiLSTM (Bidirectional long short-term memory) architecture has been proven to be relevant to model output dependencies on SLU tasks (Yao et al., 2014; Mesnil et al., 2015). To further improve the performance of our SLU model, we propose to use a BiLSTM-CNN (convolu- tional neural network) architecture (Ma, 2016) that integrates character embeddings using a convolution layer, in addition to the word embeddings. Finally, we propose to ﬁne-tune BERT (Devlin et al., 2019) on SLU task using two french models: CamemBERT (Martin et al., 2020) and FlauBERT (Le et al., 2020). The CamemBERT model is trained on the French part of the OSCAR corpus (Su´arez et al., 2019) composed of 138GB of raw text, and FlauBERT (Le et al., 2020) on 71GB of heterogeneous French corpora. 1https://github.com/XuezheMax/NeuroNLP2 2 3.3.1 Embeddings update #Layer WIKI WIKI+MEDIA Character embeddings dimensions 30 50 100 30 50 100 1 84.38 84.59 84.85 84.13 84.47 84.73 2 85.88 86.43 86.18 86.20 86.75 86.34 3 87.02 87.05 87.40 87.29 87.01 87.30 Table 2: Results on Test MEDIA in terms of F1 score using embeddings trained on both wiki and wiki+MEDIA corpora, using the BiLSTM-CNN architecture. (The word embeddings are frozen) Conﬁg. Update Freeze Train #nb. BiLSTM layers #nb. BiLSTM layers Emb. 1 2 3 1 2 3 MEDIA 84.18 84.18 85.35 72.36 79.57 80.69 WIKI 84.73 85.82 86.47 84.11 86.06 86.40 WIKI 84.84 85.35 86.00 84.08 85.74 86.69 +MEDIA Table 1: Performance on Test MEDIA in terms of F1 score of CBOW word embeddings ap- proach trained on three corpora (MEDIA, WIKI and WIKI+MEDIA), using the BiLSTM architec- ture. Table 1: Performance on Test MEDIA in terms of F1 score of CBOW word embeddings ap- proach trained on three corpora (MEDIA, WIKI and WIKI+MEDIA), using the BiLSTM architec- ture. Table 1: Performance on Test MEDIA in terms of F1 score of CBOW word embeddings ap- proach trained on three corpora (MEDIA, WIKI and WIKI+MEDIA), using the BiLSTM architec- ture. Table 2: Results on Test MEDIA in terms of F1 score using embeddings trained on both wiki and wiki+MEDIA corpora, using the BiLSTM-CNN architecture. (The word embeddings are frozen) Table 2: Results on Test MEDIA in terms of F1 score using embeddings trained on both wiki and wiki+MEDIA corpora, using the BiLSTM-CNN architecture. (The word embeddings are frozen) 3.3.2 Character embeddings evaluation In this section, we propose to use a BiLSTM-CNN architecture (Ma, 2016) that integrates character em- beddings as additional features, using a convolution layer. We experiment the use of different character embeddings dimensions, and different numbers of BiLSTM layers. Based on the results in section 3.3.1, for those experiments, we used the embeddings trained on both WIKI and WIKI+MEDIA corpora, which are frozen during the BiLSTM-CNN training. Results summarized in table 2, show that the use of character embeddings as additional features was helpful and improves the performance in comparison to the results in table 1. We observe that, both embeddings trained on WIKI and WIKI+MEDIA achieve comparable results. This shows that, we don’t need to use both a task-dependent corpus and another out-of-domain corpus to train the word embed- dings. Note that, we observed the same thing, when the embeddings trained on WIKI data are ﬁne-Tuned on MEDIA data. The best results (F1=87.40) achieved using the embeddings trained on WIKI data, us- ing the appropriate parameters: 3 BiLSTM layers and character embeddings dimension of 100. Note that, beyond these values the performance of the system drops slightly. 3.2 Word embeddings training One of the aim of our experiments is to see whether the update of the word embeddings during training of the SLU model (update) improves or not the results by mainly varying the data used to train the word embeddings and the hyper-parameters of the SLU model. Following Ghannay et al. (2020)’s results, we propose to use CBOW word embeddings approach from word2vec (Mikolov et al., 2013), which is trained using the default parameters using three different corpora setup. The ﬁrst one is a small and task-dependent corpus: training set of MEDIA corpus is used, by keeping all the words due to the small data size. A huge and out-of-domain corpus was used as second setup: the French Wikipedia dump (WIKI), which is composed of 573 million words using a vocabulary size of 923k words. Finally, both corpora (noted WIKI+MEDIA). The common parameters used to train our word embeddings are: window size=5, negative sampling=5, dimension=300. They have been selected based on previous studies (Pennington et al., 2014; Bojanowski et al., 2017). Note that the out of vocabulary (OOV) words are represented by null vectors. 3.3.1 Embeddings update Those experiments aim to observe the impact of the update (noted update) of CBOW word embeddings or their freeze (noted freeze) while training of SLU module. We proposed different training setups for the word embeddings (MEDIA, WIKI and WIKI+MEDIA), presented in section 3.2. Also, the BiLSTM number of layers is set from 1 to 3. Results summarized in Table 1 show that when the word embeddings are trained on MEDIA data, the update of the word embeddings is helpful and improves the results in terms of F1 score, whatever the size of the architecture. However, when the embeddings are trained on WIKI or WIKI+MEDIA data the update of the embeddings while training is not helpful and degrades the results. Thus, the best results are obtained using the BiLSTM architecture composed of 3 hidden layers, using one of the CBOW embeddings trained on WIKI or WIKI+MEDIA corpora, that obtain comparable results in terms of F1 score (86.40 vs. 86.69). Conﬁg. Update Freeze Train #nb. BiLSTM layers #nb. BiLSTM layers Emb. 1 2 3 1 2 3 MEDIA 84.18 84.18 85.35 72.36 79.57 80.69 WIKI 84.73 85.82 86.47 84.11 86.06 86.40 WIKI 84.84 85.35 86.00 84.08 85.74 86.69 +MEDIA Table 1: Performance on Test MEDIA in terms of F1 score of CBOW word embeddings ap- proach trained on three corpora (MEDIA, WIKI and WIKI+MEDIA), using the BiLSTM architec- ture. #Layer WIKI WIKI+MEDIA Character embeddings dimensions 30 50 100 30 50 100 1 84.38 84.59 84.85 84.13 84.47 84.73 2 85.88 86.43 86.18 86.20 86.75 86.34 3 87.02 87.05 87.40 87.29 87.01 87.30 Table 2: Results on Test MEDIA in terms of F1 score using embeddings trained on both wiki and wiki+MEDIA corpora, using the BiLSTM-CNN architecture. (The word embeddings are frozen) Conﬁg. Update Freeze Train #nb. BiLSTM layers #nb. BiLSTM layers Emb. 1 2 3 1 2 3 MEDIA 84.18 84.18 85.35 72.36 79.57 80.69 WIKI 84.73 85.82 86.47 84.11 86.06 86.40 WIKI 84.84 85.35 86.00 84.08 85.74 86.69 +MEDIA Table 1: Performance on Test MEDIA in terms of F1 score of CBOW word embeddings ap- proach trained on three corpora (MEDIA, WIKI and WIKI+MEDIA), using the BiLSTM architec- ture. 3.3.3 Comparison to French BERT ii) Last, we propose to integrate the extracted BERT’s contextual embeddings to the BiLSTM and BiLSTM-CNN architectures, instead of CBOW word embeddings. Based on the results of i) we used the embeddings extracted from CamemBERT base model trained on ccnet data. After tokenizing the ME- DIA corpus, the CamemBERT model was applied on the resulting data to extract the embeddings of 768 dimensions, for each sub-word from the last transformer layer. The token embeddings corresponds to the sum of its sub-word embeddings. A new CBOW embeddings is trained on WIKI data with dimension 768 to have comparable results. Results (last 2 lines) show that the use of CamemBERT contextual embed- dings achieves competitive results in comparison to CBOW embeddings whatever the architecture used (BiLSTM or BiLSTM-CNN). Those results corroborate the results reported by Ghannay et al. (2020), in which, CBOW and ELMo (Peters et al., 2018) embeddings obtained comparable results in terms of F1 score (86.06 vs. 86.42). Last, the results with BiLSTM and BiLSTM-CNN architectures reveals the importance of character embeddings, even when they are combined with contextual embeddings. 3.3.3 Comparison to French BERT In this section, we propose to evaluate the performance of BERT approaches on the MEDIA task through two different ways. The experimental results are summarized in table 3 using F1 score and the Concept Error Rate (CER), which is estimated like the Word Error Rate (WER). The CER is used to compare our approach with the State-of-the-Art proposed by Simonnet (2019) and noted biRNN-EDA. We also reported the results of the best system presented in table 2. i) We propose to ﬁne-tune BERT on SLU task using two French models: CamemBERT (Martin et al., 2020) and FlauBERT (Le et al., 2020) base models. Results in table 3 show the performance of BERT’s models on SLU task. The best results achieved using CamemBERT base model trained on ccnet data. It yields 29.35% of relative improvement in terms of CER reduction in comparison to the baseline (7.56 vs 10.7). In addition, it outperforms the proposed BiLSTM-CNN system and improves the prediction of Architecture Embed. Training data Embed.’s approach F1 CER biRNN-EDA – – – 10.7 BiLSTM-CNN WIKI CBOW (dim=300)† 87.40 9.88 CBOW (dim=768) 86.80 10.11 FineTune BERT (i) oscar 138 GB CamemBERT-base 89.18 7.93 ccnet 135 GB CamemBERT-base 89.37 7.56 heterogeneous corpus 71 GB FlauBERT-base 89.04 8.13 BiLSTM ccnet 135GB (ii) CamemBERT-base (dim=768) 86.59 10.45 BiLSTM-CNN CamemBERT-base (dim=768) 87.15 10.11 Table 3: Performance on Test MEDIA in terms of F1 and CER scores of the proposed systems († is the best system presented in table 2). Table 3: Performance on Test MEDIA in terms of F1 and CER scores of the proposed systems († is the best system presented in table 2). Table 3: Performance on Test MEDIA in terms of F1 and CER scores of the proposed systems († is the best system presented in table 2). some tags: “nom, chambre-fumeur, objet, ...”. We observe that the models CamemBERT base (trained on OSCAR data) and FlauBERT obtain competitive results in terms of F1 and CER scores. Note that CamemBERT and FlauBERT base models achieve better results than the large models. some tags: “nom, chambre-fumeur, objet, ...”. We observe that the models CamemBERT base (trained on OSCAR data) and FlauBERT obtain competitive results in terms of F1 and CER scores. Note that CamemBERT and FlauBERT base models achieve better results than the large models. References Fr´ed´eric B´echet and Christian Raymond. 2019. Benchmarking benchmarks: introducing new automatic indicators for benchmarking spoken language understanding corpora. In Interspeech, Graz, Austria. Piotr Bojanowski, Edouard Grave, Armand Joulin, and Tomas Mikolov. 2017. Enriching word vectors with subword information. Transactions of the Association for Computational Linguistics, 5. H´el`ene Bonneau-Maynard, Christelle Ayache, Fr´ed´eric Bechet, Alexandre Denis, Anne Kuhn, Fabrice Lefevre, Djamel Mostefa, Matthieu Quignard, Sophie Rosset, Christophe Servan, and Jeanne Vil- laneau. 2006. Results of the French Evalda-Media evaluation campaign for literal understanding. In lrec, Genoa. Jacob Devlin, Ming-Wei Chang, Kenton Lee, and Kristina Toutanova. 2019. BERT: Pre-training of deep bidirectional transformers for language understanding. In NAACL-HLT, Minneapolis, Minnesota. Association for Computational Linguistics. Marco Dinarelli, Vedran Vukotic, and Christian Raymond. 2017. Label-dependency coding in Simple Recurrent Networks for Spoken Language Understanding. In Interspeech, Stockholm, Sweden. S. Ghannay, A. Neuraz, and S. Rosset. 2020. What is best for spoken language understanding: small but task-dependant embeddings or huge but out-of-domain embeddings? In ICASSP 2020 - 2020 IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), pages 8114–8118. Daniel Guo, Gokhan Tur, Wen-tau Yih, and Geoffrey Zweig. 2014. Joint semantic utterance classiﬁcation and slot ﬁlling with recursive neural networks. In Spoken Language Technology Workshop (SLT), 2014 IEEE, pages 554–559. IEEE. Mandy Korpusik, Zoe Liu, and James Glass. 2019. A comparison of deep learning methods for language understanding. In Interspeech, September 15–19, 2019, Graz, Austria, Graz, Austria. Hang Le, Lo¨ıc Vial, Jibril Frej, Vincent Segonne, Maximin Coavoux, Benjamin Lecouteux, Alexandre Allauzen, Benoˆıt Crabb´e, Laurent Besacier, and Didier Schwab. 2020. Flaubert: Unsupervised lan- guage model pre-training for french. In Proceedings of The 12th Language Resources and Evaluation Conference, pages 2479–2490, Marseille, France. European Language Resources Association. R´emi Lebret, Jo¨el Legrand, and Ronan Collobert. 2013. Is deep learning really necessary for word embeddings? Technical report, Idiap. Eduard Ma, Xuezheand Hovy. 2016. End-to-end sequence labeling via bi-directional lstm-cnns-crf. In ACL. Association for Computational Linguistics. Louis Martin, Benjamin Muller, Pedro Javier Ortiz Su´arez, Yoann Dupont, Laurent Romary, ´Eric Ville- monte de la Clergerie, Djam´e Seddah, and Benoˆıt Sagot. 2020. Camembert: a tasty french language model. In Proceedings of the 58th Annual Meeting of the Association for Computational Linguistics. Gr´egoire Mesnil, Yann Dauphin, Kaisheng Yao, Yoshua Bengio, Li Deng, Dilek Hakkani-Tur, Xiaodong He, Larry Heck, Gokhan Tur, Dong Yu, and Geoffrey Zweig. 2015. Using recurrent neural networks for slot ﬁlling in spoken language understanding. IEEE/ACM Trans. Audio, Speech and Lang. 4 Conclusions and future work The paper presented a study focuses on French Spoken Language Understanding (SLU) task using the MEDIA corpus. First we proposed the evaluation of whether updating the word embeddings during train- ing improves or not the results, according to several scenarios.Second, we proposed to use a BiLSTM- CNN architecture that integrates character embeddings as additional features. Last, we proposed to evaluate the performance of BERT approaches on the MEDIA task through different ways. Experimental results show, that the word embeddings needed to be updated during SLU model training are the ones trained on small corpus like MEDIA. However, It is better for word embeddings trained on huge and out-of-domain to be frozen, since those word embeddings have captured enough general semantic and syntactic characteristics relevant to SLU task. More, The word embeddings trained on WIKI and WIKI+MEDIA achieve comparable results. This shows that, we don’t need to use both a task-dependent corpus and another out-of-domain corpus to train the word embeddings. In addition, we observed the usefulness of character embeddings when added as additional features. Regarding the evaluation of the performance of BERT approaches, the fune-tuning of CamemBERT and Flaubert base models show that the best results are achieved using CamemBERT base model trained on ccnet data. It yields to 29.35% of relative improvement in terms of CER reduction in comparison to the baseline (7.56 vs 10.7). Finally, the integration of the extracted CamemBERT’s contextual embeddings to the BiLSTM and BiLSTM-CNN architectures reveal that contextual embeddings achieves competitive results in comparison to CBOW word embeddings whatever the architecture, and conﬁrm the importance of character embeddings. For future work, we propose to evaluate the performance of Bert’s contextual embeddings extracted from different encoder’s layers, and to make in-depth error analysis for the different systems. Acknowledgements This work has been partially funded by the LIHLITH project (ANR-17-CHR2-0001-03), and supported by ERA-Net CHIST-ERA, and the “Agence Nationale pour la Recherche” (ANR, France). This work has been partially funded by the LIHLITH project (ANR-17-CHR2-0001-03), and supported by ERA-Net CHIST-ERA, and the “Agence Nationale pour la Recherche” (ANR, France). References Proc., 23(3):530–539. Tomas Mikolov, Kai Chen, Greg Corrado, and Jeffrey Dean. 2013. Efﬁcient estimation of word repre- sentations in vector space. In arXiv preprint arXiv:1301.3781. Jeffrey Pennington, Richard Socher, and Christopher D Manning. 2014. Glove: Global vectors for word representation. In EMNLP, volume 14. Matthew E Peters, Mark Neumann, Mohit Iyyer, Matt Gardner, Christopher Clark, Kenton Lee, and Luke Zettlemoyer. 2018. Deep contextualized word representations. arXiv preprint arXiv:1802.05365. Edwin Simonnet. 2019. Deep learning applied to spoken langage understanding. Theses, Universit´e du Maine. Edwin Simonnet, Sahar Ghannay, Nathalie Camelin, Yannick Est`eve, and Renato De Mori. 2017. ASR error management for improving spoken language understanding. In Interspeech 2017, Stockholm, Sweden. Pedro Javier Ortiz Su´arez, Benoˆıt Sagot, and Laurent Romary. 2019. Asynchronous pipeline for process- ing huge corpora on medium to low resource infrastructures. Challenges in the Management of Large Corpora (CMLC-7) 2019, page 9. Gokhan Tur and Renato De Mori. 2011. Spoken Language Understanding: Systems for Extracting Semantic Information from Speech. John Wiley & Sons. Kaisheng Yao, Baolin Peng, Yu Zhang, Dong Yu, Geoffrey Zweig, and Yangyang Shi. 2014. Spoken lan- guage understanding using long short-term memory neural networks. In Spoken Language Technology Workshop (SLT), 2014 IEEE, pages 189–194. IEEE. Xiaodong Zhang and Houfeng Wang. 2016. A joint model of intent determination and slot ﬁlling for spoken language understanding. In IJCAI, pages 2993–2999.
https://openalex.org/W2046660895	https://europepmc.org/articles/pmc3873526?pdf=render	English	null	Ventral-subgenual anterior cingulate cortex and self-transcendence	Frontiers in psychology	2,013	cc-by	2,097	Edited by: Edited by: Zoran Josipovic, New York University, USA Keywords: self-transcendence, altered state of consciousness, anterior cingulate cortex, meditation, integrative bo Self-transcendence (ST) is one of spe- ciﬁc human experiences often related to harmony with nature or feeling one- ness with others or the self as an inte- gral part of the whole universe. The Temperament and Character Inventory (TCI) is a widely used personality mea- sure, and ST is one of personality dimen- sions (Cloninger, 1994; Cloninger et al., 1994). Previous studies showed that ST has signiﬁcant positive correlation with the sgACC encompassing a ventromedial portion of the prefrontal cortex (vmPFC) using TCI and PET scan (Hakamata et al., 2013). Meanwhile, sgACC/vmPFC activ- ity has been shown to be signiﬁcantly decreased in patients with anxiety, major depression and mood disorders (Drevets et al., 2008; Shin and Liberzon, 2010; Kühn and Gallinat, 2013). Altogether, these ﬁnd- ings suggest that sgACC/vmPFC play an important role in emotion regulation and ST (Hakamata et al., 2013). Meditation often exempliﬁes positive emotion, pleasant feeling and ST expe- rience in practitioners (Cahn and Polich, 2006; Tang et al., 2007). Studies showed ST is positively related to meditation practice (Levenson et al., 2005). One meditation- category—automatic self-transcending includes techniques designed to transcend their own activity and improve ST (Travis and Shear, 2010). Substantial evidences indicate that ACC plays a key role in med- itation training (Hölzel et al., 2011). For example, compared to non-meditators, long-term Vipassana meditators showed stronger activations in the rostral ACC and adjacent medial PFC bilaterally for the meditation condition (contrasted to arithmetic task). Greater ACC and mPFC activations in meditators may reﬂect pro- cessing of distracting events and emotional processing (Hölzel et al., 2007, 2011). Compared with a memory training con- trol, compassion training elicited activity in a neural network including pregen- ual ACC, medial orbitofrontal cortex and striatum—brain regions previously asso- ciated with positive affect and afﬁliation (Klimecki et al., 2013a,b). In the same vein, 5 days of one form of meditation— integrative body–mind training (IBMT) improves vACC activity compared to same amount of relaxation training (Tang et al., 2009). Meanwhile, 5 days of IBMT also reduces stress, improves positive emotion and self-report of feeling oneness with nature (Tang et al., 2007). Further, 10 days of IBMT increases white matter connectiv- ity surrounding ACC and this brain struc- tural change correlates with emotional regulation (Tang et al., 2012a,b). Edited by: These results indicate that meditation accompa- nies positive emotion, ST experience, and ACC functional and structural changes. ST related meditation not only induces brain and behavioral changes, it often involves brain (mind) and body coop- eration indexed by central (CNS) and autonomic (ANS) nervous system inter- action (Cahn and Polich, 2006; Hölzel et al., 2011). Studies have begun to explore interaction and dynamics between CNS and ANS (Critchley et al., 2003; Tang, 2009; Tang and Posner, 2009; Tang et al., 2009; Hölzel et al., 2011; Critchley and Harrison, 2013). For instance, using heart rate variability (HRV), and high- and low-frequency power in the cardiac rhythm, ACC activity related to sym- pathetic modulation of heart rate was observed (Critchley et al., 2003). We measured the physiological and brain changes at rest before, during, and after 5 days of IBMT and relaxation train- ing. During and after training, the IBMT group showed signiﬁcantly better physio- logical reactions in heart rate, respiratory amplitude and rate, and skin conduc- tance response (SCR) than the relaxation control. Differences in HRV and EEG power suggested greater involvement of the ANS in the IBMT group during and after training. Imaging data demonstrated stronger v/sgACC activity in the IBMT group. Frontal midline ACC theta was also correlated with high-frequency HRV, sug- gesting control by the ACC over parasym- pathetic activity (Tang et al., 2009). These results indicate that brief IBMT induces better regulation of the ANS by a mid- line v/sg ACC brain system. This changed state probably reﬂects training in the coor- dination of body and mind given in the IBMT but not in the relaxation group. These results indicate body-brain works together to maintain certain consciousness states such as ST that may be related to ACC as a part of the brain’s lim- bic system, appears active in many neu- roimaging studies (Bush et al., 2000; Posner et al., 2007). In general ACC is involved in cognitive (dorsal division) and emotional (ventral/rostral part) process- ing (Bush et al., 2000). The sensitivity of the ACC to both reward and pain, and evidence for ACC coupling to cognitive and emotional areas during resting state and task performance, support the role of ACC in self-regulation or self-control including emotional, cognitive and auto- nomic control. Yi-Yuan Tang 1,2* and Rongxiang Tang 3 Edited by: Zoran Josipovic, New York University, USA Edited by: Zoran Josipovic, New York University, USA Yi-Yuan Tang 1,2* and Rongxiang Tang 3 1 Department of Psychology, Texas Tech University, Lubbock, TX, USA 2 Department of Psychology, University of Oregon, Eugene, OR, USA 3 Department of Psychology, The University of Texas at Austin, Austin, TX, USA Correspondence: yiyuan@uoregon.edu Yi-Yuan Tang 1,2 and Rongxiang Tang 3 1 Department of Psychology, Texas Tech University, Lubbock, TX, USA 2 Department of Psychology, University of Oregon, Eugene, OR, USA 3 Department of Psychology, The University of Texas at Austin, Austin, TX, USA *Correspondence: yiyuan@uoregon.edu Edited by: Particularly, v/sgACC and adjacent mPFC area involves in emotional control and autonomic regulation (Luu and Posner, 2003; Posner et al., 2007), consistent with many meditation ﬁndings (Hölzel et al., 2011; Tang et al., 2012b; Tang and Posner, 2013). December 2013 \| Volume 4 \| Article 1000 \| 1 www.frontiersin.org Ventral-subgenual anterior cingulate cortex and self-transcendence Tang and Tang different performance (Tang et al., 2007; Xue et al., 2011; Tang et al., 2012a,b). Our ﬁndings suggest meditation training could induce altered states of conscious- ness which may allow us to explore the neuroscience of consciousness based on how alterations in normal consciousness result in functional or/and structural brain changes and plasticity. These alterations in consciousness can affect long-term cog- nitive, affective and social activities, and may help understand the disease states or disorders of consciousness such as coma, vegetative state, etc. (Tang et al., 2013). Critchley, H. D., and Harrison, N. A. (2013). Visceral inﬂuences on brain and behavior. Neuron. 77, 624–638. doi: 10.1016/j.neuron.2013.02.008 Tang, Y. Y., Lu, Q., Fan, M., Yang, Y., and Posner. M. I. (2012a). Mechanisms of white matter changes induced by meditation. Proc. Natl. Acad. Sci. U.S.A. 109, 10570–10574. doi: 10.1073/pnas.1207817109 Critchley, H. D., Mathias, C. J., Josephs, O., O’Doherty, J., Zanini, S., Dewar, B. K., et al. (2003). Human cingulate cortex and autonomic control: converging neuroimaging and clinical evidence. Brain. 126(Pt 10), 2139–2152. doi: 10.1093/brain/awg216 Tang, Y. Y., Rothbart, M. K., and Posner, M. I. (2012b). Neural correlates of establishing main- taining and switching brain states. Trends Cogn. Sci. 16, 330–337. doi: 10.1016/j.tics.2012.05.001 Tang, Y. Y., Ma, Y., Fan, Y., Feng, H., Wang, J., Feng, S., et al. (2009). Central and autonomic nervous system interaction is altered by short-term medita- tion. Proc. Natl. Acad. Sci. U.S.A. 106, 8865–8870. doi: 10.1073/pnas.0904031106 Drevets, W., Savitz, J., and Trimble, M. (2008). The subgenual anterior cingulate cortex in mood dis- orders. CNS Spectr. 13, 663–681. Hakamata, Y., Iwase, M., Kato, T., Senda, K., and Inada, T. (2013). The neural correlates of mindful awareness: a possible buffering effect on anxiety- related reduction in subgenual anterior cingu- late cortex activity. PLoS ONE 8:e75526. doi: 10.1371/journal.pone.0075526 Tang, Y. Y., Ma, Y., Wang, J., Fan, Y., Feng, S., Lu, Q., et al. (2007). Short-term meditation train- ing improves attention and self-regulation. Proc. Natl. Acad. Sci. U.S.A. 104, 17152–17156. Edited by: doi: 10.1073/pnas.0707678104 In summary, growing empirical evi- dences indicate meditation has potential to develop ST—a positive relationship between self and other that tran- scends self-focused needs and increases prosocial characteristics (Hölzel et al., 2011; Tang et al., 2012a,b; Vago and Silbersweig, 2012). Future studies could examine the relationship between ST and short-term or long-term med- itation, and how meditation shapes the perspectives on the self, self- others, self-nature and its underlying mechanisms using multimodal neu- roimaging, physiological, psychosocial and genetic methods. Tang, Y. Y., and Posner, M. I. (2009). Attention train- ing and attention state training. Trends Cogn. Sci. 13, 222–227. doi: 10.1016/j.tics.2009.01.009 Hölzel, B. K., Lazar, S. W., Gard, T., Schuman-Olivier, Z., Vago, D. R., and Ott, U. (2011). How does mindfulness meditation work? Proposing mech- anisms of action from a conceptual and neural perspective. Perspect. Psychol. Sci. 6, 537–559. doi: 10.1177/1745691611419671 Tang, Y. Y., and Posner, M. I. (2013). Tools of the trade: Theory and method in mindfulness neuro- science. Soc. Cogn. Affect. Neurosci. 8, 118–120. doi: 10.1093/scan/nss112 Hölzel, B. K., Ott, U., Hempel, H., Hackl, A., Wolf, K., Stark, R., et al. (2007). Differential engagement of anterior cingulate and adjacent medial frontal cortex in adept meditators and non-meditators. Neurosci Lett. 421, 16–21. doi: 10.1016/j.neulet.2007.04.074 Tang, Y. Y., Posner, M. I., and Rothbart, M. K. (2013). Meditation improves self-regulation over the life span. Ann. N.Y. Acad. Sci. doi: 10.1111/nyas.12227. [Epub ahead of print]. Travis, F., and Shear, J. (2010). Focused attention, open monitoring and automatic self-transcending: Categories to organize meditations from Vedic, Buddhist and Chinese traditions. Conscious Cogn. 19, 1110–1118. doi: 10.1016/j.concog.2010.01.007 Klimecki, O. M., Leiberg, S., Lamm, C., and Singer, T. (2013a). Functional neural plasticity and asso- ciated changes in positive affect after compas- sion training. Cereb Cortex. 23, 1552–1561. doi: 10.1093/cercor/bhs142 Vago, D. R., and Silbersweig, D. A. (2012). Self- awareness, self-regulation, and self-transcendence (S-ART): a framework for understanding the neurobiological mechanisms of mind- fulness. Front. Hum. Neurosci. 6:296. doi: 10.3389/fnhum.2012.00296 Klimecki, O. M., Leiberg, S., Ricard, M., and Singer, T. (2013b). Differential pattern of func- tional brain plasticity after compassion and empa- thy training. Soc. Cogn. Affect. Neurosci. doi: 10.1093/scan/nst060. [Epub ahead of print]. ACKNOWLEDGMENTS We thank Michael Posner’s insightful com- ments. This work was supported by the Ofﬁce of Naval Research. Xue, S., Tang, Y. Y., and Posner, M. I. (2011). Short- term meditation increases network efﬁciency of the anterior cingulate cortex. Neuroreport 22, 570–574. doi: 10.1097/WNR.0b013e328348c750 Kühn, S., and Gallinat, J. (2013). Resting-state brain activity in schizophrenia and major depression: a quantitative meta-analysis. Schizophr. Bull. 39, 358–365. doi: 10.1093/schbul/sbr151 REFERENCES Levenson, M. R., Jennings, P. A., Aldwin, C. M., and Shiraishi, R. W. (2005). Self-transcendence: con- ceptualization and measurement. Int J Aging Hum Dev. 60(2):127–143 doi: 10.2190/XRXM-FYRA- 7U0X-GRC0 Received: 28 November 2013; accepted: 15 December 2013; published online: 27 December 2013. Citation: Tang Y-Y and Tang R (2013) Ventral-subgenual anterior cingulate cortex and self-transcendence. Front. Psychol. 4:1000. doi: 10.3389/ fpsyg.2013.01000 Received: 28 November 2013; accepted: 15 December 2013; published online: 27 December 2013. Bush, G., Luu, P., and Posner, M. I. (2000). Cognitive and emotional inﬂuences in anterior cingu- late cortex. Trends Cogn. Sci. 4, 215–222. doi: 10.1016/S1364-6613(00)01483-2 Citation: Tang Y-Y and Tang R (2013) Ventral-subgenual anterior cingulate cortex and self-transcendence. Front. Psychol. 4:1000. doi: 10.3389/ fpsyg.2013.01000 Luu, P., and Posner, M. I. (2003). Anterior cingulate cortex regulation of sympathetic activity. Brain. 126(Pt 10), 2119–2120. doi: 10.1093/brain/awg257 Cahn, B. R., and Polich, J. (2006). Meditation states and traits: EEG, ERP, and neuroimaging studies. Psychol. Bull. 132, 180–211. doi: 10.1037/0033- 2909.132.2.180 This article was submitted to Consciousness Research, a section of the journal Frontiers in Psychology. Posner, M. I., Sheese, B., Rothbart, M., and Tang, Y. Y. (2007). The anterior cingulate gyrus and the mechanism of self-regulation. Cogn Affect Behav Neurosci. 7, 391–395 doi: 10.3758/CABN.7.4.391 Copyright © 2013 Tang and Tang. This is an open- access article distributed under the terms of the Creative Cloninger, C. R. (1994). Temperament and per- sonality. Curr. Opin. Neurobiol. 4, 266–273. doi: 10.1016/0959-4388(94)90083-3 Copyright © 2013 Tang and Tang. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, dis- tribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Shin, L., and Liberzon, I. (2010). The neuro- circuitry of fear, stress, and anxiety disor- ders. Neuropsychopharmacology 35, 169–191. doi: 10.1038/npp.2009.83 Cloninger, C. R., Przybeck, T. R., Svrakic, D. M., and Wetzel, R. D. (1994). The Temperament and Character Inventory (TCI): A Guide to Its Development and Use. St. Louis: Center for Psychobiology of Personality, Washington University at St. Louis. Tang, Y. Y. (2009). Exploring the Brain, Optimizing the Life. Beijing: Science Press. December 2013 \| Volume 4 \| Article 1000 \| 2 Frontiers in Psychology \| Consciousness Research
https://openalex.org/W4387616205	https://www.mdpi.com/2076-2615/13/20/3195/pdf?version=1697177847	English	null	Seasonal Variation in the Thermoregulation Pattern of an Insular Agamid Lizard	Animals	2,023	cc-by	8,704	animals animals Article Emmanouela Karameta 1,* , Ioanna Gavriilidi 1,2 , Spyros Sfenthourakis 3 and Pana a Karameta 1,* , Ioanna Gavriilidi 1,2 , Spyros Sfenthourakis 3 and Panayiotis Paﬁlis 1,4 Emmanouela Karameta 1 Section of Zoology and Marine Biology, Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, Ilissia, 15784 Athens, Greece; ioanna.gavriilidi@uantwerpen.be (I.G.); ppaﬁl@biol.uoa.gr (P.P.) 2 Department of Biology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium 3 Department of Biological Sciences University of Cyprus Panepistimiou 1 2109 Nicosia Cyprus; 2 Department of Biology, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Belgium 3 Department of Biological Sciences, University of Cyprus, Panepistimiou 1, 2109 Nicosia, Cyprus; sfendour@ucy.ac.cy 3 Department of Biological Sciences, University of Cyprus, Panepistimiou 1, 2109 Nicosia, Cypru sfendour@ucy.ac.cy y y 4 Zoological Museum, National and Kapodistrian University of Athens, Panepistimioupolis, 15784 Athens, Greece 4 Zoological Museum, National and Kapodistrian University of Athens, Panepistimioupolis, 15784 Ath G 4 Zoological Museum, National and Kapodistrian University of Athens, Panepistimioupolis, 15784 Ath G * Correspondence: emykarameta@biol.uoa.gr Simple Summary: The ability of animals to maintain their body temperature within an optimal range, known as thermoregulation, is essential for their survival, overall health, and daily activities. Ectotherms, including reptiles, rely on external energy resources to regulate their body temperature. How well they can achieve this, heavily depends on various environmental factors, such as the climate and its seasonal changes. Islands typically have a mild climate, which is expected to favor the thermoregulation of reptiles throughout the year. In this study, we investigate the effect of seasonality on the thermoregulation efﬁciency and behavior of a population of lizards found on Naxos Island, in the Cyclades, Greece. Our results reveal that seasonal ﬂuctuations signiﬁcantly inﬂuence how easily and precisely lizards can regulate their body temperature, with summer being the most favorable period, and autumn being the least favorable. Interestingly, lizards adjusted their thermal preferences and thermoregulation efﬁciency depending on the challenges imposed by each season and thus managed to maintain stable body temperatures. Whether these adjustments represent evolutionary adaptations or simply reversible shifts, awaits further research. Understanding how lizards adapt to their changing environment can provide valuable insights into their survival strategies and how they may cope with future environmental changes. Keywords: seasonality; thermoregulation; islands; Agamidae animals animals Citation: Karameta, E.; Gavriilidi, I.; Sfenthourakis, S.; Paﬁlis, P. Seasonal Variation in the Thermoregulation Pattern of an Insular Agamid Lizard. Animals 2023, 13, 3195. https:// doi.org/10.3390/ani13203195 Citation: Karameta, E.; Gavriilidi, I.; Sfenthourakis, S.; Paﬁlis, P. Seasonal Variation in the Thermoregulation Pattern of an Insular Agamid Lizard. Animals 2023, 13, 3195. https:// doi.org/10.3390/ani13203195 Citation: Karameta, E.; Gavriilidi, I.; Sfenthourakis, S.; Paﬁlis, P. Seasonal Variation in the Thermoregulation Pattern of an Insular Agamid Lizard. Animals 2023, 13, 3195. https:// doi.org/10.3390/ani13203195 Abstract: Ectotherms, including lizards, rely on behavioral thermoregulation to maintain their body temperature within an optimal range. The benign climate of islands is expected to favor the ther- moregulation efﬁciency of reptiles throughout their activity period. In this study, we investigated the seasonal variation in thermoregulation in an insular population of the roughtail rock agama (Laudakia stellio) on Naxos Island, Greece. We measured body, operative, and preferred temperatures across three seasons (spring, summer, and autumn), and we evaluated the effectiveness of thermoregula- tion, using the Hertz index (E). Our results revealed that the effectiveness of thermoregulation was signiﬁcantly inﬂuenced by seasonality. E was quite high in summer (0.97) and spring (0.92), and lowest in autumn (0.81). Accordingly, the quality of the thermal environment was signiﬁcantly low during autumn, and maximum during summer. However, despite the environmental temperature ﬂuctuations, lizards exhibited remarkable stability in body temperatures. They also adjusted their preferred temperatures seasonally and doubled the thermal niche breadth they occupied during summer, thus enhancing thermoregulation efﬁciency. Whether or not these adjustments are plastic or ﬁxed local adaptations remains to be explored in further research across multiple years and seasons, including additional insular populations. Academic Editor: Vincent L. Bels Received: 27 July 2023 Revised: 8 October 2023 Accepted: 10 October 2023 Published: 13 October 2023 Received: 27 July 2023 Revised: 8 October 2023 Accepted: 10 October 2023 Published: 13 October 2023 Received: 27 July 2023 Revised: 8 October 2023 Accepted: 10 October 2023 Published: 13 October 2023 1. Introduction Ectothermic vertebrates regulate their body temperature (Tb) using energy stem- ming from external, environmental sources, in contrast to endotherms that rely mainly on metabolic heat [1,2]. The former, predominantly ﬁsh, amphibians, and reptiles, employ behavioral mechanisms to control their Tb, while the latter, mainly birds and mammals, use both behavioral tactics as well as changes in autonomic effector activity, such as shivering, skeletal muscle thermogenesis, etc. [3]. Regardless of the category they belong to, animals try to maintain their Tb within an optimal range, a process known as active thermoreg- ulation [4]. In reptiles and amphibians, thermoregulation includes behavioral responses (basking, retreating to shaded microhabitats, underwater submersion, etc.), allowing an- imals to gain or lose heat via convection, conduction, radiation, and evaporation [5,6]. On the other hand, very few large-bodied reptile species with low metabolism, such as the Leatherback Sea Turtles (Dermochelys coriacea), can maintain constantly high Tb in comparison to the surrounding environmental temperatures, due to inertial endothermy (gigantothermy) [7]. In contrast, the much smaller tegu lizard (Salvator merianae) can achieve facultative endothermy via metabolic thermogenesis and decreased thermal conductance during the reproductive season [8]. g p The ability of ectotherms to keep their Tb within or close to an optimum range is imperative for their survival, condition, as well as daily activities, and performance, such as locomotion, foraging ability, growth rate, and reproductive investment [5,9]. At the individual level, the efﬁciency of thermoregulation is constrained by the trade-off between the costs and beneﬁts it entails: intraspeciﬁc competition and predation risk on one hand, and the maximizing of performance and ﬁtness on the other [4,10,11]. At the environmental level, it heavily depends on the availability of operative temperatures and spatial heterogeneity of a particular habitat [12]. Seasonal variations in environmental conditions, such as rainfall, wind intensity, tem- perature, and sunlight exposure may inﬂuence many of the behavioral and physiological attributes of lizards [13,14], including thermoregulation [15,16]. For example, subtropical lizards may shift their activity and thermoregulation effort as a response to weather ﬂuctu- ations but are sensitive to extreme winter conditions [17]. Likewise, temperate species can change their thermoregulatory behavior, including daily activity and microhabitat selection, depending on the season [18], or even shift their preferred temperature range [19] in an effort to maximize thermoregulation effectiveness. 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/). Keywords: seasonality; thermoregulation; islands; Agamidae https://www.mdpi.com/journal/animals Animals 2023, 13, 3195. https://doi.org/10.3390/ani13203195 Animals 2023, 13, 3195 2 of 11 1. Introduction Besides, a compilation of meta-analyses examining the effect of various factors on thermoregulation, such as climate, body size, habitat, altitude, season, and insularity, concluded that the most important ones were altitude and seasonality [20]. In this study, we aim to examine the seasonal variation in the effectiveness of ther- moregulation in an insular population of the roughtail rock agama (Laudakia stellio), the only agamid ranging in Europe (Aegean islands, Greece). The challenges posed by seasonal variation may be buffered by island climate conditions that differ from those of the main- land [21,22]. This more benign insular climate is reﬂected in higher-quality thermal habitats that lower the thermoregulatory effort made by lizards [23,24]. However, small islets devi- ate from this general pattern. Due to their limited heterogeneity [25], they are thermally demanding habitats that may promote high thermoregulation effectiveness [26–28]. To avoid islet particularities, we focused on a population inhabiting the largest Cycladic Island, Naxos (430 km2). Just a handful of studies have assessed the effect of thermoregulatory seasonality on Mediterranean lizards [15,18,29,30] and none has focused on a large island. Here we posed a simple question: do the favorable insular environmental conditions minimize seasonal variations in thermoregulation? If the insular climate is indeed milder, there should be no seasonal variation in thermoregulation; if not, lizards should shift their thermoregulatory effectiveness in response to weather changes. Animals 2023, 13, 3195 3 of 11 2. Materials and Methods 2.1. Study System Laudakia stellio (Linnaeus, 1758) is a diurnal agamid lizard measuring up to 133 mm (maximum snout-vent length, SVL) [31]. It is widespread in the E. Mediterranean, where it is found mostly in rocky habitats and human-made constructions, in arid and semi-arid parts of Turkey and Greece [32]. It can live up to 7 years in the wild [33] and females lay 4–10 eggs from May until July [34]. It employs a combination of a “sit and wait” foraging behavior as well as an active foraging mode depending on the season and hence food availability, and its diet varies from insects to seeds and fruits, and many other species they can capture, even snails and young snakes [35–37]. Two morphological subspecies L. s. stellio and L. s. daani co-exist in the Cyclades: the former inhabiting Delos and the Mykonos archipelago, and the latter being present in Naxos, Paros, and Antiparos. This co-existence in neighboring islands is explained by ancient, human-mediated transport rather than dispersal [38]. The presence of newly-discovered populations in Corfu (Ionian Sea, Greece) [39,40], Karpathos [41] and Crete [42] highlight the role of humans in shaping the present-day distribution of this species. Fieldwork was conducted in May, July, and October 2017 in Naxos (Aegean Sea, Greece). In each season, lizards were captured in the stone walls surrounding Demetra’s temple in Sagri (37.029◦N, 25.431◦E). This area is covered by low vegetation, mainly phrygana and maquis, and extensive stonewalls serve as a refuge for this species, as well as other reptiles [43]. Naxos has a typical Mediterranean climate according to the Koppen/Geiger climate classiﬁcation, characterized by long dry summers and mild winters, and strong winds blowing during the whole year [44]. Accordingly, precipitation is fairly low (400 mm/yr on average) and mostly occurs between autumn and spring [45]. 2.2. Operative (Te) and Body (Tb) Temperatures Operative temperatures (Te) sketch out the thermal environment in which lizards live, as they correspond to the body temperatures that animals would achieve if they didn’t make any effort to actively thermoregulate [46]. To evaluate operative temperatures in autumn, spring, and summer, we used copper models sealed with plasticine and containing 2.5–3 mL of water inside. Previous research suggests that the temperatures monitored by these models as well as their heating/cooling rates showed a strong linear correlation with those exhibited by living individuals, and therefore, match the focal species’ thermal capacity [47,48]. In each season, measurements were recorded every 30 min for three consecutive days, using 20 models that were connected to five data loggers (HOBO U12 4—Channel External Data Logger—U12—008; [49]) (Table 1). To sample all of the microhabitats shaping the thermal niche of the species, models were randomly placed under full sunlight (e.g., lying on a stonewall), in the shade (e.g., inside crevices), and in semi-light exposure (e.g., on the side of a stonewall or a bush) [50]. Table 1. Thermal metrics (Te, de, Tb, db, Tpref, Tset) and thermoregulation effectiveness index (E) across seasons. Season Te de Tb db Tpref Tset E Spring Mean ± SD 27.7 ± 3.9 7.3 ± 3.6 34.7 ± 1.3 0.6 ± 0.9 36.2 ± 1.2 0.92 N 1140 1140 22 22 13 Range 19.3–47.5 0.0–15.6 31.8–37.0 0.0–3.1 33.5–37.7 34.9–37.7 Lower–Upper Q 25.6–30.3 4.6–9.3 34.0–35.8 0–0.9 35.7–36.9 Summer Mean ± SD 30.4 ± 5.0 2.8 ± 3 34.3 ± 1.2 0.1 ± 0.3 33.2 ± 2.9 0.97 N 1490 1490 30 30 11 Range 23.8–54.0 0.0–17.7 29.8–36.2 0.0–1.8 27.1–36.7 30.4–36.3 Lower–Upper Q 26.0–31.7 0.2–5.0 33.9–35.8 0–0 32.5–35.6 Autumn Mean ± SD 24.4 ± 5.2 12.6 ± 5.1 34.8 ± 2.5 2.4 ± 2.4 38.1 ± 0.6 0.81 N 1881 1881 26 26 8 Range 16.8–40.3 0.0–20.2 30.0–38.0 0.0–7.0 36.7–39.0 37.0–39.5 Lower–Upper Q 19.9–28.7 8.3–17.1 33.0–37.0 0.0–4.0 37.6–38.8 1. Thermal metrics (Te, de, Tb, db, Tpref, Tset) and thermoregulation effectiveness index (E) Table 1. Thermal metrics (Te, de, Tb, db, Tpref, Tset) and thermoregulation effectiveness index (E) across seasons. 2.2. Operative (Te) and Body (Tb) Temperatures 4 of 11 Animals 2023, 13, 3195 In each season, body temperatures (Tb) were measured in wild-caught males (with SVL > 85 mm, which is the typical adult size [51]), immediately after capture [52,53] by inserting a type K thermocouple, connected to a mini-logger (EasyLog—USB—1, Lascar Electronics Ltd., Whiteparish, UK), directly into the cloaca [54]. A total of 22 males were caught in spring, 30 in summer and 26 in autumn. Females were excluded from the study to avoid physiologically triggered Tb changes due to gravidity [55]. SVL was measured with a digital caliper (Silverline 380244, accurate to 0.01 mm) and weight with a digital balance (0.0001 g precision) (Table 1). 2.3. Preferred Temperatures (Tpref) In each season, a subset of lizards captured in the ﬁeld for Tb measurements were subsequently transferred to the laboratory facilities of the Department of Biology at the National and Kapodistrian University of Athens, where they were housed in individual terraria (60 cm × 30 cm × 40 cm). Each terrarium contained sand as substrate and a tile that served as an artiﬁcial shelter as well as a basking spot, allowing lizards to behaviourally thermoregulate under a 60 W incandescent heating lamp (operating 8 h/day). Lizards were fed every other day with mealworms (Tenebrio molitor) coated with a multivitamin powder (TerraVit Powder, JBL GmbH and Co. KG, Neuhofen, Germany) and water was provided ad libitum. Sunlight entering through two 2.5 m × 1.5 m windows allowed for a natural photoperiod. A continuously operating air conditioning system kept the room temperature at 25 ◦C. The most reliable way to determine an organism’s preferred temperature is by ob- serving its body temperature in controlled thermal environments, which typically feature connected compartments or gradients, and allow the organism to select its desired tem- perature [5]. Thus, preferred temperatures (Tpref), were estimated for each male (N = 13 in spring, N = 11 in summer, and N = 8 in autumn). Each individual was allowed to thermoregulate within a thermal gradient, ranging from 15 to 60 ◦C (Table 1). To create this gradient, two heating lamps (100 W 4 and 60 W), and two ice bags were positioned at the opposite sides of a terrarium (100 cm × 25 cm × 25 cm) [56]. Body temperatures were measured again using the same type of K thermocouple previously described, but this time it was taped on the animal’s back (without impeding locomotion) so that it could remain inside the cloaca for the whole duration of the experiment, minimizing the stress on the an- imal caused by handling that could potentially affect its body temperature [57]. Each lizard was allowed to thermoregulate for an hour prior to the beginning of the experiment [55,58]. Measurements were recorded every ﬁve minutes for a period of 5 h (from 10:00–15:00). The interquartile range (middle 50%) of the preferred body temperatures (Tpref) of each individual [4] was used to estimate the set-point temperature range (Tset) in each season, with the average values setting the upper and lower limits of Tset (Table 1). 3.1. Thermal Measurements (Te, Tb, Tpref) Mean values, ranges, and sample sizes for all thermal measurements are presented in Table 1. Mean operational temperatures differed signiﬁcantly among all seasons, with the highest temperatures being recorded in summer, the lowest in autumn, and intermediate values in spring (ANOVA, F2, 4514 = 669.29, p < 0.001, Tukey’s HSD test, p < 0.05 in all cases). The opposite pattern of signiﬁcant differences was observed in the deviation of Te from the preferred temperature range, (de), with the highest mean value observed in October, the lowest in July, and an intermediate value in April (ANOVA, F2, 4514 = 2208.5, p < 0.001, Tukey’s HSD test, p < 0.05 in all cases). Body temperatures measured in the ﬁeld were similar across seasons (p = 0.945). The same is true for both the animal weight (p = 0.882) and SVL (p = 0.481). The accuracy of thermoregulation was lower in October when db obtained its maximum value. This difference in db between autumn and the other two seasons was found to be statistically signiﬁcant (ANOVA, F2, 75 = 22.027, p < 0.001, Tukey’s HSD post-hoc test, p < 0.05). This deviation was minimal in both summer and spring when db was close to zero (Table 1). p g Lizards selected signiﬁcantly lower Tpref in the summer, higher temperatures in the autumn, and intermediate temperatures in the spring (Table 1), (ANCOVA, F2, 27 = 15.290, p < 0.001). Tukey’s HSD post-hoc tests indicated signiﬁcant differences in Tpref between summer and all other seasons (p < 0.05), but not between spring and autumn (p = 0.123). The breadth of the set-point range doubled during summer. 2.5. Statistical Analyses 2.5. Statistical Analyses All data were log10 transformed to meet the assumptions of parametric analyses after testing for normality and homogeneity of variances. Differences in operative temperatures, de, and db were explored using one-way Analysis of Variance (ANOVA). Important sta- tistical differences were identiﬁed using Tukey’s HSD post-hoc test. Seasonal differences regarding ﬁeld-measured body temperatures and lab-measured preferred temperatures (using individual mean values) were explored using a one-way Analysis of Covariance, with weight and SVL as covariates. The effectiveness of thermoregulation (E) was estimated using a bootstrap resampling method, with the E index and its 95% conﬁdence intervals being calculated by 1000 replicates [4]. Differences among seasons were again identiﬁed using Tukey’s HSD post hoc test with p ≤0.05. All statistical analyses were performed in SPSS Statistics version 27.0.1.0 (IBM 2020, Armonk, NY, USA). 2.4. Effectiveness of Thermoregulation The effectiveness of thermoregulation (E) was estimated using the widely used E index [4] which is based on the ability of an animal to achieve body temperatures (Tb) within the range of its thermal preference (Tset), and the degree to which this is enabled or impeded by the thermal habitat (Te). This interrelation is depicted in the Hertz index: E = 1—(db/de), where db is the mean deviation (absolute values) of Tb from Tset, while de is the mean deviation (absolute values) of Te from Tset. Hence, db alone shows the accuracy of thermoregulation while de reveals the thermal quality of a particular habitat (Table 1). Taken together, these deviations point to the active effort made by an animal to thermoregulate effectively. Therefore, E values close to 0 correspond to thermoconformers, animals that select a microhabitat randomly, while values close to 1 describe thermoregulators: animals actively selecting a microhabitat that is appropriate for thermoregulation [4]. Animals 2023, 13, 3195 5 of 11 3.2. Effectiveness of Thermoregulation The effectiveness of thermoregulation differed across seasons according to the boot- strap resampling method. Animals were able to thermoregulate more effectively in the summer (E = 0.97), and less in autumn (E = 0.81) (Table 1). In spring, E was also quite high (E = 0.92). All the aforementioned differences were statistically signiﬁcant (ANOVA, F2, 2997 = 7159.8, p < 0.001, Tukey’s HSD post-hoc test, p < 0.05 in all cases). 4. Discussion Insular lizards are expected to expend less energy to achieve effective thermoregulation when compared to their mainland counterparts as a result of the milder insular climate they experience [23]. Indeed, the challenging climate of the mainland is often reﬂected in the effectiveness of thermoregulation: lizards from higher altitudes and mountain ranges on the mainland are often moderate thermoregulators, such as Liolaemus tandilensis, (E = 0.50–0.69), Iberolacerta aurelioi (E = 0.74–0.83), and Abronia taeniata (E= 0.60) [59–61]. On the other hand, not all islands are characterized by equally favorable climates due to their discrete morphological attributes such as size, wind exposure, and geographic location [62]. Therefore, lizards inhabiting minuscule islets have to demonstrate exceptional thermoregulation efﬁciency to ensure their survival [15,26–28]. According to our results, this is also the case for L. stellio inhabiting the largest of the Cyclades, Naxos Island. The Animals 2023, 13, 3195 6 of 11 effectiveness of thermoregulation was maximum in the summer (E = 0.97), quite high in spring (E = 0.92), and lower in autumn (E = 0.81). These ﬁndings refute our initial hypothesis, stating that the benign climate of a larger island would allow lizards to survive with a lower thermoregulation effort across different seasons. In contrast, temperature ﬂuctuations had a signiﬁcant effect on the thermoregulation efﬁciency achieved by this species, which responded swiftly to the environmental challenges posed by seasonality, to ensure its survival. Operative environmental temperatures followed the predictable ﬂuctuations of the temperate climate. They were higher in summer, intermediate in spring, and lower in autumn (Table 1). From the animal’s perspective, the thermal quality of their habitat changed accordingly, as the environmental temperatures’ deviation from the Tset range (de) was minimum in July and maximum in October. This seasonal variation in tempera- ture, along with other predictable changes in abiotic factors such as wind intensity, and sunlight exposure, can have a profound effect on lizard physiology and behavior, and hence thermoregulation [5,6,13]. Indeed, summer, as largely expected, provided the most advantageous conditions for precise thermoregulation [20]. g p g The seasonal variation in operative temperatures was not followed by body temper- atures (Tb), which remained surprisingly constant. As ectotherms, lizards respond to climatic ﬂuctuations in an effort to maintain their body temperatures within a narrow mar- gin of preferred temperatures, so that they are able to exploit resources and optimize ﬁtness and performance [5,63–66]. Indeed, body temperatures (Tb) of L. 4. Discussion stellio were remarkably stable across seasons (ﬁxed around 34 ◦C, Table 1), and did not follow the seasonal weather variation. This is the opposite pattern of what has been observed in many lacertids, in which Tbs were signiﬁcantly higher in summer and in spring than in winter [16,29,30,67]. Moreover, lizards were able to thermoregulate with greater accuracy in summer and in spring, as expressed by the extremely low db values, which were close to zero. Accuracy of thermoregulation was much lower in autumn when mean db was quite high (db = 2.4), but lizards still managed to maintain high Tbs. This ﬁnding is in accordance with previous research on the behavioral thermoregulation patterns of the closely related Laudakia species inhabiting Israel and Egypt, which can achieve body temperatures above the environmental ones [68,69]. The ability of lizards to achieve highly constant temperatures across seasons can be attributed to (a) an adjustment in their thermoregulatory behavior, such as changes in their activity period [63,70], their posture while basking, and/or microhabitat selection and use [71,72], (b) the acclimation of their thermal physiology, i.e., a shift in preferred temperatures [5,73], or (c) a combination of the above. Although changes in microhabitat selection and activity period were not evaluated in the present study, they could contribute to the thermoregulation effort exhibited by L. stellio across seasons. Previous studies on closely related Laudakia species have shown that they occupy a wide thermal niche breadth, and they can achieve elevated body temperatures through conductive basking, and by shuttling between warmer and cooler microhabitats [68,69]. Apart from the aforemen- tioned behavioral tactics that contribute to the effectiveness of thermoregulation, our study revealed a seasonal shift in preferred temperatures. The profound effect of seasonality was observed on both mean values, as well as set- point ranges (Tset) of preferred temperatures (Tpref). Lizards selected significantly higher temperatures in October (38.1 ◦C), lower temperatures in the hot summer month of July (33.2 ◦C), and intermediate values in May (36.2 ◦C). The same tendency was observed in Tset (Figure 1), which was shifted from lower temperatures in spring (34.9–37.7 ◦C) to- wards higher temperatures in autumn (37.0–39.5 ◦C). Furthermore, its breadth doubled in summer (30.4–36.3 ◦C) and so animals could exploit a greater range of available en- vironmental temperatures, as a greater proportion of Te fell within the Tset point range (Figure 1). 4. Discussion A shift in preferred temperatures has been reported in many lizard families, such as skinks and agamids [74,75]. In many lacertids, this shift is often a response to seasonality [18,19,26,29,67,76], however, in many of these cases lizards preferred higher Animals 2023, 13, 3195 env (Fig 7 of 11 7 of 11 temperatures in the summer in comparison to spring. In the case of L. stellio, the lower Tpref values observed in summer could reﬂect the need to avoid overheating and prevent dehydration [30,77] as extremely high temperatures were recorded in July (Figure 1). The Cyclades complex is fairly dry during summer and has one of the highest numbers of consecutive dry days per year in the country [78]. Likewise, a recent study comparing three insular Greek populations of L. stellio with L. cypriaca from Cyprus showed that the latter opted for much lower temperatures, probably as an adaptation to the extremely hot summers in Cyprus [48]. 18,19,26,29,67,76], however, in many of these cases lizards preferred higher in the summer in comparison to spring. In the case of L. stellio, the lower observed in summer could reﬂect the need to avoid overheating and prevent [30,77] as extremely high temperatures were recorded in July (Figure 1). The mplex is fairly dry during summer and has one of the highest numbers of dry days per year in the country [78]. Likewise, a recent study comparing Greek populations of L. stellio with L. cypriaca from Cyprus showed that the or much lower temperatures, probably as an adaptation to the extremely hot Cyprus [48]. Figure 1. Frequency of ﬁeld body temperatures (Tb, dark colors) and operative temperatures (Te, light colors) in autumn (blue), spring (orange), and summer (green). Vertical black solid lines indi- cate the set-point range temperatures (Tset). Figure 1. Frequency of ﬁeld body temperatures (Tb, dark colors) and operative temperatures (Te, light colors) in autumn (blue), spring (orange), and summer (green). Vertical black solid lines indicate the set-point range temperatures (Tset). ency of ﬁeld body temperatures (Tb, dark colors) and operative temperatures (Te, autumn (blue), spring (orange), and summer (green). Vertical black solid lines indi- nt range temperatures (Tset). Figure 1. Frequency of ﬁeld body temperatures (Tb, dark colors) and operative temperatures (Te, light colors) in autumn (blue), spring (orange), and summer (green). Vertical black solid lines indicate the set-point range temperatures (Tset). 4. Discussion t in thermal preferences could be a response to the seasonality of temperate s in thermal preferences could facilitate thermoregulation eﬀectiveness, by deviation of Te from Tset. Or they could simply reﬂect the optimal tempera- r another physiological process, such as sprint speed or digestion [2,19,63]. e extent of behavioral thermoregulation is determined by the trade-oﬀ be- izing physiological performance and individual ﬁtness, and the relevant en- e constraints, as well as predation risk [10,11]. In our study, we could argue in Tpref and Tset favored thermoregulation eﬃciency as well as thermoreg- acy, mainly in the summer, by reducing both de and db, and hence the ener- hermoregulation. Given the limitations of this study in terms of sample sizes hat it covers only a single year, the only assumption we could make is that is plastic rather than a “ﬁxed” adaptation, as this would require further re- ng multiple seasons and years and including several other insular popula- This shift in thermal preferences could be a response to the seasonality of temperate climate. Shifts in thermal preferences could facilitate thermoregulation effectiveness, by reducing the deviation of Te from Tset. Or they could simply reﬂect the optimal temperature range for another physiological process, such as sprint speed or digestion [2,19,63]. In general, the extent of behavioral thermoregulation is determined by the trade-off between maximizing physiological performance and individual ﬁtness, and the relevant energy and time constraints, as well as predation risk [10,11]. In our study, we could argue that this shift in Tpref and Tset favored thermoregulation efﬁciency as well as thermoregulation accuracy, mainly in the summer, by reducing both de and db, and hence the energetic cost of thermoregulation. Given the limitations of this study in terms of sample sizes and the fact that it covers only a single year, the only assumption we could make is that this response is plastic rather than a “ﬁxed” adaptation, as this would require further research spanning multiple seasons and years and including several other insular populations. g p y g p p Our initial hypothesis suggested that the particularities of insular life such as the low predation pressure [79,80], and the favorable climate [21,22] would lower the costs of thermoregulation. Indeed, the thermoregulation efﬁciency of many Mediterranean lacertids is higher in the summer than in the spring, mostly driven by the availability of higher environmental temperatures [20]. 4. Discussion The favorable climatic conditions, together with the ecological release from predators and competition, allow island species to exploit their Animals 2023, 13, 3195 8 of 11 8 of 11 thermal habitats more efﬁciently and thus enjoy greater ﬁtness beneﬁts, which in turn may hinder rather than speed up the evolution of physiology in insular environments [81]. However, the positive effect of the mild island climate appears to be buffered by season- ality, which exerts a stronger effect on thermoregulation in comparison to climate and habitat type [20]. The present study demonstrates this effect, as the most decisive thermal parameter, Tpref, showed substantial seasonal variation and led to the discrepancy in the effectiveness of thermoregulation across seasons. Thus, despite the greater availability of suitable thermal habitats found on larger islands, lizards still face the challenges posed by climatic ﬂuctuations and have to actively thermoregulate to overcome them and ensure their survival. Finally, understanding these shifts in thermal physiology will provide valuable in- sights into the adaptive and evolutionary potential of lizards, which is crucial in view of the ongoing climate change. Lizard species may need to either avoid rising temperatures by moving to more favorable thermal environments, or employ behavioural and physiological plastic mechanisms, or adaptation in order to survive [82–84]. While mainland populations can potentially shift their geographic distribution, lizards with limited dispersal abilities, or those occupying a limited space, such as the insular endemics, are often more vulnerable and face a greater risk of extinction [85,86]. This study highlights a shift in the thermal preferences of a Mediterranean lizard during the hot summer period, which could poten- tially have a buffering effect against the rising temperatures. However, the extent to which this shift can ensure the future survival of the species remains unclear. Thus, effective conservation planning and management should also consider the capacity of species to adapt to these physiological challenges imposed by climate change [87,88]. References 1. Cooper, B.S. The evolution of thermal physiology in endotherms. Front. Biosci. 2010, 2, 861–881. [C 2. Angilletta, M.J.; Niewiarowski, P.H.; Navas, C.A. The evolution of thermal physiology in ectotherms. J. Therm. Biol. 2002, 27, 249–268. [CrossRef] 3. Bicego, K.C.; Barros, R.C.H.; Branco, L.G.S. Physiology of temperature regulation: Comparative aspects. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 2007, 147, 616–639. [CrossRef] 4. Hertz, P.E.; Huey, R.B.; Stevenson, R.D. Evaluating temperature regulation by ﬁeld-active ectotherms: The fallacy of the inappropriate question. Am. Nat. 1993, 142, 796–818. [CrossRef] [PubMed] pp p q 5. Angilletta, M.J. Thermal Adaptation: A Theoretical and Empirical Synthesis; Oxford biology; Oxford University Press: Oxford, UK; New York, NY, USA, 2009; ISBN 978-0-19-857087-5. ture, physiology, and the ecology of reptiles. In Biology of the Reptilia. Physiology (C); Gans, C., Pough, F.H. s: Cambridge, MA, USA, 1982; pp. 25–91. 6. Huey, R.B. Temperature, physiology, and the ecology of reptiles. In Biology of the Reptilia. Physiolog Eds.; Academic Press: Cambridge, MA, USA, 1982; pp. 25–91. g 7. James, M.C.; Mrosovsky, N. Body temperatures of leatherback turtles (Dermochelys coriacea) in temperate waters off Nova Scotia, Canada. Can. J. Zool. 2004, 82, 1302–1306. [CrossRef] J [ ] 8. Tattersall, G.J.; Leite, C.A.C.; Sanders, C.E.; Cadena, V.; Andrade, D.V.; Abe, A.S.; Milsom, W.K. Seasonal reproductive endothermy in tegu lizards. Sci. Adv. 2016, 2, e1500951. [CrossRef] g 9. Angilletta, M.J. Evolution of thermal reaction norms for growth rate and body size in ectotherms: An Introduction to the Symposium. Integr. Comp. Biol. 2004, 44, 401–402. [CrossRef] [PubMed] g p B.; Slatkin, M. Cost and beneﬁts of lizard thermoregulation. Q. Rev. Biol. 1976, 51, 363–384. [CrossRef] 10. Huey, R.B.; Slatkin, M. Cost and beneﬁts of lizard thermoregulation. Q. Rev. Biol. 1976, 51, 363–384 11. Sears, M.W.; Angilletta, M.J. Costs and beneﬁts of thermoregulation revisited: Both the heterogeneity and spatial structure of temperature drive energetic costs. Am. Nat. 2015, 185, E94–E102. [CrossRef] 12. Meiri, S.; Bauer, A.M.; Chirio, L.; Colli, G.R.; Das, I.; Doan, T.M.; Feldman, A.; Herrera, F.-C.; Novosolov, M.; Paﬁlis, P.; et al. Are lizards feeling the heat? A tale of ecology and evolution under two temperatures. Glob. Ecol. Biogeogr. 2013, 22, 834–845. [CrossRef] 13. Basson, C.H.; Clusella-Trullas, S. The behavior-physiology nexus: Behavioral and physiological compensation are relied on to different extents between seasons. Physiol. Biochem. Zool. 2015, 88, 384–394. [CrossRef] [PubMed] 14. Jessop, T.S.; Purwandana, D.; Imansyah, M.J.; Cioﬁ, C.; Jackson Benu, Y.; Arieifandy, A. 5. Conclusions This study emphasizes how seasonality impacts the thermal biology of an insular lizard population. During the summer, when the environment provides the best thermal conditions, these lizards can utilize a wider range of temperatures. However, in autumn, the thermal quality of the environment is lowest. Additionally, the efﬁciency of thermoreg- ulation is inﬂuenced by the changing environmental temperatures throughout the seasons, with the highest levels occurring in summer and the lowest in autumn. Furthermore, lizards respond to these seasonal ﬂuctuations by adjusting their thermal preferences, which allows them to maintain consistent body temperatures across seasons. Our ﬁndings stress the need for further studies that span across seasons and encompass several insular populations, in order to tease apart the plastic responses and the ﬁxed local adaptations in the thermal biology of these lizards. Author Contributions: Conceptualization, P.P., S.S. and E.K.; methodology, E.K. and I.G.; formal analy- sis, E.K.; investigation, E.K. and I.G.; resources, P.P. and S.S.; data curation, E.K.; writing—original draft preparation, E.K.; writing—review and editing, P.P., S.S., E.K. and I.G.; visualization, E.K.; supervision, P.P and, S.S.; All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: All aspects of this study were conducted in full compliance with Hellenic national law (Presidential Decree 67/81) on the humane use of animals, and the EU Directive 2010/63/EU for animal experiments guidelines. Data Availability Statement: All data in this study are available upon request from the authors and will be made publicly available pending publication of additional manuscripts. Acknowledgments: We would like to thank Maria Skordali, Danai Kouvari, and Danai Karakasi for their assistance in the ﬁeld. Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. 9 of 11 Animals 2023, 13, 3195 References The inﬂuence of tropical seasonality on breeding phenology, growth, survival and movement of a large reptile (Varanus komodoensis). Biol. J. Linn. Soc. 2022, 136, 552–565. [CrossRef] 15. Ortega, Z.; Mencía, A.; Giroux, A.; Pérez-Mellado, V. Broad seasonal changes in thermoregulation of Podarcis lilfordi (Squamata, Lacertidae) at Binicodrell islet (Menorca, Spain). Herpetozoa 2019, 32, 57–63. [CrossRef] 16. Ortega, Z.; Pérez-Mellado, V. Seasonal patterns of body temperature and microhabitat selection in a lacertid lizard. Acta Oecologica 2016, 77, 201–206. [CrossRef] 17. Vicente Liz, A.; Santos, V.; Ribeiro, T.; Guimarães, M.; Verrastro, L. Are lizards sensitive to anomalous seasonal temperatures? Long-term thermobiological variability in a subtropical species. PLoS ONE 2019, 14, e0226399. [CrossRef] 18. Diaz, J.A.; Cabezas-Diaz, S. Seasonal variation in the contribution of different behavioural mechanisms to lizard thermoregulation. Funct. Ecol. 2004, 18, 867–875. [CrossRef] 19. Díaz, J.A.; Iraeta, P.; Monasterio, C. Seasonality provokes a shift of thermal preferences in a temperate lizard, but altitude does not. J. Therm. Biol. 2006, 31, 237–242. [CrossRef] Vallejo, F.J. Main factors affecting lacertid lizard thermal ecology. Integr. Zool. 2019, 14, 293–305. [CrossRef] 20. Ortega, Z.; Martín-Vallejo, F.J. Main factors affecting lacertid lizard thermal ecology. Integr. Zool. 2 [PubMed] 21. MacArthur, R.H.; Wilson, E.O. The Theory of Island Biogeography; Princeton University Press: Princeton, NJ, USA, 1967; ISBN 0-691-08836-5. 22. Whittaker, R.J.; Fernández-Palacios, J.M. Island Biogeography: Ecology, Evolution, and Conservation; Oxford University Press: Oxford, UK, 2007; ISBN 0-19-856611-5. 23. Sagonas, K.; Valakos, E.D.; Paﬁlis, P. The impact of insularity on the thermoregulation of a Mediterranean lizard. J. Therm. Biol. 2013, 38, 480–486. [CrossRef] 24. Grbac, I.; Bauwens, D. Constraints on Temperature Regulation in Two Sympatric Podarcis Lizards during Autumn. Copeia 2001, 2001, 178–186. [CrossRef] iantis, K.A. Habitat diversity, ecological requirements of species and the Small Island Effect. Divers. Distrib CrossRef] 25. Sfenthourakis, S.; Triantis, K.A. Habitat diversity, ecological requirements of species and the Small 2009, 15, 131–140. [CrossRef] 26. Ortega, Z.; Pérez-Mellado, V.; Garrido, M.; Guerra, C.; Villa-García, A.; Alonso-Fernández, T. Seasonal changes in thermal biology of Podarcis lilfordi (Squamata, Lacertidae) consistently depend on habitat traits. J. Therm. Biol. 2014, 39, 32–39. [CrossRef] f ( q ) y p J [ ] 27. Paﬁlis, P.; Herrel, A.; Kapsalas, G.; Vasilopoulou-Kampitsi, M.; Fabre, A.-C.; Foufopoulos, J.; Donihue, C.M. Habitat shapes the thermoregulation of Mediterranean lizards introduced to replicate experimental islets. J. Therm. Biol. 2019, 84, 368–374. [CrossRef] q y p 27. References Paﬁlis, P.; Herrel, A.; Kapsalas, G.; Vasilopoulou-Kampitsi, M.; Fabre, A.-C.; Foufopoulos, J.; Donihue, C.M. Habitat shapes the thermoregulation of Mediterranean lizards introduced to replicate experimental islets. J. Therm. Biol. 2019, 84, 368–374. [CrossRef] 28. Paﬁlis, P.; Lymberakis, P.; Sagonas, K.; Valakos, E. The particularities of a remote islet shape the thermoregulatory proﬁle of an d d l d h l [ f] g p p 28. Paﬁlis, P.; Lymberakis, P.; Sagonas, K.; Valakos, E. The particularities of a remote islet shape the thermoregulatory proﬁle of an endemic Mediterranean lizard. J. Therm. Biol. 2016, 61, 55–60. [CrossRef] 29. Paﬁlis, P.; Maragou, P.; Sagonas, K.; Valakos, E. Partitioning thermal habitat on a vertical rock, a herculean task. J. Therm. Biol. 2017, 70, 54–60. [CrossRef] [PubMed] 10 of 11 10 of 11 Animals 2023, 13, 3195 30. Sannolo, M.; Civantos, E.; Martín, J.; Carretero, M.A. Variation in ﬁeld body temperature and total evaporative water loss along an environmental gradient in a diurnal ectotherm. J. Zool. 2020, 310, 221–231. [CrossRef] g 31. Ergül Kalayci, T.; Özdemir, N.; Gül, Ç.; Tosuno˘glu, M. Variation in Body Size and Age Structure of Stellagama stellio (L., 1758) (Reptilia: Agamidae) from Turkey. Acta Zool. Bulg. 2014, 66, 65–72. p g y g 32. Valakos, E.D.; Paﬁlis, P.; Sotiropoulos, K.; Lymberakis, P.; Maragou, P.; Foufopoulos, J. The Amphibians and Reptiles of Greece (Frankfurt Contributions to Natural History); Ed. Chimaira: Frankfurt am Main, Germany, 2008. 33. Kuma¸s, M.; Ayaz, D. Age determination and long bone histology in Stellagama stellio (LINNAEUS, 1758) (Squamata: Sauria: Agamidae) populations in Turkey. Vertebr. Zool. 2014, 64, 113–126. [CrossRef] p y [ ] aragou, P. (Eds.) Atlas of Amphibian and Reptiles of Greece; Broken Hill Publishers Ltd.: Nicosia, Cyprus, 2020. g ) p p y 34. Paﬁlis, P.; Maragou, P. (Eds.) Atlas of Amphibian and Reptiles of Greece; Broken Hill Publishers Ltd.: Nico 34. Paﬁlis, P.; Maragou, P. (Eds.) Atlas of Amphibian and Reptiles of Greece; Broken Hill Publishers Ltd.: Nicosia, Cyprus, 2020. 35. Dü¸sen, S.; Öz, M. A Study on the Feeding Biology of Laudakia (=Agama) stellio (L. 1758) (Lacertilia: Agamidae) Populations in the Antalya Region. Turk. J. Zool. 2001, 25, 177–181. y g 36. Karameta, E.; Papadopoulos, V.V.; Paﬁlis, P. First record of ophiophagy in the roughtail rock agama (Stellagama stellio): One of the most rare feeding behaviors among European lizards. Herpetol. Notes 2015, 8, 111–113. 37. Lo Cascio, P.; Corti, C.; Luiselli, L. References Seasonal variations of the diet of Laudakia stellio (Agamidae) from Nisyros island, Dodecanese (Greece). Herpetol. J. 2001, 11, 33–35. p 38. Karameta, E.; Lymberakis, P.; Grillitsch, H.; Ilgaz, Ç.; Avci, A.; Kumluta¸s, Y.; Candan, K.; Wagner, P.; Sfenthourakis, S.; Paﬁlis, P.; et al. The story of a rock-star: Multilocus phylogeny and species delimitation in the starred or roughtail rock agama, Laudakia stellio (Reptilia: Agamidae). Zool. J. Linn. Soc. 2022, 195, 195–219. [CrossRef] 40. Razzetti, E.; Bader, T.; Bilek, K.; Delﬁno, M.; Rita, A.; Di Cerbo, A.R.; Duda, M.; Hill, J.; Rathbauer, F.; Riegler, C.; et al. A contribution to the knowledge of the herpetofauna of the Greek island of Corfu. In Societas Herpetologica Italica: Atti del V Convegno Nazionale, Calci (Pisa), 29 Settembre–3 Ottobre 2004; Florence University Press: Firenze, Italy, 2006; pp. 207–216. y y pp 41. Kapsalas, G.; Probonas, N.; Dimalexis, A.; Paﬁlis, P. First record of Stellagama stellio (Squamata: Agamidae) from Karpathos Island, Greece. Russ. J. Herpetol. 2020, 47, 299–302. [CrossRef] p 42. Spaneli, V.; Lymberakis, P. First record of Stellagama stellio (Linnaeus, 1758) from Crete, Greece. Herpetol. Notes 2014, 7, 367–369. 43. Foufopoulos, J.; Paﬁlis, P. Biodiversity on Greek Paths of Culture in the Lesser Cyclades; ELLET, Society for the Environment and 42. Spaneli, V.; Lymberakis, P. First record of Stellagama stellio (Linnaeus, 1758) from Crete, Greece. Herpetol. Notes 2014, 7, 367–369. 43. Foufopoulos, J.; Paﬁlis, P. Biodiversity on Greek Paths of Culture in the Lesser Cyclades; ELLET, Society for the Environment and Cultural Heritage: Athens, Greece, 2021. g 44. Theocharatos, G. The Climate of Cyclades. Ph.D. Thesis, Athens University, Athens, Greece, 1978. (In Greek) 45 Gi ik l A S D ht h t i ti i C l d l G E W t 2014 47 31 43 g 44. Theocharatos, G. The Climate of Cyclades. Ph.D. Thesis, Athens University, Athens, Greece, 1978. (In G 45. Giannikopoulou, A.S. Drought characterisation in Cyclades complex, Greece. Eur. Water. 2014, 47, 31– 46. Bakken, G.S. Measurement and application of operative and standard operative temperatures in Ecology. Am. Zool. 1992, 32, 194–216. [CrossRef] 47. Lutterschmidt, W.I.; Reinert, H.K. Modeling body temperature and thermal inertia of large-bodied reptiles: Support for water- ﬁlled biophysical models in radiotelemetric studies. J. Therm. Biol. 2012, 37, 282–285. [CrossRef] 48. Karameta, E.; Sfenthourakis, S.; Paﬁlis, P. Are all islands the same? A comparative thermoregulatory approach in four insular populations. Amphibia-Reptililia 2022, 44, 59–69. [CrossRef] 49. References Evolution 2019, 73, 1241–1252. [CrossRef] 82. Sinervo, B.; Méndez-de-la-Cruz, F.; Miles, D.B.; Heulin, B.; Bastiaans, E.; Villagrán-Santa Cruz, M.; Lara-Resendiz, R.; Martínez- Méndez, N.; Calderón-Espinosa, M.L.; Meza-Lázaro, R.N.; et al. Erosion of lizard diversity by climate change and altered thermal niches. Science 2010, 328, 894–899. [CrossRef] assot, M.; Baron, J.-P.; Clobert, J. Ecological effects of climate change on European reptiles. Wildl. Conserv 79, e203. 83. Le Galliard, J.F.; Massot, M.; Baron, J.-P.; Clobert, J. Ecological effects of climate change on Europ Chang. Clim. 2012, 179, e203. g 84. Urban, M.C.; Richardson, J.L.; Freidenfelds, N.A. Plasticity and genetic adaptation mediate amphibian and reptile responses to climate change. Evol. Appl. 2014, 7, 88–103. [CrossRef] [PubMed] g 84. Urban, M.C.; Richardson, J.L.; Freidenfelds, N.A. Plasticity and genetic adaptation mediate amphibian and reptile responses to climate change. Evol. Appl. 2014, 7, 88–103. [CrossRef] [PubMed] g pp 85. Inman, R.D.; Esque, T.C.; Nussear, K.E. Dispersal limitations increase vulnerability under climate change for reptiles and amphibians in the southwestern United States. J. Wildl. Manag. 2023, 87, e22317. [CrossRef] g pp 85. Inman, R.D.; Esque, T.C.; Nussear, K.E. Dispersal limitations increase vulnerability under climate change for reptiles and amphibians in the southwestern United States. J. Wildl. Manag. 2023, 87, e22317. [CrossRef] amphibians in the southwestern United States. J. Wildl. Manag. 2023, 87, e22317. [CrossRef] 86. Chen, C.; Holyoak, M.; Xu, J.; de Oliveira Caetano, G.H.; Wang, Y. Range restriction, climate variability and human-related risks imperil lizards world-wide. Glob. Ecol. Biogeogr. 2023, 32, 780–792. [CrossRef] 87 Wik l ki M C k S J C ti h i l T d E l E l 2006 21 38 46 [C R f] [P bM d] p g 86. Chen, C.; Holyoak, M.; Xu, J.; de Oliveira Caetano, G.H.; Wang, Y. Range restriction, climate variability and human-related risks imperil lizards world-wide. Glob. Ecol. Biogeogr. 2023, 32, 780–792. [CrossRef] 87 Wik l ki M C k S J C i h i l T d E l E l 21 38 46 [C R f] [P bM d] 86. Chen, C.; Holyoak, M.; Xu, J.; de Oliveira Caetano, G.H.; Wang, Y. Range restriction, climate variability and human-related risks imperil lizards world-wide. Glob. Ecol. Biogeogr. 2023, 32, 780–792. References Living in the mountains: Thermal ecology and freezing tolerance of the lizard Abronia taeniata (Squamata: Anguidae). J. Therm. Biol. 2023, 117, 103679. [CrossRef] [PubMed] ( q g ) J , , [ ] [ ] 62. Losos, J.B.; Ricklefs, R.E. Adaptation and diversiﬁcation on islands. Nature 2009, 457, 830–836. [CrossRef] , J.B.; Ricklefs, R.E. Adaptation and diversiﬁcation on islands. Nature 2009, 457, 830–836. [CrossRef] ph, S.C.; Porter, W.P. Temperature, activity, and lizard life histories. Am. Nat. 1993, 142, 273–295. [CrossRef] 63. Adolph, S.C.; Porter, W.P. Temperature, activity, and lizard life histories. Am. Nat. 1993, 142, 273–295. [ 64. Cowles, R.B.; Bogert, C.M. A preliminary study of the thermal requirements of desert reptiles. Bull. AM 6 Co es, ; oge , C p e y s y o e e eq e e s o ese ep es , , 95 96 65. Avery, R.A. Thermoregulation, metabolism and social behaviour in Lacertidae. In Morphology and Biology of Reptiles, A. d’A. Bellairs and C.B. Cox. Ed. Linn. Soc. Symp. Ser. (3); Academic Press: Cambridge, MA, USA, 1976; Volume 3, pp. 245–259. 65. Avery, R.A. Thermoregulation, metabolism and social behaviour in Lacertidae. In Morphology and Biology and C.B. Cox. Ed. Linn. Soc. Symp. Ser. (3); Academic Press: Cambridge, MA, USA, 1976; Volume 3, pp. 66. Van Damme, R.; Bauwens, D.; Verheyen, R.F. Evolutionary rigidity of thermal physiology: The case of the cool temperate lizard Lacerta vivipara. Oikos 1990, 57, 61. [CrossRef] p 67. Ortega, Z.; Mencía, A.; Pérez-Mellado, V. Adaptive seasonal shifts in the thermal preferences of the lizard Iberolacerta galani (Squamata, Lacertidae). J. Therm. Biol. 2016, 62, 1–6. [CrossRef] rtega, Z.; Mencía, A.; Pérez-Mellado, V. Adaptive seasonal shifts in the thermal preferences of the lizard quamata Lacertidae) J Therm Biol 2016 62 1–6 [CrossRef] 67. Ortega, Z.; Mencía, A.; Pérez-Mellado, V. Adaptive seasonal shifts in the thermal preferences of the (Squamata, Lacertidae). J. Therm. Biol. 2016, 62, 1–6. [CrossRef] rtega, Z.; Mencía, A.; Pérez Mellado, V. Adaptive seasonal shifts in the thermal preferences of the lizard quamata, Lacertidae). J. Therm. Biol. 2016, 62, 1–6. [CrossRef] Nevo, E. Thermal biology of four Israeli Agamid lizards in early summer. Isr. J. Ecol. Evol. 1981, 30, 190–210. 68. Hertz, P.E.; Nevo, E. Thermal biology of four Israeli Agamid lizards in early summer. Isr. J. Ecol. Evol. 69. Saber, S.A. Preferred body temperature of free-ranging Starred Agama Laudakia stellio (Linnaeus, 1758)(Agamidae) from Egypt. Russ. J. Herpetol. References Diaz, J.A. Ecological correlates of the thermal quality of an ectotherm’s habitat: A comparison between two temperate lizard populations. Funct. Ecol. 1997, 11, 79–89. [CrossRef] 50. Dzialowski, E.M. Use of operative temperature and standard operative temperature models in th 2005, 30, 317–334. [CrossRef] se of operative temperature and standard operative temperature models in thermal biology. J. Therm. Biol CrossRef] 51. Gidiþ, M.; Baþkale, E. Age structure and life expectancy in a Stellagama stellio (LINNAEUS, 1758) population from Kutahya, Turkey. Russ. J. Herpetol. 2021, 28, 327–332. [CrossRef] y p 52. Veríssimo, C.V.; Carretero, M.A. Preferred temperatures of Podarcis vaucheri from Morocco: Intraspeciﬁc variation and interspeciﬁc comparisons. Amphibia-Reptilia 2009, 30, 17–23. [CrossRef] 53. Osojnik, N.; Žagar, A.; Carretero, M.A.; García-Muñoz, E.; Vrezec, A. Ecophysiological dissimilarities of two sympatric lizards. Herpetologica 2013, 69, 445–454. [CrossRef] 54. Taylor, E.N.; Diele-Viegas, L.M.; Gangloff, E.J.; Hall, J.M.; Halpern, B.; Massey, M.D.; Rödder, D.; Rollinson, N.; Spears, S.; Sun, B.; et al. The thermal ecology and physiology of reptiles and amphibians: A user’s guide. J. Exp. Zool. Part Ecol. Integr. Physiol. 2021, 335, 13–44. [CrossRef] 55. Carretero, M.A.; Roig, J.M.; Llorente, G.A. Variation in preferred body temperatures in an oviparous population of Lacerta (Zootoca) vivipara. Herpetol. J. 2005, 15, 51–55. 56. Van Damme, R.; Bauwens, D.; Verheyen, R.F. Selected body temperatures in the lizard Lacerta vivipara: Variation within and between populations. J. Therm. Biol. 1986, 11, 219–222. [CrossRef] p p 57. Cabanac, M.; Gosselin, F. Emotional fever in the lizard Callopistes maculatus (Teiidae). Anim. Behav. 199 57. Cabanac, M.; Gosselin, F. Emotional fever in the lizard Callopistes maculatus (Teiidae). Anim. Behav. 1993, 46, 200–202. [CrossRef] 58. Carneiro, D.; García-Muñoz, E.; Kaliontzopoulou, A.; Llorente, G.A.; Carretero, M.A. Comparing ecophysiological traits in two Podarcis wall lizards with overlapping ranges. Salamandra 2015, 51, 335–344. 58. Carneiro, D.; García-Muñoz, E.; Kaliontzopoulou, A.; Llorente, G.A.; Carretero, M.A. Comparing ecophysiological traits in two Podarcis wall lizards with overlapping ranges. Salamandra 2015, 51, 335–344. 59. Stellatelli, O.A.; Villalba, A.; Block, C.; Vega, L.E.; Dajil, J.E.; Cruz, F.B. Seasonal shifts in the thermal biology of the lizard Liolaemus tandiliensis (Squamata, Liolaemidae). J. Therm. Biol. 2018, 73, 61–70. [CrossRef] 60. Ortega, Z.; Mencía, A.; Pérez-Mellado, V. Wind constraints on the thermoregulation of high mountain lizards. Int. J. Biometeorol. 2017, 61, 565–573. [CrossRef] Animals 2023, 13, 3195 11 of 11 11 of 11 61. Fierro-Estrada, N.; Méndez-de la Cruz, F.R.; Tellez-Valdes, O. References 2012, 19, 171–176. p 70. Hertz, P.E. Temperature regulation in Puerto Rican Anolis lizards: A ﬁeld test using null hypotheses. Ecology 1992, 73, 1405–1417. [CrossRef] 71. Bauwens, D.; Hertz, P.E.; Castilla, A.M. Thermoregulation in a Lacertid lizard: The relative contributions of distinct behavioral mechanisms. Ecology 1996, 77, 1818–1830. [CrossRef] gy 72. Sears, M.W.; Angilletta, M.J., Jr.; Schuler, M.S.; Borchert, J.; Dilliplane, K.F.; Stegman, M.; Rusch, T.W.; Mitchell, W.A. Conﬁguration of the thermal landscape determines thermoregulatory performance of ectotherms. Proc. Natl. Acad. Sci. USA 2016, 113, 10595– 10600. [CrossRef] [ ] 73. Little, A.G.; Seebacher, F. Acclimation, acclimatization, and seasonal variation in amphibians and reptiles. In Amphibian and Reptile Adaptations to the Environment; CRC Press: Boca Raton, FL, USA, 2016; ISBN 978-1-315-37363-8. p p 74. Andrews, R.M. Activity and thermal biology of the Sand-Swimming Skink Neoseps reynoldsi: Diel and seasonal patterns. Copeia 1994, 1994, 91–99. [CrossRef] [ ] 75. Christian, K.A.; Bedford, G.S. Seasonal changes in thermoregulation by the Frillneck Lizard, Chlam Australia. Ecology 1995, 76, 124–132. [CrossRef] Australia. Ecology 1995, 76, 124–132. [CrossRef] 76. Huey, R.B.; Pianka, E.R. Seasonal variation in thermoregulatory behavior and body temperature of diurnal Kalahari lizards. gy , , [ ] 76. Huey, R.B.; Pianka, E.R. Seasonal variation in thermoregulatory behavior and body temperature of diurnal Kalahari lizards. Ecology 1977, 58, 1066–1075. [CrossRef] 76. Huey, R.B.; Pianka, E.R. Seasonal variation in thermoregulatory behavior and body temperature Ecology 1977, 58, 1066–1075. [CrossRef] gy 77. Megía-Palma, R.; Arregui, L.; Pozo, I.; Žagar, A.; Serén, N.; Carretero, M.A.; Merino, S. Geographic patterns of stress in insular lizards reveal anthropogenic and climatic signatures. Sci. Total Environ. 2020, 749, 141655. [CrossRef] p g g 78. Nastos, P.T.; Zerefos, C.S. Spatial and temporal variability of consecutive dry and wet days in Greece. Atmos. Res. 2009, 94, 616–628. [CrossRef] 79. Cooper, W.E., Jr.; Pérez-Mellado, V. Historical inﬂuence of predation pressure on escape by Podarcis lizards in the Balearic Islands. Biol. J. Linn. Soc. 2012, 107, 254–268. [CrossRef] 80. Itescu, Y.; Schwarz, R.; Meiri, S.; Paﬁlis, P. Intraspeciﬁc competition, not predation, drives lizard tail loss on islands. J. Anim. Ecol. 2017, 86, 66–74. [CrossRef] ario Castañeda, M.; Londoño, G.A.; Bodensteiner, B.L.; Muñoz, M.M. Physiological evolution during adaptive he island effect in Anolis lizards. Evolution 2019, 73, 1241–1252. [CrossRef] 81. Salazar, J.C.; del Rosario Castañeda, M.; Londoño, G.A.; Bodensteiner, B.L.; Muñoz, M.M. Physiologic radiation: A test of the island effect in Anolis lizards. References [CrossRef] 87 Wik l ki M C k S J C i h i l T d E l E l 2006 21 38 46 [C R f] [P bM d] p y gy 88. Chown, S.L. Trait-based approaches to conservation physiology: Forecasting environmental change risks from the bottom up. Philos. Trans. R. Soc. B Biol. Sci. 2012, 367, 1615–1627. [CrossRef] [PubMed] 88. Chown, S.L. Trait-based approaches to conservation physiology: Forecasting environmental change risks from the bottom up. Philos. Trans. R. Soc. B Biol. Sci. 2012, 367, 1615–1627. [CrossRef] [PubMed] Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
https://openalex.org/W1919930445	https://europepmc.org/articles/pmc4537000?pdf=render	English	null	Wee1 is required to sustain ATR/Chk1 signaling upon replicative stress	Oncotarget	2,015	cc-by	10,255	Wee1 is required to sustain ATR/Chk1 signaling upon replicative stress Priyanka Saini1, Yizhu Li1 and Matthias Dobbelstein1 1 Institute of Molecular Oncology, Göttingen Centre of Molecular Biosciences (GZMB), Faculty of Medicine, University of Göttingen, Göttingen, Germany Correspondence to: Matthias Dobbelstein, email: mdobbel@uni-goettingen.de Keywords: Wee1, ATR signaling pathway, replicative stress, checkpoint kinases, gemcitabine Received: December 05, 2014 Accepted: March 31, 2015 Published: April 19, 2015 Correspondence to: Matthias Dobbelstein, email: mdobbel@uni-goettingen.de Correspondence to: Matthias Dobbelstein, email: mdobbel@uni-goettingen.de Keywords: Wee1, ATR signaling pathway, replicative stress, checkpoint kinases, gemcitabine Received: December 05, 2014 Accepted: March 31, 2015 Published: April 19, 2015 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract The therapeutic efficacy of nucleoside analogues, e.g. gemcitabine, against cancer cells can be augmented by inhibitors of checkpoint kinases, including Wee1, ATR, and Chk1. We have compared the chemosensitizing effect of these inhibitors in cells derived from pancreatic cancer, a tumor entity where gemcitabine is part of the first-line therapeutic regimens, and in osteosarcoma-derived cells. As expected, all three inhibitors rendered cancer cells more sensitive to gemcitabine, but Wee1 inhibition proved to be particularly efficient in this context. Investigating the reasons for this potent sensitizing effect, we found that Wee1 inhibition or knockdown not only blocked Wee1 activity, but also reduced the activation of ATR/Chk1 in gemcitabine- treated cells. Combination of several inhibitors revealed that Wee1 inhibition requires Cyclin-dependent kinases 1 and 2 (Cdk1/2) and Polo-like kinase 1 (Plk1) to reduce ATR/Chk1 activity. Through activation of Cdks and Plk1, Wee1 inhibition reduces Claspin and CtIP levels, explaining the impairment in ATR/Chk1 activity. Taken together, these results confer a consistent signaling pathway reaching from Wee1 inhibition to impaired Chk1 activity, mechanistically dissecting how Wee1 inhibitors not only dysregulate cell cycle progression, but also enhance replicative stress and chemosensitivity towards nucleoside analogues. Oncotarget, Vol. 6, No. 15 Oncotarget, Vol. 6, No. 15 www.impactjournals.com/oncotarget/ www.impactjournals.com/oncotarget Introduction kinases. This notion initiated the design of small molecules that target and inhibit this class of enzymes [7]. Checkpoint kinases have emerged as therapeutically important targets, as their inhibition can sensitize cancer cells to DNA-damaging chemotherapeutics. In a majority of cancer cells, the G1/S checkpoint is impaired; as a consequence, these cells rely on intra S and G2/M checkpoints for DNA repair and survival [8]. Known players involved in the intra S and G2/M checkpoints include ATR, Chk1, and Wee1. Thus, combining inhibitors of these kinases with gemcitabine can sensitize tumor cells, including pancreatic, colon and breast tumors [9, 10, 11]. Gemcitabine leads to replicative stress and activates the intra S-phase checkpoint which, in turn, counteracts the damage to DNA. Therefore, inhibitors of checkpoint kinases enhance replicative stress, DNA damage, and tumor cell death. However, there is a lack of quantitative comparisons between the efficacy of inhibiting different checkpoint kinases to sensitize cells towards gemcitabine. Moreover, it remains to be determined how Wee1 and Gemcitabine (2’, 2’-difluorodeoxycytidine, dFdC), an analogue of deoxycytidine, is active against a broad spectrum of solid tumors, mostly pancreatic cancer [1], but also breast cancer [2], bladder cancer [3] or non-small cell lung cancer [4]. Pancreatic cancer is the eighth leading cause of cancer-related deaths [5]. Currently, gemcitabine is the principal compound used for its treatment, and it improves survival in a fraction of patients; however, the tumor response rate to gemcitabine monotherapy is only 5.4% [1], and the median progression-free survival under such therapy is 3.5 months [6]. Thus, in nearly all cases, pancreatic cancers display either primary or secondary resistance towards gemcitabine. This raises the need to identify strategies for improving the chemosensitivity of pancreatic cancer cells. Cancer cells can evade the normal physiological signals controlling growth and survival by deregulating www.impactjournals.com/oncotarget Oncotarget 13072 ATR/Chk1 activities affect each other in gemcitabine- treated cells. combining the Wee1 inhibitor with gemcitabine leads to more pronounced cell death in comparison to single drug treatment (Supplemental Figure 1D-1F). In our study, we found that Wee1 inhibition is particularly potent to eliminate gemcitabine-treated cancer cells, as compared to the inhibition of Chk1 or ATR. Importantly, inhibition of Wee1 in gemcitabine-treated cells hampered the ATR/Chk1 pathway, thus resulting in the impairment of at least three kinases that would otherwise attenuate replicative stress. Inhibitors of Chk1, Wee1 or ATR sensitize tumor- derived cells towards gemcitabine For comparative assessment of their chemosensitizing activities, we evaluated pharmacological inhibitors against Chk1, Wee1 and ATR (SB 218078, MK-1775,and VE-821 respectively). The efficiency of these inhibitors was confirmed through immunoblot staining of their respective substrates (Supplemental Figure 1A, 1B). Earlier studies performed using these inhibitors have shown sensitization of tumor cells towards various chemotherapeutics [9, 11, 12, 13], here, we were aiming at the direct comparison of the cytotoxic effects of these inhibitors in combination with gemcitabine. We investigated the long-term effect of the combined treatment by monitoring the growth of the cells over 1-2 weeks after treatment. Panc1 (pancreatic adenocarcinoma) and U2OS (osteocarcinoma) cells were treated with the inhibitors in the presence or absence of gemcitabine for 24 h. After removal of all the drugs, the growth of the cells was followed using bright field microscopy and automated image analysis (Celigo cytometer) for 8-13 days. The length of the experiments was chosen as to allow control-treated cells to reach confluence. We observed that combining inhibitors of either Wee1 or ATR with gemcitabine retards the growth of the cells to a higher extent than the Chk1 inhibitor in both Panc1 and U2OS cells (Figure 1A-1D). Similarly, MiaPaCa2 (pancreatic adenocarcinoma) cells were found to be sensitized towards gemcitabine upon inhibition of Wee1 or ATR (Supplemental Figure 1C). Furthermore, cell viability assays in these cell lines revealed that For comparative assessment of their chemosensitizing activities, we evaluated pharmacological inhibitors against Chk1, Wee1 and ATR (SB 218078, MK-1775,and VE-821 respectively). The efficiency of these inhibitors was confirmed through immunoblot staining of their respective substrates (Supplemental Figure 1A, 1B). Earlier studies performed using these inhibitors have shown sensitization of tumor cells towards various chemotherapeutics [9, 11, 12, 13], here, we were aiming at the direct comparison of the cytotoxic effects of these inhibitors in combination with gemcitabine. Introduction Inhibition of Cyclin- dependent kinases (Cdks) along with Wee1 rescued the ATR/Chk1 activity, thus identifying Cdks as mediators of ATR/Chk1 inactivation in this system. Furthermore, we observed that increased activity of Cdks upon inhibition of Wee1 caused activation of Polo-like kinase1 (Plk1). Plk1, in turn, led to the reduction of Claspin and CtIP levels, thereby attenuating the ATR/Chk1 pathway. These findings thus identify a cross-talk between Wee1 and ATR/ Chk1 activities and a role of Wee1 in sustaining ATR/ Chk1 activation during replicative stress. In parallel, we determined the phosphorylation of (the histone variant) H2AX, an established marker of DNA damage response, directly after treatment with the drugs for 24 h. We used quantitative immunofluorescence to measure the amount of phosphorylated H2AX (γH2AX). We found that the inhibition of each of the three kinases cooperates with gemcitabine in potentiating the DNA damage signal as determined by increased average γH2AX intensity (Figure 1E, 1F). To rule out that the appearance of γH2AX is a result of apoptosis [14] rather than the direct consequence of DNA damage, we performed similar experiments in the presence of Z-VAD.fmk, a pan caspase inhibitor that prevents apoptosis. However, caspase inhibition did not interfere with the accumulation of γH2AX in this context (Supplemental Figure 1G). Wee1 inhibition increased γH2AX levels even on its own (Figure 1E, 1F) and it also proved to impair survival to a particularly large extent (Figure 1A-1D). In contrast, we observed only a mild cooperative effect on γH2AX accumulation when combining the inhibitor of Chk1 with Wee1 inhibition (Fig.1G, 1H). This observation held true even in the presence of Z-VAD.fmk (Supplemental Figure 1H). This raised the question whether the Wee1-dependent signaling pathways might be epistatic to the ATR/Chk1 pathway, or vice-versa. Wee1 inhibition attenuates Chk1 phosphorylation in gemcitabine-treated cells Panc1 and U2OS cells were treated with 2.5µM SB 218078, 0.5µM MK-1775 and 5µM VE-821 (referred to as Chk1i, Wee1i, and ATRi, respectively, for their target kinases), in the absence or presence of gemcitabine (Gem) at the indicated concentrations. After 24 h, all drugs were removed and fresh medium was added. Cells were incubated for 8-13 days, and confluency was measured each day using brightfield microscopy (Celigo cell cytometer). Error bars represent the SD, n = 3. p-values (based on Student’s t-test, 2-sided, assuming different variances) were determined for the last measurement of respective cell line. E, F. Cells were treated for 24 h with gemcitabine, followed by treatment with checkpoint kinase inhibitors (5µM Chk1i; 1µM (Panc1) or 0.5µM (U2OS) Wee1i; 10µM ATRi) and gemcitabine for another 20 h. Cells were then fixed and stained for γH2AX. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Error bars represent the SD, n = 3. Images of γH2AX stainings are shown in (Supplemental Figure S4 A, B). G, H. Cells were treated with 1µM Wee1i, 5µM Chk1i or DMSO in the presence of 300nM gemcitabine for 24 h. As a control, cells were treated with DMSO without gemcitabine. The cells were then processed as described in (E-F). Figure 1: Three checkpoint kinase inhibitors cooperate with gemcitabine to enhance cytotoxicity. A.-D. Panc1 and U2OS cells were treated with 2.5µM SB 218078, 0.5µM MK-1775 and 5µM VE-821 (referred to as Chk1i, Wee1i, and ATRi, respectively, for their target kinases), in the absence or presence of gemcitabine (Gem) at the indicated concentrations. After 24 h, all drugs were removed and fresh medium was added. Cells were incubated for 8-13 days, and confluency was measured each day using brightfield microscopy (Celigo cell cytometer). Error bars represent the SD, n = 3. p-values (based on Student’s t-test, 2-sided, assuming different variances) were determined for the last measurement of respective cell line. E, F. Cells were treated for 24 h with gemcitabine, followed by treatment with checkpoint kinase inhibitors (5µM Chk1i; 1µM (Panc1) or 0.5µM (U2OS) Wee1i; 10µM ATRi) and gemcitabine for another 20 h. Cells were then fixed and stained for γH2AX. Detection and analysis was performed using automated immunofluorescence microscopy (BD Pathway). Error bars represent the SD, n = 3. Images of γH2AX stainings are shown in (Supplemental Figure S4 A, B). G, H. Wee1 inhibition attenuates Chk1 phosphorylation in gemcitabine-treated cells To analyze the signaling pathways involved in the DNA damage response upon Wee1 inhibition, we detected DNA damage signaling intermediates through immunoblot analysis. Cells were treated with the Wee1 inhibitor and/ or gemcitabine for 24 h, followed by detection of DNA damage response factors (Figure 2A, 2B). The activity of the inhibitor was verified by detecting the phosphorylation of Cdk1 at Tyr15, a known Wee1 phosphorylation site [15]. As expected, this phosphorylation was decreased upon treatment with the Wee1 inhibitor (Figure 2A, 2B). Next, we determined the activity of the ATR-Chk1 signaling pathway upon Wee1 inhibition. Phosphorylation of Chk1 at Ser317 is mediated by ATR and activates Chk1 [16]. Strikingly, we observed that Chk1 phosphorylation (Ser317) decreased upon Wee1 inhibition in gemcitabine- treated cells. To our knowledge, this is the first time that an impact of Wee1 on Chk1 activation is reported. γH2AX intensity did not decrease by Wee1 inhibition. This experiment was also performed after removing Wee1 using two distinct siRNAs, and this also reduced the phosphorylation of Chk1 in gemcitabine-treated U2OS and Panc1 cells (Figure 2C and Supplemental Figure 2A). This decreased activation of Chk1 was www.impactjournals.com/oncotarget Oncotarget 13073 Three checkpoint kinase inhibitors cooperate with gemcitabine to enhance cytotoxicity. A.-D. Panc1 treated with 2.5µM SB 218078, 0.5µM MK-1775 and 5µM VE-821 (referred to as Chk1i, Wee1i, and ATRi, respe t kinases), in the absence or presence of gemcitabine (Gem) at the indicated concentrations. After 24 h, all drugs wer medium was added. Cells were incubated for 8-13 days, and confluency was measured each day using brightfield m ll cytometer). Error bars represent the SD, n = 3. p-values (based on Student’s t-test, 2-sided, assuming different varia d for the last measurement of respective cell line. E, F. Cells were treated for 24 h with gemcitabine, followed by trea t kinase inhibitors (5µM Chk1i; 1µM (Panc1) or 0.5µM (U2OS) Wee1i; 10µM ATRi) and gemcitabine for another fixed and stained for γH2AX. Detection and analysis was performed using automated immunofluorescence micro Error bars represent the SD, n = 3. Images of γH2AX stainings are shown in (Supplemental Figure S4 A, B). G, H. h 1µM Wee1i, 5µM Chk1i or DMSO in the presence of 300nM gemcitabine for 24 h. As a control, cells were treated w Figure 1: Three checkpoint kinase inhibitors cooperate with gemcitabine to enhance cytotoxicity. A.-D. Wee1 inhibition attenuates Chk1 phosphorylation in gemcitabine-treated cells Cells were treated with 1µM Wee1i, 5µM Chk1i or DMSO in the presence of 300nM gemcitabine for 24 h. As a control, cells were treated with DMSO without gemcitabine. The cells were then processed as described in (E-F). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 13074 down p53 in U2OS cells and treated them with Wee1 inhibitor, with or without gemcitabine. Eliminating p53 led to somewhat higher levels of total Chk1, in agreement with the notion that Chk1 is negatively regulated by p53 independent of the p53 status of the cells, since both U2OS (p53 wild type) and Panc1 (p53 mutant; R273H) [17] cells showed reduced phospho-Chk1 upon Wee1 inhibition. To further rule out a role of p53, we knocked Oncotarget 13075 w impactjournals com/oncotarget dependent of the p53 status of the cells, since both 2OS (p53 wild type) and Panc1 (p53 mutant; R273H) 7] cells showed reduced phospho-Chk1 upon Wee1 hibition. To further rule out a role of p53, we knocked down p53 in U2OS cells and treated them with Wee1 inhibitor, with or without gemcitabine. Eliminating p53 led to somewhat higher levels of total Chk1, in agreement with the notion that Chk1 is negatively regulated by p53 igure 2: Inhibition of Wee1 decreases the phosphorylation of Chk1 in gemcitabine-treated cells. A, B. Panc1 and 2OS cells were treated with 1µM Wee1i or DMSO, with or without 300nM gemcitabine, for 24 h. Blots of cell lysates were stained r phosphorylation of the ATR-substrate Chk1. HSC 70 or β-Actin was stained as loading controls. C. Cells were depleted of Wee1 by ansfection with 10nM siRNA for 48h, followed by gemcitabine treatment for 24 h and immunoblot analysis as in (A, B). Scrambled RNA was used as control. D. Cells were transfected with siRNA against p53 and scrambled siRNA was used as control. After 48 h (for ch condition), cells were exposed to Wee1 inhibitor in the presence or absence of gemcitabine. 24 h later, cells were harvested and mmunoblotting was performed. β-Actin was stained as loading control. E, F. Cells were treated with Wee1i or DMSO, with or without mcitabine, in the presence or absence of the pan-caspase inhibitor Z-VAD.fmk at the indicated concentrations. After 24 h, the cells were bjected to immunoblot analysis. Figure 2: Inhibition of Wee1 decreases the phosphorylation of Chk1 in gemcitabine-treated cells. A, B. Panc1 and U2OS cells were treated with 1µM Wee1i or DMSO, with or without 300nM gemcitabine, for 24 h. Blots of cell lysates were stained for phosphorylation of the ATR-substrate Chk1. HSC 70 or β-Actin was stained as loading controls. C. Wee1 is required for sustained ATR-Rad17 signaling in gemcitabine-treated cells Besides Chk1, we also detected the phosphorylation of another ATR substrate, Rad17 (Ser645) [22] as a function of Wee1 activity. We performed quantitative immunofluorescence analysis of the phosphorylation of Rad17 upon combining the inhibition of checkpoint kinases with gemcitabine. Panc1 and U2OS cells were treated with the 1µM Wee1 inhibitor and gemcitabine for 24 h, followed by analysis of phospho-Rad17 staining intensity. The inhibition/ removal of Wee1 sharply decreased phospho-Rad17 accumulation in gemcitabine- treated cells (Figure 3A, 3B and Supplemental Figure 2B, 2C). PARP cleavage was increased when Wee1 inhibition was combined with gemcitabine, indicating caspase activity in these cells (Figure 2A-2D). To exclude that apoptosis may lead to a loss in the phosphorylation of Chk1, e.g. by general removal of phosphate groups from proteins [19] or PP2A-mediated Chk1 dephosphorylation [20, 21], we performed the treatment of the cells with gemcitabine and/or Wee1 inhibitor in the presence of Z-VAD.fmk. Analysis of the blots showed that the loss of Chk1 phosphorylation by Wee1 inhibition occurred igure 3: ATR activity is hampered upon inhibition of Wee1 in the presence of gemcitabine. A, B. Panc1 and U2OS cells ere treated with 1µM Wee1i or DMSO in the presence or absence of 300nM gemcitabine for 24 h. Cells were then fixed and stained r phosphorylated Rad17 (another ATR-substrate). Fluorescence intensities were determined by automated microscopy (BD Pathway). ror bars represent the SD, n = 3. Images of phospho-Rad17 staining are shown in (Supplemental Figure S4C, D). C, D. Panc1 and 2OS cells were treated with 1µM Wee1i or DMSO in the presence or absence of 300nM gemcitabine for 24 h. Cells were harvested and mmunoprecipitation (IP) of ATR was performed. Phosphorylated ATR (Thr1989) was stained on immunoblots (IB), in the cell lysates nput, C), and after ATR IP D. The Immunoglobulin G heavy chain (IgG-H) of the precipitating antibody was detected by the secondary antibody and shown as a loading control. Figure 3: ATR activity is hampered upon inhibition of Wee1 in the presence of gemcitabine. A, B. Panc1 and U2OS cells were treated with 1µM Wee1i or DMSO in the presence or absence of 300nM gemcitabine for 24 h. Cells were then fixed and stained for phosphorylated Rad17 (another ATR-substrate). Fluorescence intensities were determined by automated microscopy (BD Pathway). Error bars represent the SD, n = 3. Images of phospho-Rad17 staining are shown in (Supplemental Figure S4C, D). C, D. www.impactjournals.com/oncotarget Cells were depleted of Wee1 by transfection with 10nM siRNA for 48h, followed by gemcitabine treatment for 24 h and immunoblot analysis as in (A, B). Scrambled siRNA was used as control. D. Cells were transfected with siRNA against p53 and scrambled siRNA was used as control. After 48 h (for each condition), cells were exposed to Wee1 inhibitor in the presence or absence of gemcitabine. 24 h later, cells were harvested and immunoblotting was performed. β-Actin was stained as loading control. E, F. Cells were treated with Wee1i or DMSO, with or without gemcitabine, in the presence or absence of the pan-caspase inhibitor Z-VAD.fmk at the indicated concentrations. After 24 h, the cells were subjected to immunoblot analysis. www.impactjournals.com/oncotarget Oncotarget 13075 independent of caspase activation (Figure 2E, 2F). Thus, active caspases are not required for this impairment of the ATR/Chk1 signaling axis. [18]. Chk1 phosphorylation was induced by gemcitabine, regardless of the p53-knockdown. Importantly, however, the phosphorylation of Chk1 was still reduced when gemcitabine-treated cells were additionally incubated with a Wee1 inhibitor, regardless of the p53 knockdown (Figure 2D). We conclude that the inhibition or removal of Wee1 hampers the Chk1 signaling pathway and leads to diminished activation of Chk1 in cells that are undergoing replicative stress. Wee1 is required for sustained ATR-Rad17 signaling in gemcitabine-treated cells Panc1 and U2OS cells were treated with 1µM Wee1i or DMSO in the presence or absence of 300nM gemcitabine for 24 h. Cells were harvested and immunoprecipitation (IP) of ATR was performed. Phosphorylated ATR (Thr1989) was stained on immunoblots (IB), in the cell lysates (Input, C), and after ATR IP D. The Immunoglobulin G heavy chain (IgG-H) of the precipitating antibody was detected by the secondary IB antibody and shown as a loading control. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 13076 ATR (Thr1989). Phospho-ATR levels, as expected, were increased upon gemcitabine treatment, but when gemcitabine was combined with the Wee1 inhibitor, the levels of ATR phosphorylation were reduced (Figure 3C, 3D), suggesting impaired activity of ATR. These results suggest that Wee1 activity sustains the activation of ATR pathway upon induction of DNA damage by gemcitabine. To address whether Wee1 inhibition leads to the inactivation of ATR, we detected ATR phosphorylation at Thr-1989; phosphorylation of this site has earlier been described as a marker of ATR activity [23]. Upon treatment of cells with Wee1 inhibitor and/or gemcitabine, ATR was immunoprecipitated to concentrate this protein and then immunoblotted to detect phospho- Oncotarget 13077 actjournals com/oncotarget nt of cells with Wee1 inhibitor and/or gemcitabine, was immunoprecipitated to concentrate this and then immunoblotted to detect phospho- 3D), suggesting impaired activity of ATR. These results suggest that Wee1 activity sustains the activation of ATR pathway upon induction of DNA damage by gemcitabine. 4: Cdks mediate the attenuation of the ATR-Chk1 pathway by Wee1 inhibition. A. Panc1 and U2OS cells were with Wee1i or DMSO, with or without gemcitabine, in the presence or absence of Roscovitine (an inhibitor of Cdk1, 2 and 5) at the d concentrations for 24 h. Blots of the cell lysates were stained for phosphorylation of the ATR substrate Chk1. HSC 70 or β-Actin ned as loading control. B, C. Panc1 and U2OS cells were treated as mentioned in (A). Blots of the cell lysates were stained for rylation of the ATR. HSC 70 was stained as loading control. D. Panc1 and U2OS cells were treated with Wee1i or DMSO, with ut gemcitabine, in the presence or absence of RO-3306 (a Cdk1 inhibitor) at the indicated concentrations for 24 h. Cells were d and processed as in (A). Figure 4: Cdks mediate the attenuation of the ATR-Chk1 pathway by Wee1 inhibition. A. Panc1 and U2OS cells were treated with Wee1i or DMSO, with or without gemcitabine, in the presence or absence of Roscovitine (an inhibitor of Cdk1, 2 and 5) at the indicated concentrations for 24 h. Blots of the cell lysates were stained for phosphorylation of the ATR substrate Chk1. HSC 70 or β-Actin was stained as loading control. B, C. Panc1 and U2OS cells were treated as mentioned in (A). Blots of the cell lysates were stained for phosphorylation of the ATR. HSC 70 was stained as loading control. Wee1 inhibition impairs ATR-Chk1 signaling activity through Cyclin-dependent kinases also restored Chk1 phosphorylation upon simultaneous knock down of Wee1 in the presence of gemcitabine (Supplemental Figure 2D, 2E). In conclusion, Cdk1 is specifically required for inactivating the ATR-Chk1 pathway upon Wee1 inhibition. Wee1 directly phosphorylates and inhibits Cdk1 and Cdk2 at the conserved Tyr15 residue [24]. Thus, Wee1 inhibition can lead to Cdk1/2 activation. To test whether the impairment of the ATR-Chk1 pathway by Wee1 inhibition is due to Cdk activation, we inhibited Cdks using Roscovitine, along with Wee1 inhibition and gemcitabine exposure. Western blot analysis showed rescue of decreased Chk1 as well as ATR phosphorylation when Cdks were inhibited in gemcitabine-treated cells, despite the presence of Wee1 inhibitor (Figure 4A-4C). These findings imply that the inactivation of the ATR/ Chk1 pathway is mediated through Cdks upon Wee1 inhibition. Functional inactivation of the Retinoblastoma protein (also referred as pRb) has been found to be controlled by distinct Cyclin-cdk complexes, namely Cyclin D-Cdk4/6, Cyclin E-Cdk2 and Cyclin A-Cdk2/1 [27]. As Cdks negatively regulate pRb, we tested whether pRb might be involved in maintaining the activation of the ATR signaling pathway, e.g. through E2F-mediated transcription of ATR and/or its signaling intermediates. However, the mRNA levels of ATR did not significantly change upon knockdown of Wee1 (Supplemental Figure 2F). Moreover, we analyzed the effects of Wee1 inhibition in Hela cells that contain the E7 protein from human papilloma virus 18, which can bind and inactivate pRb [28]. We treated this cell line with Cdk inhibitor, Wee1 inhibitor, and gemcitabine, alone or in combinations. We observed that even in Hela cells, Cdk inhibition could rescue the phosphorylation of Chk1 (Supplemental Figure 2G). This suggests that Wee1 inhibition interferes with ATR/Chk1 activity through Cdk1, but independently of pRb. Roscovitine is a potent inhibitor of Cdks and binds competitively to the ATP binding domain of these kinases [25]. To further specify the Cdk(s) involved, we used a selective inhibitor of Cdk1, RO-3306. This inhibitor has nearly 10-fold selectivity for Cdk1, as compared to Cdk2 [26]. We found that RO-3306, when combined with Wee1 inhibition and gemcitabine, could restore the phosphorylation of Chk1 (Figure 4D). In line with these observations, the removal of Cdk1 by siRNAs Figure 5: Targeting Plk1 rescues ATR-Chk1 activity in the context of Wee1 inhibition. A, B. www.impactjournals.com/oncotarget D. Panc1 and U2OS cells were treated with Wee1i or DMSO, with or without gemcitabine, in the presence or absence of RO-3306 (a Cdk1 inhibitor) at the indicated concentrations for 24 h. Cells were harvested and processed as in (A). www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 13077 Wee1 inhibition impairs ATR-Chk1 signaling activity through Cyclin-dependent kinases Panc1 and U2OS cells were treated with combinations of Wee1i, gemcitabine, and the Plk1 inhibitor, GSK 461364 (referred to as Plk1i) at 100nM for 24 h, followed by immunoblot analysis. C, D. Panc1 and U2OS cells were treated with Wee1i, Plk1i, and/or Roscovitine, in the presence of gemcitabine at the indicated concentrations for 8 h. Immunoblots were stained for phosphorylation of Plk1 (Thr210), an indicator of Plk1 activity. Figure 5: Targeting Plk1 rescues ATR-Chk1 activity in the context of Wee1 inhibition. A, B. Panc1 and U2OS cells were treated with combinations of Wee1i, gemcitabine, and the Plk1 inhibitor, GSK 461364 (referred to as Plk1i) at 100nM for 24 h, followed by immunoblot analysis. C, D. Panc1 and U2OS cells were treated with Wee1i, Plk1i, and/or Roscovitine, in the presence of gemcitabine at the indicated concentrations for 8 h. Immunoblots were stained for phosphorylation of Plk1 (Thr210), an indicator of Plk1 activity. Plk1 activation and Claspin/CtIP reduction precede the inactivation of ATR/Chk1 upon Wee1 inhibition In order to understand the chronological order of the events regulating ATR/Chk1 activity, we treated Panc1 and U2OS cells with the Wee1 inhibitor and/or gemcitabine and harvested at different time points for immunoblot analysis. We observed that inhibition of Wee1 in gemcitabine-treated cells initially activated Plk1, reduced Claspin levels and altered the electrophoretic mobility of CtIP (compatible with a posttranslational modification). At a later time, the phosphorylations of ATR and Chk1 were reduced (Figure 8A; cf. Figure 2A and 2B, for 24 h treatment results). This sequence of phosphorylation events is compatible with a model depicted in Figure 8B, reaching from Wee1 inhibition through Cdk and Plk1 activation to a reduction in the activating phosphorylations of ATR and Chk1. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 13078 Wee1 inhibition diminishes levels of CtIP in a Cdk-dependent manner, and this hampers ATR activation upon replicative stress Plk1 activation and Claspin/CtIP reduction precede the inactivation of ATR/Chk1 upon Wee1 inhibition Upon Wee1 inhibition, Plk1 mediates inactivation of Chk1 through reduction in the levels of Claspin protein Next, we investigated whether Wee1 inhibition diminishes Chk1 activity by altering levels of Claspin, a cofactor of Chk1 activation. We determined the levels of Claspin while inhibiting Wee1 as well as Plk1 or Cdks. Indeed, Claspin levels were decreased upon Wee1 inhibition in the presence of gemcitabine, but the original amount of Claspin was restored when inhibitors of Plk1 or Cdks were added (Figure 6A, 6B). We further observed that the decrease in the protein levels of Claspin was due to proteasomal degradation, since exposure to MG132 (a proteasome inhibitor) could reinstate the normal amount of this protein (Supplemental Figure 3A). At the same time, mRNA levels of Claspin did not change significantly upon Wee1 inhibition (Supplemental Figure 3B, 3C). Moreover, the siRNA-mediated removal of Claspin reduced Chk1 phosphorylation as well (Figure 6C, 6D). The knockdown efficiency of siRNAs was determined using immunoblot analysis (Supplemental Figure 3D). Thus, the reduction of Claspin occurs through Cdks and Plk1, and it contributes to the attenuation of Chk1 activity upon Wee1 inhibition. Wee1 inhibition diminishes levels of CtIP in a Cdk-dependent manner, and this hampers ATR activation upon replicative stress The yeast homolog of Polo-like kinase 1 (Plk1), cdc5, is activated by the Cdk1 homolog, cdc28, in budding yeast [29, 30]. On the other hand, Plk1 is known to down- regulate the ATR/Chk1 pathway at different levels. Plk1 phosphorylates Claspin and marks it for degradation by SCFbetaTrCP, thereby restraining Chk1 activation [31] [32]. Furthermore, Plk1 interferes with CtIP activity [33]. To investigate the role of Plk1 in the negative regulation of ATR/Chk1 activity, we incubated cells with a Plk1 inhibitor (GSK 461364) or siRNA against Plk1, in the presence of the Wee1 inhibitor and gemcitabine; through immunoblot analysis, it was found that the inhibition or removal of Plk1 could recover the phosphorylation of Chk1 (Figure 5A, 5B and Supplemental Figure 2H, 2I). Hence, Plk1 activity is required for the attenuation of ATR/Chk1 signaling upon Wee1 inhibition. The CtIP protein can act as a cofactor in ATR activation [35]. On the other hand, at least in budding yeast, CtIP has been found regulated by Plk1 [33], suggesting that Plk1 may govern ATR activity through CtIP. To test this, we determined the levels of CtIP upon Wee1 inhibition in the presence of gemcitabine. Indeed, CtIP levels decreased when Wee1 was inhibited in gemcitabine-treated cells. This was found by immunofluorescence (Figure 7A, 7B) as well as immunoblot analysis (Figure 7C, 7D). The decrease in CtIP levels could be rescued by simultaneous inhibition of Cdks through Roscovitine (Figure 7C, 7D), suggesting a role of Cdks in the reduction of CtIP levels. We further tested if removal of CtIP was sufficient to attenuate ATR activation in this context. Knockdown of CtIP using siRNAs in the presence of gemcitabine decreased ATR activation (Figure 7E). Thus, CtIP is indeed required to maintain the activity of ATR. In conclusion, the decrease in CtIP in response to Wee1 inactivation contributes to the impairment of ATR activity. To validate the activation of Plk1 upon Wee1 inhibition, and its dependence on Cdks, we performed western blot analysis to detect the phosphorylation of Plk1 at Thr210, a hallmark of Plk1 activation [34]. Phosphorylated Plk1 (Thr210) increased with Wee1 inhibition, but this phosphorylation vanished when inhibitors of Plk1 or Cdks were added (Figure 5C, 5D). We conclude that Plk1 activity is increased upon Wee1 inhibition in the presence of gemcitabine, and that this activation is a necessity for impeding the ATR/Chk1 pathway. Discussion The Wee1 inhibitor, MK-1775, sensitizes tumor cells towards gemcitabine with particular efficiency, even when compared to inhibitors of ATR and Chk1. MK-1775 increased H2AX phosphorylation and markedly reduced long-term survival of the cells. Mechanistic analyses then revealed that Wee1 signaling is epistatic in relation to ATR/Chk1 activity in gemcitabine-treated cells. www.impactjournals.com/oncotarget Oncotarget 13079 Oncotarge 13080 w.impactjournals.com/oncotarget Figure 6: Reduced Claspin levels interfere with Chk1 activation. A, B. Panc1 and U2OS cells were treated with Wee1i, Plk1 nd/or Roscovitine, in the presence of gemcitabine, for 8 h. Blots were stained for total levels of Claspin. HSC 70 was stained as loading ontrol. C, D. Claspin was knocked down by transfecting the cells with 10nM siRNAs for 48 h, followed by treatment with 300nM emcitabine. The cells were harvested at 0 h, 6 h, 10 h and 12 h after gemcitabine addition. Immunoblots were stained for Chk1 and Rad17 hosphorylation. β-Actin was stained as a loading control. Figure 6: Reduced Claspin levels interfere with Chk1 activation. A, B. Panc1 and U2OS cells were treated with Wee1i, Plk1i and/or Roscovitine, in the presence of gemcitabine, for 8 h. Blots were stained for total levels of Claspin. HSC 70 was stained as loading control. C, D. Claspin was knocked down by transfecting the cells with 10nM siRNAs for 48 h, followed by treatment with 300nM gemcitabine. The cells were harvested at 0 h, 6 h, 10 h and 12 h after gemcitabine addition. Immunoblots were stained for Chk1 and Rad17 phosphorylation. β-Actin was stained as a loading control. www.impactjournals.com/oncotarget Oncotarget 13080 gure 7: Reduction in CtIP protein levels attenuates ATR activation. A, B. Panc1 cells were treated with combinations of ee1 inhibitor and gemcitabine for 24 h. The cells were fixed and stained for CtIP by immunofluorescence. Images were taken using nfocal microscopy A. Quantitative analysis was done by evaluating at least 100 cells per sample B. Error bars represent the SD. Scale r represents 20µm. C, D. Panc1 and U2OS cells were treated with combinations of Wee1 inhibitor, Roscovitine and gemcitabine for 24 h ots were stained for CtIP. HSC 70 was stained as a loading control. E. Panc1 and U2OS cells were transfected with two different siRNAs ainst CtIP and negative control siRNA. After 48 h, cells were treated with 300nM gemcitabine and harvested at 24 h after gemcitabine dition. Immunoblots were stained for ATR phosphorylation. Discussion HSC 70 was used as a loading control. In the figure, immunoblots with and Figure 7: Reduction in CtIP protein levels attenuates ATR activation. A, B. Panc1 cells were treated with combinations of Wee1 inhibitor and gemcitabine for 24 h. The cells were fixed and stained for CtIP by immunofluorescence. Images were taken using confocal microscopy A. Quantitative analysis was done by evaluating at least 100 cells per sample B. Error bars represent the SD. Scale bar represents 20µm. C, D. Panc1 and U2OS cells were treated with combinations of Wee1 inhibitor, Roscovitine and gemcitabine for 24 h. Blots were stained for CtIP. HSC 70 was stained as a loading control. E. Panc1 and U2OS cells were transfected with two different siRNAs against CtIP and negative control siRNA. After 48 h, cells were treated with 300nM gemcitabine and harvested at 24 h after gemcitabine addition. Immunoblots were stained for ATR phosphorylation. HSC 70 was used as a loading control. In the figure, immunoblots with and without gemcitabine for each cell line belong to the same blot. Figure 7: Reduction in CtIP protein levels attenuates ATR activation. A, B. Panc1 cells were treated with combinations of Wee1 inhibitor and gemcitabine for 24 h. The cells were fixed and stained for CtIP by immunofluorescence. Images were taken using confocal microscopy A. Quantitative analysis was done by evaluating at least 100 cells per sample B. Error bars represent the SD. Scale bar represents 20µm. C, D. Panc1 and U2OS cells were treated with combinations of Wee1 inhibitor, Roscovitine and gemcitabine for 24 h. Blots were stained for CtIP. HSC 70 was stained as a loading control. E. Panc1 and U2OS cells were transfected with two different siRNAs against CtIP and negative control siRNA. After 48 h, cells were treated with 300nM gemcitabine and harvested at 24 h after gemcitabine addition. Immunoblots were stained for ATR phosphorylation. HSC 70 was used as a loading control. In the figure, immunoblots with and without gemcitabine for each cell line belong to the same blot. www.impactjournals.com/oncotarget Oncotarget 13081 Thus, we observed attenuation of the ATR/ Chk1 pathway upon Wee1 inhibition. This provides an attractive explanation for the observed increase in the DNA damage response when combining gemcitabine with a Wee1 inhibitor. ATR and Chk1 activity, at least in general, attenuate replicative stress [36]. Discussion Therefore, if Wee1 inhibition impairs ATR/Chk1 activity, the expected consequence is that replicative stress is enhanced, especially in the presence of a false-incorporated nucleoside analogue. In the absence of sufficient ATR/ Chk1 activity, DNA replication forks tend to stall and eventually collapse [34, 37]. In such a scenario, the intermediates of incomplete DNA replication trigger a DNA damage response, e.g. through activation of ATM and/or DNA-PK. As a result, phosphorylated H2AX accumulates and cell survival is impaired. We therefore propose that attenuated ATR/Chk1 represents at least one of the reasons why Wee1 inhibitors can synergize with a number of chemotherapeutics to trigger cancer cell death [38]. Oncotarget 13082 w impactjournals com/oncotarget onsequence is that replicative stress is enhanced, specially in the presence of a false-incorporated ucleoside analogue. In the absence of sufficient ATR/ of the reasons why Wee1 inhibitors can synergize with a number of chemotherapeutics to trigger cancer cell death [38]. Figure 8: Kinetics of ATR/Chk1 attenuation upon Wee1 inhibition. A. Panc1 and U2OS cells were treated with 1µM Wee1i or DMSO, with or without 300nM gemcitabine, for 4, 8 or 12 h. Blots of cell lysates were stained for phospho-ATR, Claspin, CtIP, phospho- lk1, phospho-Chk1 and γH2AX, HSC 70 or β-Actin was stained as loading controls. B. Schematic representation of the mechanisms by hat Wee1 inhibition impairs ATR-Chk1 signaling, as suggested by the results of our study. Inhibition of Wee1 activates Cdks, which in turn ncreases the activity of Plk1. Plk1 destabilizes Claspin and thereby impairs Chk1 activity. Cdks also mediate a reduction in CtIP levels, hus attenuating ATR activation and further contributing to the loss in Chk1 activation. Figure 8: Kinetics of ATR/Chk1 attenuation upon Wee1 inhibition. A. Panc1 and U2OS cells were treated with 1µM Wee1i or DMSO, with or without 300nM gemcitabine, for 4, 8 or 12 h. Blots of cell lysates were stained for phospho-ATR, Claspin, CtIP, phospho- Plk1, phospho-Chk1 and γH2AX, HSC 70 or β-Actin was stained as loading controls. B. Schematic representation of the mechanisms by that Wee1 inhibition impairs ATR-Chk1 signaling, as suggested by the results of our study. Inhibition of Wee1 activates Cdks, which in turn increases the activity of Plk1. Plk1 destabilizes Claspin and thereby impairs Chk1 activity. Cdks also mediate a reduction in CtIP levels, thus attenuating ATR activation and further contributing to the loss in Chk1 activation. Figure 8: Kinetics of ATR/Chk1 attenuation upon Wee1 inhibition. A. Discussion Panc1 and U2OS cells were treated with 1µM Wee1i or DMSO, with or without 300nM gemcitabine, for 4, 8 or 12 h. Blots of cell lysates were stained for phospho-ATR, Claspin, CtIP, phospho- Plk1, phospho-Chk1 and γH2AX, HSC 70 or β-Actin was stained as loading controls. B. Schematic representation of the mechanisms by that Wee1 inhibition impairs ATR-Chk1 signaling, as suggested by the results of our study. Inhibition of Wee1 activates Cdks, which in turn increases the activity of Plk1. Plk1 destabilizes Claspin and thereby impairs Chk1 activity. Cdks also mediate a reduction in CtIP levels, thus attenuating ATR activation and further contributing to the loss in Chk1 activation. www.impactjournals.com/oncotarget www.impactjournals.com/oncotarget Oncotarget 13082 Regulation of Wee1 by Chk1 has been studied, revealing that Chk1 phosphorylates Wee1 to inhibit Cdc2 phosphorylation at Tyr15 [39]. Vice versa, however, it has hitherto not been known whether and how Wee1 supports ATR signaling. Since Wee1 sustains Chk1 activity upon replicative stress (our study), whereas Chk1 diminishes Wee1 activity [39], it is tempting to speculate that a negative feedback loop limits the activation of Chk1 by Wee1. number of studies still found cooperative effects when using inhibitors of Chk1 and Wee1 simultaneously for cancer treatment [22, 45]. We propose that the reason for this cooperativity might consist in the timing of the enzymatic activities. For optimum sensitization, it may be advantageous to block Chk1 immediately when cells are exposed to nucleoside analogues. In any case, however, our observations suggest that Chk1 and ATR are eventually attenuated by Wee1-inhibitors alone in the context of gemcitabine treatment. Our study revealed that Cdks are required for negatively regulating the ATR/Chk1 pathway upon Wee1 inhibition. This is conceivable since Wee1, when active, mediates an inhibitory phosphorylation on Cdk1/2. But how would enhanced Cdk activity attenuate ATR/ Chk1 signaling? Our results show that this is mediated through Plk1. In human cells, Cdk1 has been reported to ‘prime’ the Plk1 substrates by phosphorylating them. The ‘primed’ substrates, e.g. Vimentin, are then recognized and phosphorylated by Plk1 [40]. In S. cerevisiae, Cdk1 has been proposed to maintain the stability of Plk1 by phosphorylation at Thr23 [30], but it is currently unknown whether such a mechanism exists in the human system as well. However, we observed that phosphorylation of Plk1 at Thr210, a marker for its activation [34], increases upon Wee1 inhibition in gemcitabine-treated cells. It thus appears conceivable that Cdk activity may support Plks by more than one mechanism, enhancing its general activity as well as priming specific substrates. Once activated, we propose that Plk1 attenuates ATR/Chk1 signaling. Plk1 has been implicated in the phosphorylation and subsequent degradation of Claspin, thereby preventing the activation of Chk1 in response to replicative stress [31, 39, 32, 40]. Wee1 inhibitors represent promising anti-cancer drug candidates [11, 46] and are currently being tested in clinical trials of phases I and II (NCI Clinical Trials). Our results strongly suggest that Wee1 inhibition eliminates cancer cells not only by premature activation of chromosome separation [13] but also by enhancing replicative stress through impairment of ATR/Chk1 signaling. Culturing of human cancer cell lines Panc1 (human pancreatic epithelioid carcinoma) and U2OS (human osteosarcoma) cells were cultured in DMEM (Gibco, Life Technologies) with 10% FCS (Gibco, Life Technologies), 200µM L-glutamine (Gibco, Life Technologies) and antibiotics – 50U/ml Penicillin and Streptomycin (Gibco, Life Technologies), 20µg/ml Tetracycline (Gibco, Life Technologies) and 10µg/ml Ciprofloxacin (Bayer). The removal of Claspin provides an explanation for attenuated Chk1, but not for diminished ATR activity upon Wee1 inhibition. In recent studies, Wee1 inhibition has been demonstrated to impair homologous recombination [43, 44] and CtIP plays a key role in this mode of DNA repair [45]. In agreement with these findings, we observed that CtIP was degraded when Wee1 was inhibited in gemcitabine-treated cells. CtIP is phosphorylated by Cdks [46] and, at least in yeast, also by Plk1 [33]. This phosphorylation mediates binding of the peptidyl-prolyl isomerase Pin1 to CtIP. Pin1-catalyzed isomerization of CtIP facilitates the degradation of the latter [47]. Moreover, Plk1 phosphorylates and stabilizes Pin1 [47]. Therefore, we propose that upon inhibition of Wee1, hyperactive Cdks phosphorylate CtIP, while activated Plk1 stabilizes Pin1, which together facilitates proteasomal degradation of CtIP. On the other hand, CtIP is required for sustained ATR/Chk1 signaling and for keeping up the intra-S phase checkpoint [48]. As a net result, Wee1 inhibition attenuates the activities of ATR and Chk1. Taken together, our analyses reveal a pathway that leads from Wee1 inhibition to the impairment of ATR and Chk1 in the context of replicative stress (Figure 8B). Despite the attenuation of Chk1 by Wee1 inhibition, a www.impactjournals.com/oncotarget This unique combination of cytotoxic mechanisms, triggered through a single target, provides an attractive explanation for the remarkable cytotoxic efficacy of Wee1 inhibitors. Immunofluorescence analysis For immunofluorescence microscopy, the automated microscope Pathway 855 (Becton Dickinson, Franklin Lakes, NJ, United States) was used to read fluorescence intensity in 96-well plates. For confocal microscopy, LSM 510 laser scanning microscope (Carl Zeiss, Germany) was used. Transfection of cells with siRNA, and inhibitor treatment To knock down genes of interest, reverse transfection was performed in 6-well plates with 10nM siRNA and Lipofectamine 2000 (Life Technologies). Cells were either harvested or treated with chemicals after 48 h. siRNAs to Wee1 (s21, silencer select), Claspin #1 (s34330, silencer select), Claspin #2 (s34331, silencer select), CtIP #1 (s11849, silencer select), CtIP #2 (s11851, silencer select), p53 (s605, silencer select), Wee1 (404, silencer), Cdk1 #1 (s464, silencer select), Cdk1 #2 (s465, silencer select), Plk1 (s449, silencer select) and Negative Control No.1 siRNA (silencer select, silencer) were obtained from Ambion, Life Technologies. The following chemical inhibitors were used: Wee1 inhibitor MK-1775 (Selleckchem), ATR inhibitor VE-821 (Selleckchem), Chk1 inhibitor SB 218078 (Calbiochem, Merck), Cdk1, 2 and 5 inhibitor Roscovitine (Cell Signaling), Cdk1 www.impactjournals.com/oncotarget Oncotarget 13083 inhibitor RO-3306 (Sigma Aldrich). Kerafast), PARP (9542, Cell Signaling Technology), total Rad17 (sc-17761, Santa Cruz Biotechnology), total Chk1 (2360, Cell Signaling Technology), total Cdk1 (9116, Cell Signaling Technology), total ATR (sc-1887, Santa Cruz Biotechnology), HSC 70 (sc-7298, Santa Cruz Biotechnology), Wee1 (4936, Cell Signaling Technology), beta-Actin (ab6276-100, abcam), Claspin (2800, Cell Signaling Technology), phosphorylated Thr 210 Plk1 (558400, BD Pharmigen), total Plk1 (37-7000, Life Technologies), CtIP (61142, Active Motif), phospho-H3 (3377, Cell signaling). Secondary antibodies coupled to horseradish peroxidase (Jackson Immunoresearch) were used for chemiluminescent detection (Millipore). CellTiter-Glo® luminescent cell viability assay Cells were seeded in a 10 cm petri dish (8 x 105 cells per dish). 24 h after seeding, the cells were treated with the indicated inhibitors in the presence or absence of gemcitabine for 24 h. Protease inhibitors (complete (mini) inhibitor mix from Roche) and phosphatase inhibitors (10mM NaF, 2mM Na-pyrophosphate, 1mM Na-orthovanadate) were added to the IP-lysis buffer (50mM Tris- HCl, pH 7.5, 300mM NaCl, 1% NP-40, 0.1% Na-deoxycholate) just before its use. Cells in IP- lysis buffer were scraped off the plate and transferred to an Eppendorf tube, followed by homogenization with a 26G syringe, sonication and centrifugation. 2 µg of antibody was added and incubated overnight at 4°C on a rotor. 30 µl of equilibrated Protein G sepharose beads were put in the lysates and incubated 1 h at 4°C. After 5 washes in 800 µl IP-lysis buffer, 30µl of 6 X Laemmli buffer was added to the pellet and boiled at 95°C for 5 min. The samples were subjected to SDS-PAGE and immunoblot analysis. This assay (Promega) was performed to determine the amount of metabolically active cells present in a culture. It is based on the activity of luciferase, which uses ATP from cells to generate a luminescent signal, quantified by a DLReady™Centro LB 960 luminometer. Cells were seeded in opaque-walled 96-well plates (3000 cells per well) and exposed to drugs after 24h. 72 hours later, cell lysates were prepared and luminescence was recorded. Preparation of whole cell lysates for SDS-PAGE Cells were seeded in 6-well plates (1.6 x 105 cells per well) for the drug treatment. Cell lysates were prepared on ice. The cells were scraped off into the medium and pelleted by centrifugation at 1500xg for 3 min at 4°C, followed by one wash in PBS. The cells were resuspended in 100µl RIPA lysis buffer (1% Triton X, 1% Desoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM EDTA, 20 mM Tris-HCl pH 7.5, 100.000KIE Aprotinin) freshly supplemented with 2M urea, 1mg/ml leupeptine/ aprotinine, 0.1M pepstatin A, 0.1M pefabloc. After 20 min of shaking at 4°C, the lysates were centrifuged at 15,700xg for 10min. Bicinchoninic acid (BCA) assay was used to normalize the concentration of proteins in the supernatant. The samples were then boiled with Laemmli buffer, followed by SDS-PAGE. Immunoprecipitation was performed to concentrate ATR using the anti-ATR (N-19) antibody from Santa Cruz and then immunoblotted to determine the levels of phospho-ATR (T1989) (Kerafast). Cell proliferation assay To track cell proliferation, the Celigo cell cytometer (Cyntellect, San Diego, CA, United States) was used; the confluency of the cells was measured by transmission microscopy. Cells were seeded in 96- well plates (5000 cells per well). After 24 h, the confluency of the cells was measured (labeled as Day 0), followed by treatment with 0.5µM MK-1775 / 2.5µM SB 218078 / 5µM VE-821 without or with gemcitabine at the indicated concentrations. After 24 h, all the drugs were removed. Subsequent measurements of cell confluency were made after every 24 h, and media was changed every 48 h. References Cells were seeded in 6-well plates and treated with the Wee1 inhibitor in the presence or absence of gemcitabine. After fixation in ethanol, the cells were washed in wash solution (0.05% Triton-X in PBS), followed by incubation in staining solution (2% FCS, 0.2% Triton-X in PBS) with phospho-H3 antibody (3377, Cell signaling) for 2 h and then with secondary antibody (Alexa-Fluor 488) for one hour. Subsequently, the cells were resuspended in 0.5 mg/ml RNAse A solution andincubated for 30 min at 37°C. Directly before measurement, propidium iodide (final concentration: 30 µg/ml) was added. Samples were measured using a FACS machine Guava PCA-96 Base System (Millipore, Merck, Darmstadt, Germany). 1. Burris HA, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, Cripps MC, Portenoy RK, Storniolo AM, Tarassoff P, Nelson R, Dorr FA, Stephens CD, Von Hoff DD. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol Off J Am Soc Clin Oncol. 1997 Jun;15:2403–13. 1. Burris HA, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, Cripps MC, Portenoy RK, Storniolo AM, Tarassoff P, Nelson R, Dorr FA, Stephens CD, Von Hoff DD. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol Off J Am Soc Clin Oncol. 1997 Jun;15:2403–13. 2. Albain KS, Nag SM, Calderillo-Ruiz G, Jordaan JP, Llombart AC, Pluzanska A, Rolski J, Melemed AS, Reyes-Vidal JM, Sekhon JS, Simms L, O’Shaughnessy J. Gemcitabine Plus Paclitaxel Versus Paclitaxel Monotherapy in Patients With Metastatic Breast Cancer and Prior Anthracycline Treatment. J Clin Oncol. 2008 Aug 20;26:3950–7. Western blot analysis The cells were fixed in 3.7% paraformaldehyde for 20 min, followed by permeabilization with 0.5% triton-X in PBS for 15 min and blocking for 15 min using blocking solution (3% BSA in PBS). The primary antibody to phospho-H2AX (05-636, Millipore)/ phospho-Rad17 (6981, Cell Signaling Technology)/ CtIP (61142, Active Motif), diluted in blocking solution, was added for 1 h, followed by incubation with a secondary antibody (Alexa- Blots on nitrocellulose or PVDF membranes were stained with the following antibodies. phosphorylated Ser 139 H2AX (05-636, Millipore), phosphorylated Ser 317 Chk1 (2344, Cell Signaling Technology), phosphorylated Ser 645 Rad17 (6981, Cell Signaling Technology), phosphorylated Tyr 15 Cdk1 (ab47594, abcam), phosphorylated Thr 1989 ATR (EVU001, www.impactjournals.com/oncotarget Oncotarget 13084 Fluor 546/488) and Hoechst 33342 (Invitrogen) diluted in blocking solution for 45 min. Cancer Aid. P. S. was a PhD student of the Göttingen Graduate School for Neurosciences, Biophysics, and Molecular Biosciences (GGNB). Images were captured and analyzed using the BD Pathway software, wherein the region of interest (ROI), in this case the cell nuclei, were defined by Hoechst stain, and the average intensity of the antibody-coupled fluorescence within each ROI was determined. Statistical analysis 5. Ferlay J, Shin H-R, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer J Int Cancer. 2010 Dec 15;127:2893–917. Statistical significance was determined using the unpaired, two-tailed Student’s T-test. Significance was assumed for p-values below 0.05. Asterisks in figures indicate resulting p-values as follows: p < 0.05, p < 0.01, **p < 0.001. n.s. = not significant. n in figure legends indicates the number of independent experiments. 6. Moore MJ, Hamm J, Dancey J, Eisenberg PD, Dagenais M, Fields A, Hagan K, Greenberg B, Colwell B, Zee B, Tu D, Ottaway J, Humphrey R, Seymour L, National Cancer Institute of Canada Clinical Trials Group. Comparison of gemcitabine versus the matrix metalloproteinase inhibitor BAY 12-9566 in patients with advanced or metastatic adenocarcinoma of the pancreas: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol Off J Am Soc Clin Oncol. 2003 Sep 1;21:3296– 302. Conflicts of interest The authors disclose no potential conflicts of interest. Reverse transcription and real time quantitative polymerase chain reaction (qRT-PCR) 3. Von der Maase H, Hansen SW, Roberts JT, Dogliotti L, Oliver T, Moore MJ, Bodrogi I, Albers P, Knuth A, Lippert CM, Kerbrat P, Sanchez Rovira P, Wersall P, Cleall SP, Roychowdhury DF, Tomlin I, Visseren-Grul CM, Conte PF. Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer: results of a large, randomized, multinational, multicenter, phase III study. J Clin Oncol Off J Am Soc Clin Oncol. 2000 Sep;18:3068–77. Total RNA from human cells was isolated. Reverse transcription was performed using M-MuLV Reverse transcriptase (New England Biolabs) and a mixture of anchored dT primers (dT23VN) and random nonamers. Quantitative PCR was carried out using thermostable Taq DNA polymerase (Fermentas, Thermo Scientific) in the presence of Sybr green. Fluorescence intensities were measured to determine the Ct values. The relative concentrations of mRNAs were calculated by the 2-∆∆Ct method, using GAPDH or 36B4 mRNAs as references. 4. Sandler AB, Nemunaitis J, Denham C, von Pawel J, Cormier Y, Gatzemeier U, Mattson K, Manegold C, Palmer MC, Gregor A, Nguyen B, Niyikiza C, Einhorn LH. Phase III trial of gemcitabine plus cisplatin versus cisplatin alone in patients with locally advanced or metastatic non-small- cell lung cancer. J Clin Oncol Off J Am Soc Clin Oncol. 2000 Jan;18:122–30. Acknowledgments We are grateful to Dr. Norman Erytch for providing Plk1 inhibitor and Phospho-Plk1 antibody. This work was supported by the Wilhelm Sander Stiftung, the German José Carreras Leukemia foundation, and the German www.impactjournals.com/oncotarget Oncotarget 13085 in human lymphoid tumor lines. J Immunol. 1992 Mar 15;148:1949–54. 7. Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer. 2009 Jan ;9:28–39. 20. Leung-Pineda V, Ryan CE, Piwnica-Worms H. Phosphorylation of Chk1 by ATR Is Antagonized by a Chk1-Regulated Protein Phosphatase 2A Circuit. Mol Cell Biol. 2006 Oct 15;26:7529–38. 8. Ashwell S, Zabludoff S. DNA Damage Detection and Repair Pathways—Recent Advances with Inhibitors of Checkpoint Kinases in Cancer Therapy. Clin Cancer Res. 2008 Jul 1;14:4032–7. 21. Santoro MF, Annand RR, Robertson MM, Peng YW, Brady MJ, Mankovich JA, Hackett MC, Ghayur T, Walter G, Wong WW, Giegel DA. Regulation of protein phosphatase 2A activity by caspase-3 during apoptosis. J Biol Chem. 1998 May 22;273:13119–28. 9. Prevo R, Fokas E, Reaper PM, Charlton PA, Pollard JR, McKenna WG, Muschel RJ, Brunner TB. The novel ATR inhibitor VE-821 increases sensitivity of pancreatic cancer cells to radiation and chemotherapy. Cancer Biol Ther. 2012 Sep 1;13:1072–81. 22. Bao S, Tibbetts RS, Brumbaugh KM, Fang Y, Richardson DA, Ali A, Chen SM, Abraham RT, Wang X-F. ATR/ ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses. Nature. 2001 Jun 21; 411:969–74. 10. Zabludoff SD, Deng C, Grondine MR, Sheehy AM, Ashwell S, Caleb BL, Green S, Haye HR, Horn CL, Janetka JW, Liu D, Mouchet E, Ready S, Rosenthal JL, Queva C, Schwartz GK, Taylor KJ, Tse AN, Walker GE, White AM. AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies. Mol Cancer Ther. 2008 Sep 1;7:2955–66. 23. Nam EA, Zhao R, Glick GG, Bansbach CE, Friedman DB, Cortez D. Thr-1989 Phosphorylation Is a Marker of Active Ataxia Telangiectasia-mutated and Rad3-related (ATR) Kinase. J Biol Chem. 2011 Aug 19;286:28707–14. 11. Rajeshkumar NV, De Oliveira E, Ottenhof N, Watters J, Brooks D, Demuth T, Shumway SD, Mizuarai S, Hirai H, Maitra A, Hidalgo M. MK-1775, a potent Wee1 inhibitor, synergizes with gemcitabine to achieve tumor regressions, selectively in p53-deficient pancreatic cancer xenografts. Clin Cancer Res. 2011 May 1;17:2799–806. 24. Guertin AD, Martin MM, Roberts B, Hurd M, Qu X, Miselis NR, Liu Y, Li J, Feldman I, Benita Y, Bloecher A, Toniatti C, Shumway SD. Acknowledgments Unique functions of CHK1 and WEE1 underlie synergistic anti-tumor activity upon pharmacologic inhibition. Cancer Cell Int. 2012 Nov 13;12:45. 12. Azorsa DO, Gonzales IM, Basu GD, Choudhary A, Arora S, Bisanz KM, Kiefer JA, Henderson MC, Trent JM, Hoff DDV, Mousses S. Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer. J Transl Med. 2009 Jun 11;7:43. 25. Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N, Inagaki M, Delcros JG, Moulinoux JP. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem FEBS. 1997 Jan 15;243:527–36. 13. Aarts M, Sharpe R, Garcia-Murillas I, Gevensleben H, Hurd MS, Shumway SD, Toniatti C, Ashworth A, Turner NC. Forced mitotic entry of S-phase cells as a therapeutic strategy induced by inhibition of WEE1. Cancer Discov. 2012 Jun;2:524–39. 26. Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H, Heimbrook DC, Chen L. Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci U S A. 2006 Jul 11;103:10660–5. 27. Lundberg AS, Weinberg RA. Functional Inactivation of the Retinoblastoma Protein Requires Sequential Modification by at Least Two Distinct Cyclin-cdk Complexes. Mol Cell Biol. 1998 Feb 1;18:753–61. 14. Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. J Biol Chem. 2000 Mar 31;275:9390–5. 28. Gonzalez SL, Stremlau M, He X, Basile JR, Münger K. Degradation of the Retinoblastoma Tumor Suppressor by the Human Papillomavirus Type 16 E7 Oncoprotein Is Important for Functional Inactivation and Is Separable from Proteasomal Degradation of E7. J Virol. 2001 Aug 15;75:7583–91. 15. Parker LL, Piwnica-Worms H. Inactivation of the p34cdc2- cyclin B complex by the human WEE1 tyrosine kinase. Science. 1992 Sep; 257:1955–7. 16. Zhao H, Piwnica-Worms H. ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1. Mol Cell Biol. 2001 Jul 1;21:4129–39. 29. Mortensen EM, Haas W, Gygi M, Gygi SP, Kellogg DR. Cdc28-Dependent Regulation of the Cdc5/Polo Kinase. Curr Biol. 2005 Nov;15:2033–7. 17. Butz J, Wickstrom E, Edwards J. Characterization of mutations and loss of heterozygosity of p53 and K-ras2 in pancreatic cancer cell lines by immobilized polymerase chain reaction. BMC Biotechnol. 2003 Jul 23;3:11. 30. Simpson-Lavy KJ, Brandeis M. Phosphorylation of Cdc5 regulates its accumulation. Cell Div. 2011 Dec 28;6:23. 18. Acknowledgments Syljuåsen RG, Sørensen CS, Hansen LT, Fugger K, Lundin C, Johansson F, Helleday T, Sehested M, Lukas J, Bartek J. Inhibition of Human Chk1 Causes Increased Initiation of DNA Replication, Phosphorylation of ATR Targets, and DNA Breakage. Mol Cell Biol. 2005 May 1;25:3553–62. 48. Kousholt AN, Fugger K, Hoffmann S, Larsen BD, Menzel T, Sartori AA, Sørensen CS. CtIP-dependent DNA resection is required for DNA damage checkpoint maintenance but not initiation. J Cell Biol. 2012 Jun 25;197:869–76. 37. Feijoo C, Hall-Jackson C, Wu R, Jenkins D, Leitch J, Gilbert DM, Smythe C. Activation of mammalian Chk1 during DNA replication arrest a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J Cell Biol. 2001 Sep 3;154:913–24. 49. Davies KD, Cable PL, Garrus JE, Sullivan FX, von Carlowitz I, Huerou YL, Wallace E, Woessner RD, Gross S. Chk1 inhibition and Wee1 inhibition combine synergistically to impede cellular proliferation. Cancer Biol Ther. 2011 Nov 1;12:788–96. 38. Hirai H, Iwasawa Y, Okada M, Arai T, Nishibata T, Kobayashi M, Kimura T, Kaneko N, Ohtani J, Yamanaka K, Itadani H, Takahashi-Suzuki I, Fukasawa K, Oki H, Nambu T, Jiang J, Sakai T, Arakawa H, Sakamoto T, Sagara T, Yoshizumi T, Mizuarai S, Kotani H. Small- molecule inhibition of Wee1 kinase by MK-1775 selectively sensitizes p53-deficient tumor cells to DNA-damaging agents. Mol Cancer Ther. 2009 Nov 1;8:2992–3000. 50. Do K, Doroshow JH, Kummar S. Wee1 kinase as a target for cancer therapy. Cell Cycle Georget Tex. 2013 Oct 1;12:3159–64. 51. Köpper F, Bierwirth C, Schön M, Kunze M, Elvers I, Kranz D, Saini P, Menon MB, Walter D, Sørensen CS, Gaestel M, Helleday T, Schön MP, Dobbelstein M. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity. Proc Natl Acad Sci U S A. 2013 Oct 15;110:16856–61. 39. O’Connell MJ, Raleigh JM, Verkade HM, Nurse P. Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation. EMBO J. 1997 Feb 1;16:545–54. 40. Yamaguchi T, Goto H, Yokoyama T, Silljé H, Hanisch A, Uldschmid A, Takai Y, Oguri T, Nigg EA, Inagaki M. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis. J Cell Biol. 2005 Nov 7;171:431–6. 52. Tiedje C, Ronkina N, Tehrani M, Dhamija S, Laass K, Holtmann H, Kotlyarov A, Gaestel M. The p38/MK2- Driven Exchange between Tristetraprolin and HuR Regulates AU–Rich Element–Dependent Translation. PLoS Genet. 2012 Sep 27;8:e1002977. 41. Acknowledgments Löhr K, Möritz C, Contente A, Dobbelstein M. p21/ CDKN1A mediates negative regulation of transcription by p53. J Biol Chem. 2003 Aug 29;278:32507–16. 31. Mailand N, Bekker-Jensen S, Bartek J, Lukas J. Destruction of Claspin by SCFbetaTrCP restrains Chk1 activation and facilitates recovery from genotoxic stress. Mol Cell. 2006 Aug 4;23:307–18. 19. Baxter GD, Lavin MF. Specific protein dephosphorylation in apoptosis induced by ionizing radiation and heat shock 32. Peschiaroli A, Dorrello NV, Guardavaccaro D, Venere www.impactjournals.com/oncotarget Oncotarget 13086 RI, Silljé HHW, de Vries EGE, van Vugt MATM. Forced activation of Cdk1 via wee1 inhibition impairs homologous recombination. Oncogene. 2013 Jun 13;32:3001–8. M, Halazonetis T, Sherman NE, Pagano M. SCFβTrCP- Mediated Degradation of Claspin Regulates Recovery from the DNA Replication Checkpoint Response. Mol Cell. 2006 Aug 4;23:319–29. , Halazonetis T, Sherman NE, Pagano M. SCFβTrCP 44. Karnak D, Engelke CG, Parsels LA, Kausar T, Wei D, Robertson JR, Marsh KB, Davis MA, Zhao L, Maybaum J, Lawrence TS, Morgan MA. Combined Inhibition of Wee1 and PARP1/2 for Radiosensitization in Pancreatic Cancer. Clin Cancer Res Off J Am Assoc Cancer Res. 2014 Oct 1;20:5085–96. 33 33. Donnianni RA, Ferrari M, Lazzaro F, Clerici M, Tamilselvan Nachimuthu B, Plevani P, Muzi-Falconi M, Pellicioli A. Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway. PLoS Genet. 2010 Jan 22;6:e1000763. 45. Yun MH, Hiom K. CtIP-BRCA1 modulates the choice of DNA double-strand-break repair pathway throughout the cell cycle. Nature. 2009 May 21;459:460–3. 34. Seki A, Coppinger JA, Jang C-Y, Yates JR, Fang G. Bora and the Kinase Aurora A Cooperatively Activate the Kinase Plk1 and Control Mitotic Entry. Science. 2008 Jun 20;320:1655–8. 46. Huertas P, Jackson SP. Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair. J Biol Chem. 2009 Apr 3;284:9558–65. 35. Sartori AA, Lukas C, Coates J, Mistrik M, Fu S, Bartek J, Baer R, Lukas J, Jackson SP. Human CtIP promotes DNA end resection. Nature. 2007 Nov 22;450:509–14. 47. Steger M, Murina O, Hühn D, Ferretti LP, Walser R, Hänggi K, Lafranchi L, Neugebauer C, Paliwal S, Janscak P, Gerrits B, Del Sal G, Zerbe O, Sartori AA. Prolyl Isomerase PIN1 Regulates DNA Double-Strand Break Repair by Counteracting DNA End Resection. Mol Cell. 2013 May 9;50:333–43. 36. Acknowledgments Mamely I, van Vugt MA, Smits VAJ, Semple JI, Lemmens B, Perrakis A, Medema RH, Freire R. Polo-like kinase-1 controls proteasome-dependent degradation of Claspin during checkpoint recovery. Curr Biol CB. 2006 Oct 10;16:1950–5. 42. Bassermann F, Frescas D, Guardavaccaro D, Busino L, Peschiaroli A, Pagano M. The Cdc14B-Cdh1-Plk1 axis controls the G2 DNA-damage-response checkpoint. Cell. 2008 Jul 25;134:256–67. 43. Krajewska M, Heijink AM, Bisselink YJWM, Seinstra www.impactjournals.com/oncotarget Oncotarget 13087
https://openalex.org/W4365512030	https://qmro.qmul.ac.uk/xmlui/bitstream/123456789/85964/2/Woodley%20Mannose%20metabolism%20inhibition%20sensitizes%202023%20Published.pdf	English	null	Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death	Nature communications	2,023	cc-by	20,770	Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death Keith Woodley1, Laura S. Dillingh2, George Giotopoulos 2,3, Pedro Madrigal 2,3,7, Kevin M. Rattigan4, Céline Philippe 1, Vilma Dembitz 1, Aoife M. S. Magee1, Ryan Asby3, Louie N. van de Lagemaat 1, Christopher Mapperley 1, Sophie C. James1, Jochen H. M. Prehn5, Konstantinos Tzelepis 2,3,6, Kevin Rouault-Pierre 1, George S. Vassiliou 2,3, Kamil R. Kranc 1, G. Vignir Helgason 4, Brian J. P. Huntly 2,3 & Paolo Gallipoli 1 Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukaemia (AML) and is often driven by metabolic adapta- tions which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the ﬁrst enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our ﬁndings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a connection between two apparently independent meta- bolic pathways and support further efforts to achieve eradication of therapy- resistant AML cells by sensitizing them to ferroptotic cell death. Resistance to therapy leading to disease relapse is the most frequent cause of treatment failure in acute myeloid leukaemia (AML)1 and commonly results from the emergence of genetic mutations, often within the therapeutic target2,3. However clinical and preclinical stu- dies, in both solid4 and haematological malignancies5 have shown that early non-genetic adaptations might allow some cancer cells to with- stand therapeutic stress, while allowing the development of a fully resistant phenotype through either established adaptive changes or the subsequent acquisition of genetic mutations6. Adaptive changes can be driven by metabolic rewiring and metabolism has emerged as a therapeutically actionable vulnerability in AML7, where speciﬁc meta- bolic adaptations arising as a result of driver mutations or in response to therapy have been reported8–13. D-Mannose is the 2-epimer of glucose and is a six-carbon sugar mostly used by cells for production of glycoconjugates rather than as an energy source. Article https://doi.org/10.1038/s41467-023-37652-0 1Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK. 2Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK. 3Department of Haematology, University of Cambridge, Cambridge, UK. 4Wolfson Wohl Cancer Research Centre, Institute of Cancer Sciences, University of Glasgow, Glasgow, UK. 5Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland University of Medicine and Health Sciences, Dublin, Ireland. 6Milner Therapeutics Institute, University of Cambridge, Cambridge, UK. 7Present address: European Molecular Biology Laboratory, European Bioinformatics Institute, EMBL-EBI, Hinxton CB10 1SD, UK. e-mail: p.gallipoli@qmul.ac.uk MPI KO causes increased lipid uptake in AML cells Given its high millimolar plasma concentration glucose generates the majority of cellular Man-6-P via MPI15. Our previous9 liquid chromatography-mass spectrometry (LC/MS) experiments with uniformly-labelled 13Carbon(U-13C6)-glucose in FLT3ITD mutant cells treated with FLT3-TKI show that upon FLT3 inhibition, glucose metabolism is blocked at the level of Fru-6-P (Supplementary Fig. 1a and ref. 9.). This suggests that while glycolysis is inhibited, metabolic pathways branching from Fru-6-P, such as MM are still active and might play a cytoprotective role. Corroborating this notion, it is noticeable that other enzymes downstream of MPI in MM, i.e. GMPPB, were depleted in our published screen9 (Supplementary Fig. 1a). Interestingly analysis of 2 published gene expression proﬁles of newly diagnosed AML cases associated with patient outcome shows that higher levels of MPI expression correlated with signiﬁcant or borderline signiﬁcant worse patient outcome (Fig. 1a and Sup- plementary Fig. 1b). MPI expression levels are higher in AML mono- nuclear cells (MNC) compared to normal bone marrow MNC (Fig.1b and Supplementary Fig. 1c-d) and particularly in FLT3ITD compared to FLT3WT AML (Fig.1c and Supplementary Fig. 1e) suggesting that MPI might have a prominent role in this AML subtype. When speciﬁcally focusing on FLT3ITD mutant patients we observed a trend towards MPI KO causes increased lipid uptake in AML cells p p To understand how MPI inhibition increases sensitivity to AML thera- pies, we pursued an unbiased complementary metabolomic and transcriptional analysis (Supplementary Data 1 and 2). We focused on FLT3ITD mutant cells treated with FLT3-TKI because MPI expression is higher in this AML subtype and adaptive resistance to FLT3-TKI is less well described thus necessitating a better understanding given the increasing clinical use of FLT3-TKI. Principal component analysis of both RNA-seq and untargeted metabolomics highlighted that MPI KO cells treated with FLT3-TKI separated from their respective Control and that the separation was partially rescued by addition of mannose to the media (Supplementary Fig. 3a, b). We identiﬁed over 600 metabolites across different pathways through untargeted metabo- lomics. Unexpectedly, the most striking change detected was the sig- niﬁcant increase in the intracellular levels of both long-chain saturated fatty acids (SFA) and PUFA in MPI KO cells treated with FLT3-TKI which was partially rescued by mannose supplementation (Fig. 3a). Inter- estingly we also noted that both long and medium-chain acylcarnitines increase in AC220 treated MPI KO cells (Fig. Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death Interestingly, MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensiti- zers to the clinical grade FLT3-tyrosine kinase inhibitor (TKI) AC220 (quizartinib) in AML carrying activating FLT3 internal tandem duplica- tion (ITD) mutations9 and more recently was identiﬁed amongst 81 United States Food and Drug Administration (FDA) druggable genetic dependencies of a murine model of FLT3ITD AML24. MPI inhibition sensitizes both wild-type and FLT3ITD mutant AML cells to novel targeted and standard therapies To test the functional role of MPI in AML, we generated MPI KO and respective control non-targeting(NT)gRNA (hereafter Control) AML cells by CRISPR editing (Supplementary Fig. 2a) and conﬁrmed that MPI KO sensitized FLT3ITD AML cells to FLT3-TKI (Fig. 2a). Moreover, both FLT3ITD and FLT3WT MPI KO AML cells were sensitized to standard cytarabine (AraC) chemotherapy in competition assays (Fig. 2b). These effects were due to both reduced viability and proliferation and were rescued by adding mannose to the media thus conﬁrming the speci- ﬁcity of MPI KO (Fig. 2c, d and Supplementary Fig. 2b-c). LC/MS ana- lysis conﬁrmed that mannose levels were reduced in MPI KO cells (Supplementary Fig. 2d). Similar phenotypic effects were observed using the MPI inhibitor MLS0315771 (hereafter MLS)27 and following genetic silencing by shRNA (Fig.2e and Supplementary Fig. 2e–g). The speciﬁcity of MLS was conﬁrmed by the lack of further toxicity in MPI KO cells (Supplementary Fig. 2h). Finally, in in vivo experiments, mice transplanted with MPI KO cells were more sensitive to AC220 and AraC resulting in both signiﬁcant prolonged survival and reduction in leu- kaemia burden (Fig. 2f and Supplementary Fig. 2i–m). Taken together these results show that MPI plays a role in resistance to both FLT3-TKI and AraC in AML cells. In this work, we therefore aim to test if inhibition of MPI and MM sensitizes AML cells to FLT3-TKI and standard chemotherapy and characterise the mechanistic consequences of MPI inhibition. We show that indeed MPI inhibition sensitizes AML cells to both cytarabine and FLT3-TKI therapy by priming them to ferroptotic cell death. Mechan- istically we show this being secondary to the activation of the ATF6 arm of the unfolded protein response (UPR) which in turn impairs fatty acid metabolism in AML cells and leads to intracellular accumulation of polyunsaturated fatty acids (PUFA). Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death Recent reports have highlighted a role for MPI in cancer cell proliferation or survival through varied mechanisms; reg- ulation of TP53 O-linked-N-acetylglucosaminylation (O-GlcNAcylation) via modulation of HBP activity21, reduction of cell surface receptor glycosylation and signalling22 or modulating the susceptibility of several cancers to the toxic effects of high mannose diets23. Regardless of the mechanism, high MPI activity appears to provide a survival advantage in several cancer types. Interestingly, MPI was amongst the top drop-out genes in our published CRISPR-Cas9 screen aiming to identify sensiti- zers to the clinical grade FLT3-tyrosine kinase inhibitor (TKI) AC220 (quizartinib) in AML carrying activating FLT3 internal tandem duplica- tion (ITD) mutations9 and more recently was identiﬁed amongst 81 United States Food and Drug Administration (FDA) druggable genetic dependencies of a murine model of FLT3ITD AML24. (MPI) or directed into N-glycosylation via phosphomannomutase (PMM2)14. Since mannose plasma concentration is about 100-fold less than glucose concentration, the bidirectional MPI enzyme also plays a role in channelling glucose into the mannose metabolism (MM) path- way to feed the production of GDP-Mannose, a sugar donor for N-glycosylation reactions. Indeed, most of the mannose in N-glycans derives from glucose15 and as a result MPI sits at a branching point between N-glycosylation and energy metabolism pathways, such as glycolysis and the hexosamine biosynthetic pathway (HBP) (Supple- mentary Fig. 1a). MPI plays an important role in embryonic development as Mpi knockout mice are embryonic lethal16 and only hypomorphs rather than completely inactivating mutations are described in patients17,18. However partial loss of MPI function causes a congenital disorder of glycosylation (CDG; MPI-CDG) in humans which is suc- cessfully treated with mannose supplementation19 while hypomorphic Mpi mice are viable20. Recent reports have highlighted a role for MPI in cancer cell proliferation or survival through varied mechanisms; reg- ulation of TP53 O-linked-N-acetylglucosaminylation (O-GlcNAcylation) via modulation of HBP activity21, reduction of cell surface receptor glycosylation and signalling22 or modulating the susceptibility of several cancers to the toxic effects of high mannose diets23. Regardless of the mechanism, high MPI activity appears to provide a survival advantage in several cancer types. Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death Mannose is transported into cells via transporters of the SLC2A group (GLUT) family and once within the cell is phosphorylated by hexokinase (HK) to produce mannose-6-phosphate (Man-6-P), which can mostly be either interconverted to the glycolytic intermediate fructose-6-phosphate (Fru-6-P) by mannose-6-phosphate isomerase Nature Communications\| (2023) 14:2132 Nature Communications\| (2023) 14:2132 Nature Communications\| (2023) 14:2132 1 1 https://doi.org/10.1038/s41467-023-37652-0 Article improved survival in patients expressing low MPI levels (Supple- mentary Fig. 1f). However this was not signiﬁcant possibly because of the small numbers. Gene-set enrichment analysis (GSEA) from several published gene expression datasets of AML samples at diagnosis highlighted oxidative phosphorylation, fatty acid oxidation and MYC targets gene signatures as being consistently upregulated in patients expressing high levels of MPI (Fig.1d and Supplementary Fig. 1g). Enhanced oxidative phosphorylation and fatty acid oxidation are known features of AML cells resistant to both current and novel therapies10,13. Further supporting the role of MPI in therapy resis- tance, analysis of paired diagnosis and relapsed samples following standard chemotherapy from 2 independent datasets25,26 shows that MPI expression levels increase upon relapse (Fig.1e). Taken together these data suggest that MM and particularly MPI expression levels might play a role in resistance to both FLT3-TKI and standard therapies in AML. While this phenotype might be particularly asso- ciated with FLT3ITD mutations, higher MPI expression also correlates with gene signatures associated with drug resistance in AML beyond the presence of FLT3ITD mutations. (MPI) or directed into N-glycosylation via phosphomannomutase (PMM2)14. Since mannose plasma concentration is about 100-fold less than glucose concentration, the bidirectional MPI enzyme also plays a role in channelling glucose into the mannose metabolism (MM) path- way to feed the production of GDP-Mannose, a sugar donor for N-glycosylation reactions. Indeed, most of the mannose in N-glycans derives from glucose15 and as a result MPI sits at a branching point between N-glycosylation and energy metabolism pathways, such as glycolysis and the hexosamine biosynthetic pathway (HBP) (Supple- mentary Fig. 1a). MPI plays an important role in embryonic development as Mpi knockout mice are embryonic lethal16 and only hypomorphs rather than completely inactivating mutations are described in patients17,18. However partial loss of MPI function causes a congenital disorder of glycosylation (CDG; MPI-CDG) in humans which is suc- cessfully treated with mannose supplementation19 while hypomorphic Mpi mice are viable20. MPI KO causes increased lipid uptake in AML cells a Kaplan-Meier curve comparing survival of patients from the TCGA AML cohort separated by the top 50% and bottom 50% of MPI expression (data obtained from https://www.cbioportal.org/). Log-rank (Mantel-Cox) test; (b) Violin plots of normalised MPI expression in AML samples compared to normal, healthy bone marrow mononuclear cells (MNC) from the BeatAML dataset (data obtained http://www.vizome.org/). Unpaired t test, two sided. N = 437 AML, N = 19 for healthy BM MNC; (c) Violin plots comparing MPI expression in FLT3ITD and FLT3WT AML samples from the TCGA dataset (left) and E-TABM1029 dataset (right). Unpaired t test, two sided. N = 28 FLT3ITD and N = 127 FLT3WT for TCGA, N = 38 FLT3ITD and N = 79 FLT3WT for E-TABM1029; (d) 3 signiﬁcantly enriched gene signatures in MPI high expressing samples in 6 AML datasets (GSE6891, E-TABM1029, GSE12417, GSE15434, TCGA, and GSE13159); (e) Relative expression of MPI at diagnosis and relapse in paired samples from GSE83533 dataset and data from manuscript 10.1056/NEJMoa1808777 (NEJM WUSM), N = 28. Paired t-test, two sided. Source data are provided as a Source Data ﬁle. All data points presented. Violin plots presented as median and quartiles. Fig. 1 \| High MPI expression correlates with worse prognosis in AML and a gene signature associated with enhanced oxidative phosphorylation and fatty acid metabolism. a Kaplan-Meier curve comparing survival of patients from the TCGA AML cohort separated by the top 50% and bottom 50% of MPI expression (data obtained from https://www.cbioportal.org/). Log-rank (Mantel-Cox) test; (b) Violin plots of normalised MPI expression in AML samples compared to normal, healthy bone marrow mononuclear cells (MNC) from the BeatAML dataset (data obtained http://www.vizome.org/). Unpaired t test, two sided. N = 437 AML, N = 19 for healthy BM MNC; (c) Violin plots comparing MPI expression in FLT3ITD and FLT3WT AML MPI KO cells showed higher increase in their PUFA and PUFA con- taining lipid species levels compared to MUFA and SFA (Supplemen- tary Fig. 3e). Interestingly CD36, a fatty acid transporter with prognostic signiﬁcance in AML13,28, was transcriptionally upregulated in MPI KO cells (Supplementary Fig. 3f). To test if CD36 played a role in lipid uptake, we used its irreversible inhibitor sulfosuccinimidyl oleate (SSO)29 and observed that, while CD36 inhibition reduced arachidonic acid uptake in MPI KO cells, it did not affect the uptake of the saturated lipid probe C1-Bodipy 500/510 C12 (Supplementary Fig. 3g-h). MPI KO causes increased lipid uptake in AML cells 3b) and this was coupled with a reduction in tricarboxylic acid (TCA) cycle intermediates in the same conditions (Supplementary Fig. 3c). This metabolomic pattern Nature Communications\| (2023) 14:2132 2 https://doi.org/10.1038/s41467-023-37652-0 Article Fig. 1 \| High MPI expression correlates with worse prognosis in AML and a gene signature associated with enhanced oxidative phosphorylation and fatty acid metabolism. a Kaplan-Meier curve comparing survival of patients from the TCGA AML cohort separated by the top 50% and bottom 50% of MPI expression (data obtained from https://www.cbioportal.org/). Log-rank (Mantel-Cox) test; (b) Violin plots of normalised MPI expression in AML samples compared to normal, healthy bone marrow mononuclear cells (MNC) from the BeatAML dataset (data obtained http://www.vizome.org/). Unpaired t test, two sided. N = 437 AML, N = 19 for healthy BM MNC; (c) Violin plots comparing MPI expression in FLT3ITD and FLT3WT AML samples from the TCGA dataset (left) and E-TABM1029 dataset (right). Unpa test, two sided. N = 28 FLT3ITD and N = 127 FLT3WT for TCGA, N = 38 FLT3ITD an N = 79 FLT3WT for E-TABM1029; (d) 3 signiﬁcantly enriched gene signatures i high expressing samples in 6 AML datasets (GSE6891, E-TABM1029, GSE1241 GSE15434, TCGA, and GSE13159); (e) Relative expression of MPI at diagnosis relapse in paired samples from GSE83533 dataset and data from manuscript 10.1056/NEJMoa1808777 (NEJM WUSM), N = 28. Paired t-test, two sided. Sour data are provided as a Source Data ﬁle. All data points presented. Violin plot presented as median and quartiles. Article https://doi.org/10.1038/s41467-023-376 samples from the TCGA dataset (left) and E-TABM1029 dataset (right). Unpaired t test, two sided. N = 28 FLT3ITD and N = 127 FLT3WT for TCGA, N = 38 FLT3ITD and N = 79 FLT3WT for E-TABM1029; (d) 3 signiﬁcantly enriched gene signatures in MPI high expressing samples in 6 AML datasets (GSE6891, E-TABM1029, GSE12417, GSE15434, TCGA, and GSE13159); (e) Relative expression of MPI at diagnosis and relapse in paired samples from GSE83533 dataset and data from manuscript 10.1056/NEJMoa1808777 (NEJM WUSM), N = 28. Paired t-test, two sided. Source data are provided as a Source Data ﬁle. All data points presented. Violin plots presented as median and quartiles. Fig. 1 \| High MPI expression correlates with worse prognosis in AML and a gene signature associated with enhanced oxidative phosphorylation and fatty acid metabolism. MPI KO causes increased lipid uptake in AML cells Toge- ther these data are consistent with MPI KO cells having an increased lipid uptake which, at least for PUFAs, is driven by increased CD36 expression. Interestingly CD36 is known to preferentially favour uptake of long chain PUFAs30. suggests both an increase in uptake of long-chain fatty acids and defective mitochondrial fatty acid oxidation (FAO) in treated MPI KO cells. To speciﬁcally test for fatty acid uptake, we labelled the cells with the saturated lipid probe C1-Bodipy 500/510 C12 and showed that MPI KO cells signiﬁcantly increased fatty acid uptake (Fig. 3c and Supple- mentary Fig. 3d). To clarify if there was a preferential uptake of SFAs, monounsaturated fatty acids (MUFAs) or PUFAs, we used neutral Bodipy 493/503 to stain cells fed speciﬁcally palmitate (16:0 SFA), oleate (18:1 MUFA) and arachidonic acid (20:4 PUFA). While we observed signiﬁcant increase in uptake of all subtypes of fatty acids in MPI KO cells, arachidonic acid uptake appeared to be particularly enhanced in the KO cells and was also rescued by mannose (Fig. 3d). This ﬁnding was corroborated by the metabolomic analysis as treated Nature Communications\| (2023) 14:2132 3 MPI KO results in gene expression and metabolic changes con- sistent with reduced FAO in AML cells both in the absence and, even more, in the presence of AC220. Simila genesets were enriched in AC220 treated MPI KO cells supplemente Article https://doi.org/10.1038/s41467-023-37652- Article https://doi.org/10.1038/s41467-023-37652-0 MPI KO results in gene expression and metabolic changes con- stent with reduced FAO in AML cells We then turned our attention to FAO, given our metabolomic proﬁle as suggestive of a defective FAO. GSEA analysis of RNA-seq data emonstrated that oxidative phosphorylation and fatty acid metabo- sm signatures were upregulated in Control compared to MPI KO cells both in the absence and, even more, in the presence of AC220. Sim genesets were enriched in AC220 treated MPI KO cells supplemen with mannose when compared to AC220 treated MPI KO cells (Fig and Supplementary Fig. 4a). Consistent with this, MPI expression lev were positively correlated with genes involved in fatty acid metaboli across both TCGA and BeatAML datasets (Supplementary Fig. 4 ature Communications\| (2023)14:2132 MPI KO results in gene expression and metabolic changes con- sistent with reduced FAO in AML cells We then turned our attention to FAO, given our metabolomic proﬁle was suggestive of a defective FAO. MPI KO causes increased lipid uptake in AML cells GSEA analysis of RNA-seq data demonstrated that oxidative phosphorylation and fatty acid metabo- lism signatures were upregulated in Control compared to MPI KO cells both in the absence and, even more, in the presence of AC220. Similar genesets were enriched in AC220 treated MPI KO cells supplemented with mannose when compared to AC220 treated MPI KO cells (Fig. 4a and Supplementary Fig. 4a). Consistent with this, MPI expression levels were positively correlated with genes involved in fatty acid metabolism across both TCGA and BeatAML datasets (Supplementary Fig. 4b). Nature Communications\| (2023)14:2132 4 MPI KO results in gene expression and metabolic changes con- sistent with reduced FAO in AML cells We then turned our attention to FAO, given our metabolomic proﬁle was suggestive of a defective FAO. GSEA analysis of RNA-seq data demonstrated that oxidative phosphorylation and fatty acid metabo- lism signatures were upregulated in Control compared to MPI KO cells both in the absence and, even more, in the presence of AC220. Similar genesets were enriched in AC220 treated MPI KO cells supplemented with mannose when compared to AC220 treated MPI KO cells (Fig. 4a and Supplementary Fig. 4a). Consistent with this, MPI expression levels were positively correlated with genes involved in fatty acid metabolism across both TCGA and BeatAML datasets (Supplementary Fig. 4b). MPI KO results in gene expression and metabolic changes con- sistent with reduced FAO in AML cells We then turned our attention to FAO, given our metabolomic proﬁle was suggestive of a defective FAO. GSEA analysis of RNA-seq data demonstrated that oxidative phosphorylation and fatty acid metabo- lism signatures were upregulated in Control compared to MPI KO cells Nature Communications\| (2023) 14:2132 4 4 Article https://doi.org/10.1038/s41467-023-37652-0 indicated. Treated for 6 days, N = 4 for MV411, N = 3 for MOLM13, 1 way Anova with Tukey’s correction for multiple comparisons; (d) Percentage of live cells of NT gRNA and MPI gRNA5 THP1 (left) and MOLM13 (right) cells treated with vehicle, AraC (1 µM) or AraC (1 µM) and mannose (100 µM). Treated for 3 days, N = 4 for MOLM13, N = 5 for THP1, 1 way Anova with Tukey’s correction for multiple com- parisons; (e) Percentage of live cells of WT MV411 (left) and WT MOLM13 (right) cells treated with vehicle, MLS0315771 (1 µM), AC220 (1 nM), mannose (100 µM) or combinations of these. MPI KO causes increased lipid uptake in AML cells Treatment with Ceapin A7 phe- nocopied mannose supplementation by reversing ATF6 cleavage and nuclear localisation (Fig. 5c, d and Supplementary Fig. 6a). Ceapin A7 also rescued viability and OCR of MPI KO cells treated with AC220 to the same degree of mannose and resensitized MPI KO cells to the effects of etomoxir on OCR (Fig. 5e, f). Similar effects were observed following silencing of ATF6 (Supplementary Fig. 6b, c). Conversely both the UPR activator with reported preferential activity on ATF6 AA14736 and the canonical glycosylation inhibitor and UPR activator tunicamycin mimicked the effects of MPI KO on cell viability and prevented mannose rescue (Supplementary Fig. 6d, e). Finally inhibi- tors of the other UPR branches did not affect viability of treated MPI KO cells (Supplementary Fig. 6f). Therefore despite the fact that the UPR response is often non-selective and there is evidence that ATF6 can lead to activation of other arms of the UPR response37,38, we observe in our system a preferential activation of ATF6. This might be due to low level and chronic endoplasmic reticulum stress activating preferentially the ATF6 arm of the UPR. Interestingly this is consistent with previous literature showing that ATF6 is able to refold proteins and prevent further stress and activation of the other arms of UPR which happens only when the refolding ability driven by ATF6 is overwhelmed37,39. Activation of the ATF6 arm of the UPR has been reported to inhibit the transcription/activity of the master regulator of lipid catabolism PPARA40 and interestingly both Ceapin A7 and AA147, beside modulating the expression of canonical ATF6 targets ERO1B p y p g To conﬁrm reduction in FAO in MPI deﬁcient cells, we grew cells in U-13C16-palmitate and found reduced palmitate labelling of several TCA intermediates in MPI KO cells, a phenotype rescued by mannose (Fig. 4b and Supplementary Fig. 4d). Real-time metabolic ﬂux analysis also showed that MPI KO cells had reduced oxygen consumption rate (OCR) and ATP-linked OCR compared to Control, with the OCR further reduced in the presence of AC220 and rescued in the presence of mannose (Fig. 4c). Similar ﬁndings were observed in THP1 cells treated with AraC (Supplementary Fig. 4f). To speciﬁcally assess FAO we measured OCR using substrate limiting media in the presence of only palmitate. MPI KO causes increased lipid uptake in AML cells We ﬁrst observed that with palmitate as the sole substrate, mannose supplementation rescued the respiration defect in treated MPI KO cells suggesting that MPI KO cells can engage efﬁciently in FAO only when supplemented with mannose (Supplementary Fig. 4e). Moreover, in substrate limiting media with palmitate, both basal respiration and ATP production were signiﬁcantly reduced in KO cells compared to Control following AC220 treatment with the effects res- cued by mannose (Fig. 4d). These data suggest MPI KO cells do not use palmitate efﬁciently as a source of energy, particularly in the presence of AC220, with this phenotype reversed by mannose. Indeed adding the CPT1A and FAO inhibitor etomoxir11 to AC220 or AraC did not further reduce basal respiration or ATP production in MPI KO cells. However, the re-addition of mannose resensitized MPI KO cells to the effects of etomoxir on OCR (Fig. 4e and Supplementary Fig. 4g). These effects correlated with changes in viability in response to etomoxir in MPI KO cells, as no reduction in viability beyond the effects of AC220 was observed in response to etomoxir, unless cells were cultured in the presence of mannose (Fig. 4f). Conversely viability of AC220 or AraC treated MPI KO was rescued by the PPARA agonist and FAO activator fenoﬁbrate31 to a degree similar to mannose (Supplementary Fig. 4h). Finally we did not rescue viability of treated MPI KO cells using the cell permeable TCA cycle intermediate dimethyl-alpha-ketoglutarate (DMaKG) (Supplementary Fig. 4i). These data suggest that MPI KO cells have defective FAO but the effects on viability of MPI KO are driven by both defective FAO and enhanced lipid uptake causing an increase in intracellular fatty acid levels rather than simply a reduction in TCA cycle activity and oxidative phosphorylation. MPI KO causes increased lipid uptake in AML cells Interestingly this is consistent with previous literature showing that ATF6 is able to refold proteins and prevent further stress and activation of the other arms of UPR which happens only when the refolding ability driven by ATF6 is overwhelmed37,39. Activation of the ATF6 arm of the UPR has been reported to inhibit the transcription/activity of the master regulator of lipid catabolism PPARA40 and interestingly both Ceapin A7 and AA147, beside modulating the expression of canonical ATF6 targets ERO1B Moreover, RT-QPCR showed reduction in expression levels of CPT1A and PPARA, a master regulator of lipid catabolism, in MPI KO compared to Control cells (Supplementary Fig. 4c). Therefore, gene expression analysis suggest that treated MPI KO cells have defective FAO likely contributing to intracellular accumulation of both acylcarnitines and upstream fatty acids observed in metabolomic proﬁling. concurrent accumulation of misfolded protein (Supplementary Fig. 5a, b). These effects correlated, as expected, with upregulation of UPR genes in MPI KO cells although in particular the ATF6 arm of the UPR, which is particularly sensitive to changes in glycosylation32, was upre- gulated (Fig. 5a).To validate this, we transduced cells with an ATF6-YFP reporter lentivirus (Supplementary Fig. 5c)33 and observed signiﬁcant ATF6 activation in MPI KO cells which was reversed by mannose (Fig.5b). We then used immunoﬂuorescence to track localisation of ATF6 in MPI KO cells and showed that ATF6 localised in the nucleus of MPI KO cells and this was reversed by mannose (Fig. 5c, d and Sup- plementary Fig. 5d). Similarly western blot showed higher levels of cleaved ATF6 consistent with its activation in MPI KO cells compared to Control cells which was rescued by mannose (Supplementary Fig. 5e). Interestingly both in the ATF6 reporter assay and by western blot, AC220 treatment appeared also to reduce ATF6 activation although this was not seen by immunoﬂuorescence. This discrepancy might reﬂect sensitivity of the assay and/or be related to the effects of AC220 on protein translation/ER load and ATF6 activation at the assessed timepoint. Conversely we did not observe consistent sig- niﬁcant changes in the activation of the other branches of the UPR in MPI KO cells (Supplementary Fig. 5f) To functionally test the effects of ATF6 activation in MPI KO cells, we used the highly speciﬁc ATF6 inhibitor Ceapin A7 which retains ATF6 in the endoplasmic reticulum thus preventing its nuclear translocation and ability to activate tran- scription of its target genes34,35. MPI KO causes increased lipid uptake in AML cells Treated for 6 days N = 3, 1 way Anova with Tukey’s correc- tion for multiple comparisons; (f) Kaplan-Meier survival curve showing survival time of mice after transplantation with MOLM13 NT gRNA or MPI gRNA5 cells, with or without AC220 treatment (5 mg/kg, top) and THP1 NT gRNA or MPI gRNA5 cells, with or without cytarabine treatment (50 mg/kg, bottom). Log-rank (Mantel-Cox) test. For all panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, *=p < 0.001. Source data are provided as a Source Data ﬁle. All data presented as mean values ± SEM. Fig. 2 \| MPI inhibition sensitizes both wild-type and FLT3ITD mutant AML cells to novel targeted and standard therapies. a Competition growth assays mea- suring the ratio of mCherry positive to mCherry negative cells between WT MV411 and NT gRNA or MPI gRNA2 or gRNA5 (mCherry positive) with and without AC220 (1 nM) treatments (left) and WT MOLM13 (mCherry negative) and NT gRNA or MPI gRNA2 or gRNA5 (mCherry positive) with and without AC220 (1 nM) treatments (right) over 10 days. N = 3, 2 way Anova with Bonferroni correction for multiple comparisons; (b) Competition growth assays measuring the ratio of mCherry positive to mCherry negative cells between WT THP1 (mCherry negative) and NT gRNA or MPI gRNA5 (mCherry positive) with and without AraC (1 µM) treatments (left) and WT MOLM13 (mCherry negative) and NT gRNA or MPI gRNA5 (mCherry positive) with and without AraC (1 µM) treatments (right) over 6 days. N = 3, 2 way Anova with Bonferroni correction for multiple comparisons; (c) Percentage of live cells NT gRNA, MPI gRNA2 and MPI gRNA5 MV411 (left) and MOLM13 (right) cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM), as concurrent accumulation of misfolded protein (Supplementary Fig. 5a, b). These effects correlated, as expected, with upregulation of UPR genes in MPI KO cells although in particular the ATF6 arm of the UPR, which is particularly sensitive to changes in glycosylation32, was upre- gulated (Fig. 5a).To validate this, we transduced cells with an ATF6-YFP reporter lentivirus (Supplementary Fig. 5c)33 and observed signiﬁcant ATF6 activation in MPI KO cells which was reversed by mannose (Fig.5b). MPI KO causes increased lipid uptake in AML cells We then used immunoﬂuorescence to track localisation of ATF6 in MPI KO cells and showed that ATF6 localised in the nucleus of MPI KO cells and this was reversed by mannose (Fig. 5c, d and Sup- plementary Fig. 5d). Similarly western blot showed higher levels of cleaved ATF6 consistent with its activation in MPI KO cells compared to Control cells which was rescued by mannose (Supplementary Fig. 5e). Interestingly both in the ATF6 reporter assay and by western blot, AC220 treatment appeared also to reduce ATF6 activation although this was not seen by immunoﬂuorescence. This discrepancy might reﬂect sensitivity of the assay and/or be related to the effects of AC220 on protein translation/ER load and ATF6 activation at the assessed timepoint. Conversely we did not observe consistent sig- niﬁcant changes in the activation of the other branches of the UPR in MPI KO cells (Supplementary Fig. 5f) To functionally test the effects of ATF6 activation in MPI KO cells, we used the highly speciﬁc ATF6 inhibitor Ceapin A7 which retains ATF6 in the endoplasmic reticulum thus preventing its nuclear translocation and ability to activate tran- scription of its target genes34,35. Treatment with Ceapin A7 phe- nocopied mannose supplementation by reversing ATF6 cleavage and nuclear localisation (Fig. 5c, d and Supplementary Fig. 6a). Ceapin A7 also rescued viability and OCR of MPI KO cells treated with AC220 to the same degree of mannose and resensitized MPI KO cells to the effects of etomoxir on OCR (Fig. 5e, f). Similar effects were observed following silencing of ATF6 (Supplementary Fig. 6b, c). Conversely both the UPR activator with reported preferential activity on ATF6 AA14736 and the canonical glycosylation inhibitor and UPR activator tunicamycin mimicked the effects of MPI KO on cell viability and prevented mannose rescue (Supplementary Fig. 6d, e). Finally inhibi- tors of the other UPR branches did not affect viability of treated MPI KO cells (Supplementary Fig. 6f). Therefore despite the fact that the UPR response is often non-selective and there is evidence that ATF6 can lead to activation of other arms of the UPR response37,38, we observe in our system a preferential activation of ATF6. This might be due to low level and chronic endoplasmic reticulum stress activating preferentially the ATF6 arm of the UPR. MPI KO cells activate the ATF6 arm of the UPR to inhibit FAO in AML cells MPI deﬁciency leads to a defect in protein glycosylation17 and as pre- dicted MPI KO cells showed reduced protein glycosylation as con- ﬁrmed by their lower lectin staining. This was also associated with Nature Communications\| (2023) 14:2132 5 Fig. 3 \| MPI KO causes increased lipid uptake in AML cells. a Heat maps of Z-scores for relative changes of long chain polyunsaturated fatty acids (PUFA - n3, n6 and n9, left) and long chain saturated fatty acids (SFA, right), with box plots of selected lipids of each type below the heat maps from global metabolomics of MOLM13 NT gRNA or MPI gRNA5 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) as indicated for 48 h, N = 4; (b) Heat maps of Z-scores for relative changes of fatty acid oxidation metabolism related fatty acid species, with box plots of selected lipids below the heat maps from global metabolomics of MOLM13 NT gRNA or MPI gRNA5 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) as indicated for 48 h, N = 4; (c) Uptake of ﬂuorescent C1-Bodipy 500/510 C12 by NT gRNA or MPI gRNA5 MOLM13 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) for 24 h, with a representative ﬂow cytometry plot (right). N = 3, 1 way Anova with Tukey’s cor- rection for multiple comparisons; (d) Uptake of palmitate (50 µM, left), oleate (50 µM, middle) and arachidonic acid (1 µM, right) by NT gRNA or MPI gRNA5 MOLM13 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) for 24 h as indicated and measured by Bodipy 493/503 neutral lipid stain with representative ﬂow cytometry plots comparing uptake by NT gRNA and MPI gRNA5 cells (with unstained sample, to the right of each graph). N = 3 for palmitate and oleate, N = 4 for arachidonic acid, 1 way Anova with Tukey’s correction for multiple comparisons. For all panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, **=p < 0.001. Source data are provided as a Source Data ﬁle. All data presented as mean values ± SEM, box plots presented as median with upper and lower quartiles as bounds of box and whiskers as max and min of distribution. MPI KO cells activate the ATF6 arm of the UPR to inhibit FAO in AML cells Article https://doi.org/10.1038/s41467-023-37652-0 Article https://doi.org/10.1038/s41467-023-37652-0 p // g/ / representative ﬂow cytometry plot (right). N = 3, 1 way Anova with Tukey’s cor- rection for multiple comparisons; (d) Uptake of palmitate (50 µM, left), oleate (50 µM, middle) and arachidonic acid (1 µM, right) by NT gRNA or MPI gRNA5 MOLM13 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) for 24 h as indicated and measured by Bodipy 493/503 neutral lipid stain with representative ﬂow cytometry plots comparing uptake by NT gRNA and MPI gRNA5 cells (with unstained sample, to the right of each graph). N = 3 for palmitate and oleate, N = 4 for arachidonic acid, 1 way Anova with Tukey’s correction for multiple comparisons. For all panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, **=p < 0.001. Source data are provided as a Source Data ﬁle. All data presented as mean values ± SEM, box plots presented as median with upper and lower quartiles as bounds of box and whiskers as max and min of distribution. Fig. 3 \| MPI KO causes increased lipid uptake in AML cells. a Heat maps of representative ﬂow cytometry plot (right). N = 3, 1 way Anova with Tukey’s cor- rection for multiple comparisons; (d) Uptake of palmitate (50 µM, left), oleate (50 µM, middle) and arachidonic acid (1 µM, right) by NT gRNA or MPI gRNA5 MOLM13 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) for 24 h as indicated and measured by Bodipy 493/503 neutral lipid stain with representative ﬂow cytometry plots comparing uptake by NT gRNA and MPI gRNA5 cells (with unstained sample, to the right of each graph). N = 3 for palmitate and oleate, N = 4 for arachidonic acid, 1 way Anova with Tukey’s correction for multiple comparisons. For all panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, **=p < 0.001. Source data are provided as a Source Data ﬁle. All data presented as mean values ± SEM, box plots presented as median with upper and lower quartiles as bounds of box and whiskers as max and min of distribution. Fig. 3 \| MPI KO causes increased lipid uptake in AML cells. MPI KO cells activate the ATF6 arm of the UPR to inhibit FAO in AML cells a Heat maps of Z-scores for relative changes of long chain polyunsaturated fatty acids (PUFA - n3, n6 and n9, left) and long chain saturated fatty acids (SFA, right), with box plots of Z-scores for relative changes of long chain polyunsaturated fatty acids (PUFA - n3, n6 and n9 left) and long chain saturated fatty acids (SFA right) with box plots of selected lipids of each type below the heat maps from global metabolomics of MOLM13 NT gRNA or MPI gRNA5 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) as indicated for 48 h, N = 4; (b) Heat maps of Z-scores for relative changes of fatty acid oxidation metabolism related fatty acid species, with box plots of selected lipids below the heat maps from global metabolomics of MOLM13 NT gRNA or MPI gRNA5 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) as indicated for 48 h, N = 4; (c) Uptake of ﬂuorescent C1-Bodipy 500/510 C12 by NT gRNA or MPI gRNA5 MOLM13 cells treated with vehicle, AC220 (1 nM) or AC220 (1 nM) and mannose (100 µM) for 24 h, with a and HERPUD1 as expected, respectively increased and decreased the expression of PPARA and CPT1A, the rate limiting step in FAO, thus phenocopying the transcriptional consequences of MPI KO and man- nose rescue (Supplementary Fig. 6g, h). Interestingly, MPI expression levels were negatively correlated with ERO1B and HERPUD1 expression across both TCGA and BeatAML datasets (Supplementary Fig. 6i). Taken together these data support a model where MPI KO cells pre- ferentially activate the ATF6 arm of the UPR leading to reduced FAO. Nature Communications\| (2023) 14:2132 Nature Communications\| (2023) 14:2132 6 https://doi.org/10.1038/s41467-023-37652-0 Article KO drives lipid peroxidation and ferroptotic cell death in L cells metabolic phenotype of MPI KO cells with accumulation of PUFA reduced FAO is reminiscent of that observed in cancer cells prone erroptotic cell death, i.e. clear cell renal carcinoma41,42. Moreover KO cells had lower levels of intracellular cysteine and higher levels of 4-hydroxy-nonenal-glutathione and ophthalmate (Fig. 6a) marke oxidative stress and lipid peroxidation43,44 which was further incre by AC220 a known driver of oxidative stress in these cells9. https://doi.org/10.1038/s41467-023-37652-0 FBS and glutamine treated with vehicle, AC220, mannose or palmitate (50 µM) as indicated. N = 3, 2-way Anova with Sidak’s correction (two left panels). Baseline OCR and ATP production of NTgRNA and MPIgRNA5 cells treated with palmitate (50 µM) and vehicle, AC220 or mannose as indicated, N = 3, 1-way Anova with Tukey’s cor- rection (2 right panels); (e) SEAHORSE MitoStress tests showing OCR comparing MPIgRNA5 cells treated with AC220, mannose after 72 h in combinations as indi- cated with or without etomoxir (50 µM), N = 3, 2-way Anova with Sidak’s correction (left panel). Baseline OCR and ATP production of MPI gRNA5 cells treated with vehicle, AC220, mannose after 72 h in combinations as indicated with or without etomoxir (50 µM). N = 3, 1-way Anova with Tukey’s correction (2 right panels); (f) Percentage of live MPIgRNA5 MOLM13 cells treated with vehicle, etomoxir, AC220, mannose or in combinations 6 days after treatment. N = 4,1-wayAnova with Tukey’s correction. For all panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, **=p < 0.001, data presented as mean values ± SEM unless stated. Fig. 4 \| MPI KO results in gene expression and metabolic changes consistent with reduced FAO in AML cells. a GSEA for Oxidative phosphorylation and Fatty acid oxidation signatures from RNA sequencing data comparing MOLM13 NTgRNA and MPIgRNA5 treated with AC220 (left) and MOLM13 MPIgRNA5 treated with AC220 or AC220 and mannose (right), FDR and NES from 1000 permutations; (b) Percentage of TCA cycle intermediates and associated metabolites labelled with ¹³C from ¹³C16-palmitate in MPI KO and NT MOLM13 cells treated with AC220 and mannose as indicated for 24 h along with 50 µM ¹³C16-palmitate. N = 5, 1-way anova with Tukey’s correction; (c) SEAHORSE MitoStress tests showing oxygen con- sumption rate (OCR) of NTgRNA and MPIgRNA5 MOLM13 cells treated with vehicle, AC220 or AC220 and mannose as indicated after 72 h of treatment, N = 3, 2-way Anova with Sidak’s correction (two left panels). Baseline OCR and ATP production of NTgRNA and MPIgRNA5 MOLM13 cells treated with vehicle, AC220 or AC220 and mannose as indicated after 72 h of treatment, N = 3, 1-way Anova with Tukey’s correction (2 right panels); (d) SEAHORSE MitoStress tests showing OCR compar- ing MPIgRNA5 cells cultured overnight in substrate limited RPMI media without driven by lipid peroxidation and ferroptosis. https://doi.org/10.1038/s41467-023-37652-0 Indeed Bodipy 581/591 C11 staining of MPI KO and knock-down (KD) cells conﬁrmed higher levels of lipid peroxidation in treated MPI KO/KD cells, that was rescued by mannose (Fig. 6c and Supplementary Fig. 7a, b). Moreover, cell death and induction of lipid peroxidation in both AC220 and AraC treated MPI KO cells was rescued by the radical-trapping antioxidant ferrostatin and liproxstatin, known anti-ferroptosis agents46 (Fig. 6d and Supplemen- tary Data Fig. 7c–e). Interestingly Ceapin A7 and AA147 respectively mimicked and inhibited the effects of mannose on lipid peroxidation supporting the role of ATF6 activation in driving the ferroptotic phe- notype (Supplementary Fig. 7f, g). Conversely neither PERK or IRE1 inhibition had signiﬁcant effects on lipid peroxidation (Supplementary Fig. 7h, i). MPI KO cells were more sensitive to PUFA (arachidonic acid) toxicity which is rescued by mannose and SSO thus reinforcing the role of CD36 in PUFAs uptake (Supplementary Fig. 8a, b). Conversely growing MPI KO cells in delipidated media reduced their sensitivity to AC220 (Supplementary Fig. 8c). However SSO did not affect lipid per- oxidation (Supplementary Fig. 8d) probably because it affects uptake of only some fatty acid species (Supplementary Fig. 3g, h) and has no effects on FAO. This also suggests that sensitivity to arachidonic acid toxicity is not just due to increased lipid peroxidation. MPI KO cells were also more sensitive to the ferroptotic inducing agents erastin and RSL3 (Supplementary Fig. 8e). Interestingly, and consistent with the reduced cysteine levels in MPI KO cells, surface expression of the cysteine transporter and erastin target SLC7A11/xCT was downregulated in MPI KO cells further explaining their tendency to undergo ferroptosis (Supplementary Fig. 8f). We also did not detect signiﬁcant upregulation of cleaved PARP in treated MPI KO cells and neither the apoptosis/pan caspase inhibitor Z-VAD or necroptosis inhibitor Necrostatin-1 rescued cell death in both AC220 and AraC treated MPI KO cells (Supplementary Fig. 8g, h). Interestingly Necrostatin-1 has been shown to also rescue ferroptosis in other cellular models47 but we did not observe that and therefore we conclude necroptosis is not driving cell death in our sys- tem. Finally to validate the role of ferroptosis induction in driving cell death in vivo mice transplanted with THP1 Control and MPI KO cells were treated with AraC ± liproxstatin. https://doi.org/10.1038/s41467-023-37652-0 As expected liproxstatin treat- ment reduced disease latency in AraC treated mice transplanted with MPI KO cells and this correlated with reduced staining of the leukaemic cells for 4-HNE, a marker of lipid peroxidation, in vivo (Fig. 6e, f and Supplementary Fig. 8i–j). Overall these data show that, following chemo/targeted therapy treatment the metabolic rewiring caused by MPI KO primes the cells to lipid peroxidation and ferroptotic cell death. (Supplementary Fig. 9a, b), FLT3ITD primary AML MNC were sensitized to FLT3-TKI therapy and this could be rescued by mannose (Fig. 7a). Conversely normal CD34+ cells were not sensitive to MPI KD both in the absence or presence of AC220 (Fig. 7b). Consistent with observations in AML cell lines, MPI KD primed FLT3ITD AML MNC to lipid peroxida- tion following FLT3-TKI therapy and led to higher levels of nuclear ATF6, with both effects rescued by the addition of mannose (Fig. 7c, d). Conversely a smaller induction of lipid peroxidation was observed in normal CD34+ cells upon MPI KD which was not enhanced by FLT3-TKI or rescued by mannose (Supplementary Fig. 9c). Moreover the viability of primary MPI KD FLT3ITD AML MNC treated with AC220 was rescued by ferrostatin and liproxstatin conﬁrming that the mechanism of death in primary AML MNC was also due to ferroptosis (Fig. 7e). These experiments validate the phenotype driven by MPI inhibition in pri- mary AML MNC while demonstrating that MPI KD is tolerated by normal hematopoietic stem/progenitor cells (HSPC). MPI KO cells activate the ATF6 arm of the UPR to inhibit FAO in AML cells Interest a ferroptotic gene signature45 was upregulated in treated MPI KO compared to both Control and MPI KO mannose treated cells (Fig These observations led us to test if cell death of treated MPI KO cells MPI KO drives lipid peroxidation and ferroptotic cell death in AML cells MPI KO drives lipid peroxidation and ferroptotic cell death in AML cells The metabolic phenotype of MPI KO cells with accumulation of PUFA and reduced FAO is reminiscent of that observed in cancer cells prone to ferroptotic cell death, i.e. clear cell renal carcinoma41,42. Moreover MPI KO ll h d l l l f i ll l i d hi h l l of 4-hydroxy-nonenal-glutathione and ophthalmate (Fig. 6a) markers of oxidative stress and lipid peroxidation43,44 which was further increased by AC220 a known driver of oxidative stress in these cells9. Interestingly a ferroptotic gene signature45 was upregulated in treated MPI KO cells compared to both Control and MPI KO mannose treated cells (Fig. 6b). These observations led us to test if cell death of treated MPI KO cells was MPI KO drives lipid peroxidation and ferroptotic cell death in AML cells The metabolic phenotype of MPI KO cells with accumulation of PUFA and reduced FAO is reminiscent of that observed in cancer cells prone to ferroptotic cell death, i.e. clear cell renal carcinoma41,42. Moreover MPI KO cells had lower levels of intracellular cysteine and higher levels The metabolic phenotype of MPI KO cells with accumulation of PUFA and reduced FAO is reminiscent of that observed in cancer cells prone to ferroptotic cell death, i.e. clear cell renal carcinoma41,42. Moreover MPI KO cells had lower levels of intracellular cysteine and higher levels Nature Communications\| (2023) 14:2132 7 7 Article https://doi.org/10.1038/s41467-023-37652-0 Discussion Our data show that targeting MPI and MM sensitizes AML cells to AraC and FLT3-TKI. Recent reports, including one in leukaemic cells48, suggest that MPI could be a therapeutic target in cancer therapy22,23. Most of these reports however suggest that MPI inhibi- tion would limit the ability of cancer cells to withstand the toxicity of an exogenous high-dose mannose diet. Our data instead support the role of MPI, at physiological concentrations of mannose, as an enzyme important in supporting AML cells survival following therapy through its ability to modulate MM, protein glycosylation and activity of the ATF6 arm of the UPR. It is worth noting that the effects of MPI inhibition in vivo could be blunted by the endogenous man- nose production and dietary mannose and this could partly limit the role of MPI as a therapeutic target. Conversely, given that glucose, which is 100-fold more abundant than mannose in human plasma14, is the biggest source of mannose within the cells15 and considering the higher expression levels of MPI in AML samples, it is plausible that MPI inhibition will be detrimental to AML cells survival and its effects not rescued sufﬁciently by the relatively low levels of mannose in plasma. This outcome would be expected particularly if associated with reduced glucose metabolism, such as when combined with FLT3-TKI. Interestingly congenital MPI defects in humans are suc- cessfully treated with mannose supplements suggesting that phy- siological levels of mannose are unable to overcome glycosylation defects in MPI deﬁcient patients. An ideal drug target requires a robust therapeutic window in order to be exploitable. Although the embryonic lethality of Mpi in animal models is a reason for concern, our preclinical data suggest that HSPC are less reliant on MPI activity than AML cells. Moreover the lack of hematopoietic defects in humans carrying MPI mutation (albeit in a hypomorphic state) suggest that MPI inhibition might be tolerable for MPI depletion in primary FLT3ITD AML samples causes ATF6 activation, lipid peroxidation and sensitization to FLT3-TKI therapy We ﬁnally validated the effects of MPI depletion in primary FLT3ITD AML samples (Supplementary Table 1). Following MPI KD Nature Communications\| (2023) 14:2132 8 8 MPI KO cells activate the ATF6 arm of the unfolded protein response o inhibit oxidative phosphorylation metabolism in AML cells. a Top ed score gene signatures from RNA sequencing enriched in MPI gRNA5 3 cells compared to NT gRNA MOLM13 cells. Discussion Analysis performed with Enrichr; b Mean ﬂuorescence intensity of YFP from MOLM13 NT gRNA or MPI gRNA5 cells expressing a YFP linked ATF6 reporter treated with vehicle, AC220 (1 nM), mannose (100 µM) or combinations as indicated (left panel) with a repre- sentative ﬂow cytometry plot (left right panel). N = 3, 1 way Anova with Tukey’s correction for multiple comparisons; (c) Confocal microscopy images of NT gRNA and MPI gRNA5 MOLM13 cells stained for DAPI (blue, 1st column) and ATF6 (green, 2nd column) with a merged image (3rd column), treated with vehicle, AC220 (1 nM), mannose (100 µM), Ceapin A7 (1 µM) or in combinations as indicated 72 h after treatment. Experiment performed 3 times independently; (d) Colocalisation analysis from immunoﬂuorescence images of NTgRNA (left) and MPIgRNA5 (right) MOLM13 cells treated with vehicle, AC220 (1 nM), mannose (100 µM) and Ceapin A7 (1 µM) in combinations as indicated. Analysis performed with Coloc2 plugin in ImageJ, ordinary 1 way Anova with Tukey’s correction for multiple comparisons, Fig. 5 \| MPI KO cells activate the ATF6 arm of the unfolded protein response (UPR) to inhibit oxidative phosphorylation metabolism in AML cells. a Top ImageJ, ordinary 1 way Anova with Tukey’s correction for multiple comparisons, N = 13 for NTgRNA, N = 14 for MPIgRNA5; (e) Percentage of live cells of MPI gRNA5 MOLM13 cells treated with vehicle, AC220 (1 nM), mannose (100 µM), Ceapin A7 (1 µM) or in combinations as indicated 72 h after treatment. N = 3, 1 way Anova with Tukey’s correction for multiple comparisons; (f) SEAHORSE MitoStress assay comparison of MPI gRNA5 MOLM13 cells, treated with AC220 (1 nM), Ceapin A7 (1 µM), etomoxir (10 µM) or in combinations as indicated 72 h after treatment. N = 3, 2 way Anova with Sidak’s correction for multiple comparisons, with MPI gRNA5 AC220 as control population (2 left panels). Baseline OCR and ATP production of NT gRNA and MPI gRNA5 cells treated with vehicle, AC220 (1 nM), Ceapin A7 (1 µM), etomoxir (50 µM) or in combinations as indicated, N = 3, ns = not signiﬁcant, 1 way Anova with Tukey’s correction for multiple comparisons (2 right panels). For all panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, **=p < 0.001. Source data are provided as a Source Data ﬁle. All data presented as mean values ± SEM. Fig. Discussion Analysis performed with b Mean ﬂuorescence intensity of YFP from MOLM13 NT gRNA or MPI cells expressing a YFP linked ATF6 reporter treated with vehicle, AC220 mannose (100 µM) or combinations as indicated (left panel) with a repre- e ﬂow cytometry plot (left right panel). N = 3, 1 way Anova with Tukey’s on for multiple comparisons; (c) Confocal microscopy images of NT gRNA gRNA5 MOLM13 cells stained for DAPI (blue, 1st column) and ATF6 (green, umn) with a merged image (3rd column), treated with vehicle, AC220 mannose (100 µM), Ceapin A7 (1 µM) or in combinations as indicated 72 h atment. Experiment performed 3 times independently; (d) Colocalisation from immunoﬂuorescence images of NTgRNA (left) and MPIgRNA5 (right) 3 cells treated with vehicle AC220 (1 nM) mannose (100 M) and Ceapin A7 ImageJ, ordinary 1 way Anova with Tukey’s correction for multiple compari N = 13 for NTgRNA, N = 14 for MPIgRNA5; (e) Percentage of live cells of MPI MOLM13 cells treated with vehicle, AC220 (1 nM), mannose (100 µM), Ceap (1 µM) or in combinations as indicated 72 h after treatment. N = 3, 1 way Anov Tukey’s correction for multiple comparisons; (f) SEAHORSE MitoStress assa comparison of MPI gRNA5 MOLM13 cells, treated with AC220 (1 nM), Ceapi (1 µM), etomoxir (10 µM) or in combinations as indicated 72 h after treatment 2 way Anova with Sidak’s correction for multiple comparisons, with MPI gR AC220 as control population (2 left panels). Baseline OCR and ATP product NT gRNA and MPI gRNA5 cells treated with vehicle, AC220 (1 nM), Ceapin A7 etomoxir (50 µM) or in combinations as indicated, N = 3, ns = not signiﬁcant Anova with Tukey’s correction for multiple comparisons (2 right panels). Fo panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, **=p < 0.0 Source data are provided as a Source Data ﬁle. All data presented as mean values ± SEM e https://doi.org/10.1038/s41467-023-376 Article https://doi.org/10.1038/s41467-023-37652-0 Fig. 5 \| MPI KO cells activate the ATF6 arm of the unfolded protein response (UPR) to inhibit oxidative phosphorylation metabolism in AML cells. a Top combined score gene signatures from RNA sequencing enriched in MPI gRNA5 MOLM13 cells compared to NT gRNA MOLM13 cells. Discussion 5 \| MPI KO cells activate the ATF6 arm of the unfolded protein response (UPR) to inhibit oxidative phosphorylation metabolism in AML cells. a Top combined score gene signatures from RNA sequencing enriched in MPI gRNA5 MOLM13 cells compared to NT gRNA MOLM13 cells. Analysis performed with Enrichr; b Mean ﬂuorescence intensity of YFP from MOLM13 NT gRNA or MPI gRNA5 cells expressing a YFP linked ATF6 reporter treated with vehicle, AC220 (1 nM), mannose (100 µM) or combinations as indicated (left panel) with a repre- sentative ﬂow cytometry plot (left right panel). N = 3, 1 way Anova with Tukey’s correction for multiple comparisons; (c) Confocal microscopy images of NT gRNA and MPI gRNA5 MOLM13 cells stained for DAPI (blue, 1st column) and ATF6 (green, 2nd column) with a merged image (3rd column), treated with vehicle, AC220 (1 nM), mannose (100 µM), Ceapin A7 (1 µM) or in combinations as indicated 72 h after treatment. Experiment performed 3 times independently; (d) Colocalisation analysis from immunoﬂuorescence images of NTgRNA (left) and MPIgRNA5 (right) MOLM13 cells treated with vehicle, AC220 (1 nM), mannose (100 µM) and Ceapin A7 (1 µM) in combinations as indicated. Analysis performed with Coloc2 plugin in (UPR) to inhibit oxidative phosphorylation metabolism in AML cells. a Top combined score gene signatures from RNA sequencing enriched in MPI gRNA5 MOLM13 cells compared to NT gRNA MOLM13 cells. Analysis performed with Enrichr; b Mean ﬂuorescence intensity of YFP from MOLM13 NT gRNA or MPI gRNA5 cells expressing a YFP linked ATF6 reporter treated with vehicle, AC220 (1 nM), mannose (100 µM) or combinations as indicated (left panel) with a repre- sentative ﬂow cytometry plot (left right panel). N = 3, 1 way Anova with Tukey’s correction for multiple comparisons; (c) Confocal microscopy images of NT gRNA and MPI gRNA5 MOLM13 cells stained for DAPI (blue, 1st column) and ATF6 (green, 2nd column) with a merged image (3rd column), treated with vehicle, AC220 (1 nM), mannose (100 µM), Ceapin A7 (1 µM) or in combinations as indicated 72 h after treatment. Experiment performed 3 times independently; (d) Colocalisation analysis from immunoﬂuorescence images of NTgRNA (left) and MPIgRNA5 (right) MOLM13 cells treated with vehicle, AC220 (1 nM), mannose (100 µM) and Ceapin A7 (1 µM) in combinations as indicated. Discussion Analysis performed with Coloc2 plugin in Nature Communications\| (2023) 14:2132 9 Article https://doi.org/10.1038/s41467-023-37652-0 https://doi.org/10.1038/s4146 evated mannose metabolism may be more critical for g tissues, as suggested by a recent report using hypo- leles of PMM2, the enzymatic step following MPI in MM, that ated that, while mannose sugar donors are necessary for c development, they are only needed at very low levels after ent49. Thus, the activity of MPI might be crucial for rapidly ells but less essential in adult established tissues, a ﬁnding mmon in drug development where often embryonically lethal/essential genes are shown to be good drug targets with accep- table toxicity in the adult/homeostatic context50. MPI validity as a therapeutic target has been related to its ability to detoxify exogenous high levels of mannose23,51. However our mechanistic studies show that, even in the presence of physiological mannose concentrations, MPI inhibition can increase the toxicity of FLT3-TKI or AraC to AML cells due to defective fatty acid metabolism. While other reports on the role of MPI in cancer have established its mmunications\| (2023) 14:2132 10 HSPC18. Elevated mannose metabolism may be more critical for developing tissues, as suggested by a recent report using hypo- morphic alleles of PMM2, the enzymatic step following MPI in MM, that demonstrated that, while mannose sugar donors are necessary for embryonic development, they are only needed at very low levels after development49. Thus, the activity of MPI might be crucial for rapidly dividing cells but less essential in adult established tissues, a ﬁnding not uncommon in drug development where often embryonically lethal/essential genes are shown to be good drug targets with accep- table toxicity in the adult/homeostatic context50. lethal/essential genes are shown to be good drug targets with accep- table toxicity in the adult/homeostatic context50. MPI validity as a therapeutic target has been related to its ability to detoxify exogenous high levels of mannose23,51. However our mechanistic studies show that, even in the presence of physiological mannose concentrations, MPI inhibition can increase the toxicity of FLT3-TKI or AraC to AML cells due to defective fatty acid metabolism. While other reports on the role of MPI in cancer have established its MPI validity as a therapeutic target has been related to its ability to detoxify exogenous high levels of mannose23,51. https://doi.org/10.1038/s41467-023-37652-0 Representative plots of ﬂow cytometry ﬂuorescence intensity plot of MPI gRNA5 with AC220 (1 nM), mannose (100 µM) or in combinations as indicated along with an unstained MPI gRNA5 control at 510 nm (right); (d) Percentage of live NT gRNA and MPI gRNA5 MOLM13 cells treated with vehicle, AC220 (1 nM), role in supporting or rewiring central carbon/glucose metabolism, none had previously unveiled a connection between MM and fatty acid metabolism, although the ability of exogenous mannose to upregulate FAO has been observed in T cells52. We show that the effects on fatty acid metabolism are prompted by reduction in protein N-glycosyla- tion, a known consequence of MPI depletion, which likely drives the preferential activation of the ATF6 arm of the UPR32. Interestingly reduced protein N-Glycosylation has already been shown to be a therapeutic vulnerability in solid cancers22 and leukaemia53 and speci- ﬁcally in FLT3ITD AML due to reduced surface FLT3 expression upon glycosylation inhibition54. While we could show a pattern of reduced protein glycosylation in our models, we did not detect consistent ﬁndings for reduced FLT3 expression (Supplementary Fig. 10) thus making this mechanism less likely. On the other hand, we observed that activation of the ATF6 arm of the UPR led to downregulation of FAO in AML MPI KO cells through transcriptional downregulation of PPARA and other key FAO genes. FAO has emerged as a therapeutic target in AML11,55 and is important in resistance to both standard13 and novel therapies56 via its ability to support oxidative phosphorylation. Therefore our ﬁndings support the role of FAO as a resistance mechanism in AML. Although FLT3-TKI and AraC have different mode of actions, resistance to both these drugs relies on enhanced FAO13,57,58 and likely explains why MPI KO by causing defectiveFAO and increased PUFAs levels leads to sensitization to both therapies.Moreover the role of reduced glycosylation and ATF6 activation in driving these effects further suggest that the main role of MPI in our systems is the pro- duction of mannose from glucose, rather than the opposite, and explains why MPI inhibition was able to cause cell death in the absence of high mannose concentration. lipid uptake can enrich cancer cells membranes in PUFA since in contrast to de-novo synthetisized FA, diet and stroma derived FA are enriched for PUFA thus increasing susceptibility to ferroptosis65. Moreover, there is evidence that PUFA are most readily beta-oxidised in mammalian cells compared to SFA66. Ethics statement All work involving mice was performed under UK Home Ofﬁce Personal Project License Number PP4153210 (Protocol 4) with further ethical approval for individual studies granted by our in vivo work establish- ment (BSU) Charterhouse Square Queen Mary University of London. In all experiments the maximum severity allowed under the project license was not exceeded. All work involving human samples was covered under ethics approval granted by East of England Cambridge Research Ethics Committee under the title “Barts Haemato-Oncology Research Tissue Bank” REC ref. 21/EE/0123, IRAS project ID 283103. Although the effects of MPI KO on cell death could be partly driven by defective TCA cycle activity leading to ATP depletion, we did not rescue the effects of MPI KO by supplementing the TCA cycle intermediate DMaKG. Instead AML MPI KO cells, particularly in the presence of FLT3-TKI, displayed features consistent with a state primed for ferroptotic cell death. These included reduced FAO, increased CD36 expression and PUFA uptake, reduced expression of SLC7A11, the cysteine transporter and target of the ferroptotic indu- cing agent erastin59 and increased sensitivity to ferroptosis inducing agents and PUFA-induced toxicity. Ferroptosis is a non-apoptotic, iron dependent form of regulated cell death that is speciﬁcally char- acterised by the accumulation of lipid peroxides60. Susceptibility to ferroptosis has been shown in solid cancer models to be a feature of cells in a therapy resistant state because of their high dependency on the lipid hydroperoxides quenching proteins such as glutathione peroxidase 461. Reduced FAO and downregulation of PPARA the master regulator of lipid catabolism have also been linked to susceptibility to ferroptosis in other cancer models41,42,62 while increased uptake of PUFA via CD36 has been shown to trigger ferroptotic cell death/sus- ceptibility in T cells63,64. In this respect, it is worth noting that increased https://doi.org/10.1038/s41467-023-37652-0 Although FLT3-TKI and AraC have different mode of actions, resistance to both these drugs relies on enhanced FAO13,57,58 and likely explains why MPI KO by causing defectiveFAO and increased PUFAs levels leads to sensitization to both therapies.Moreover the role of reduced glycosylation and ATF6 activation in driving these effects further suggest that the main role of MPI in our systems is the pro- duction of mannose from glucose, rather than the opposite, and explains why MPI inhibition was able to cause cell death in the absence of high mannose concentration. In conclusion, our work supports the role of metabolic rewiring in driving therapy resistance in AML and demonstrates that targeting MPI and MM sensitizes AML cells to AraC and FLT3-TKI. Mechanistically we unveil a connection between MM and fatty acid metabolism, via pre- ferential activation of the ATF6 arm of the UPR, leading to cellular PUFA accumulation, lipid peroxidation and ferroptotic cell death (Fig. 8). Finally, our ﬁndings also suggest that triggering ferroptosis could be used therapeutically to eradicate therapy-resistant AML cells. https://doi.org/10.1038/s41467-023-37652-0 mannose (100 µM), ferrostatin-1 (5 µM, left), liproxstatin (1 µM, right) or combina- tions as indicated for 6 days. N = 3 for ferrostatin, N = 2 for liproxstatin, 1 way Anova with Tukey’s correction for multiple comparisons. e Kaplan-Meier survival curve showing survival time of mice after transplantation with THP1 NT gRNA or MPI gRNA5 cells, treated with cytarabine (50 mg/kg) for 7 days concurrent with or without liproxstatin treatment (10 mg/kg) for 10 days. Log-rank (Mantel-Cox) test; (f) Mean ﬂuorescence intensity of 4-hydroxynonenal normalised to DAPI ﬂuores- cence from immunoﬂuorescence images of THP-1 cells sorted from mice after transplantation with THP1 NT gRNA or MPI gRNA5 cells and soon after treatment with cytarabine (50 mg/kg) for 7 days concurrent with or without liproxstatin treatment (10 mg/kg) for 10 days. N = 2 ﬁelds of view from 4 animals in all samples except MPIgRNA5 with AraC where 5 animals were used. 1 way Anova with Tukey’s correction. For all panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, **=p < 0.001. Source data are provided as a Source Data ﬁle. All data presented as mean ± SEM. Fig. 6 \| MPI KO drives lipid peroxidation and ferroptotic cell death in AML cells. a Levels of ROS and lipid peroxidation linked metabolites from global metabo- lomics proﬁling of NT gRNA and MPI gRNA5 treated with vehicle, AC220 (1 nM), mannose (100 µM) or in combinations as indicated for 48 h; (b) GSEA for an erastin induced (ferroptotic) gene signature45 from RNA-seq comparing MPI gRNA5 with AC220 (1 nM) to MPI gRNA5 with AC220 (1 nM) and mannose (100 µM) (left) and MPI gRNA5 with AC220 to NT gRNA with AC220 (right). FDR and NES from 1000 permutations; (c) Mean ﬂuorescence intensity at 510 nm of Bodipy 581/591 C11, which shows level of lipid peroxidation, in NT gRNA and MPI gRNA5 MOLM13 cells treated with AC220 (1 nM), mannose (100 µM) or in combinations as indicated 72 h after treatment. N = 3, 1 way Anova with Tukey’s correction for multiple compar- isons (left). Discussion However our mechanistic studies show that, even in the presence of physiological mannose concentrations, MPI inhibition can increase the toxicity of FLT3-TKI or AraC to AML cells due to defective fatty acid metabolism. While other reports on the role of MPI in cancer have established its Nature Communications\| (2023) 14:2132 10 Article https://doi.org/10.1038/s41467-023-37652-0 https://doi.org/10.1038/s41467-023-37652-0 Therefore, a combination of increased lipid uptake and reduced FAO will lead to preferential accumulation of PUFA with an increased tendency towards lipid per- oxidation, as seen in our MPI KO cells. Our rescue experiments with radical trapping agents conﬁrm that ferroptosis is mostly driving cell death in MPI KO cells and future studies will be needed to further deﬁne the molecular mechanisms through which MPI KO drives increased PUFA uptake, CD36 expression and reduced cysteine uptake and SLC7A11 expression. Moreover, our ﬁndings also suggest that, similarly to persistent cells in solid cancers61, triggering ferroptosis can be leveraged to drive cell death in therapy-resistant AML cells. How- ever, the role of ferroptosis susceptibility in AML therapy resistant/ persister cells will require further studies. role in supporting or rewiring central carbon/glucose metabolism, none had previously unveiled a connection between MM and fatty acid metabolism, although the ability of exogenous mannose to upregulate FAO has been observed in T cells52. We show that the effects on fatty acid metabolism are prompted by reduction in protein N-glycosyla- tion, a known consequence of MPI depletion, which likely drives the preferential activation of the ATF6 arm of the UPR32. Interestingly reduced protein N-Glycosylation has already been shown to be a therapeutic vulnerability in solid cancers22 and leukaemia53 and speci- ﬁcally in FLT3ITD AML due to reduced surface FLT3 expression upon glycosylation inhibition54. While we could show a pattern of reduced protein glycosylation in our models, we did not detect consistent ﬁndings for reduced FLT3 expression (Supplementary Fig. 10) thus making this mechanism less likely. On the other hand, we observed that activation of the ATF6 arm of the UPR led to downregulation of FAO in AML MPI KO cells through transcriptional downregulation of PPARA and other key FAO genes. FAO has emerged as a therapeutic target in AML11,55 and is important in resistance to both standard13 and novel therapies56 via its ability to support oxidative phosphorylation. Therefore our ﬁndings support the role of FAO as a resistance mechanism in AML. Cell culture MOLM13, MV411 and THP-1 cells were obtained from the Sanger Institute cancer cell collection and were grown in 1640-RPMI (Gibco, 21875034) supplemented with 10% FBS (Sigma, F4135) and 1% peni- cillin/streptomycin (Gibco, 15140122) at 37 °C with 5% COO2, unless otherwise stated. For SEAHORSE analysis, media comprised of SEA- HORSE RPMI (Agilent), without bicarbonate and FBS, supplemented with 11 mM glucose and 2mM L-Glutamine (SEAHORSE media), unless otherwise stated. HEK-293T Phoenix cells were obtained from the Huntly lab and grown in DMEM (Gibco, 41966029) supplemented with 10% FBS (Sigma, F4135) and 1% penicillin/streptomycin (Gibco, Nature Communications\| (2023) 14:2132 11 Article https://doi.org/10.1038/s41467-023-37652-0 15140122) at 37 °C with 5% COO2 unless otherwise stated. For experi- ments where delipidated FBS was used, this was done by the adding of 2 g of fumed silica to 100 ml of heat inactivated FBS (Sigma, F4135) and stirring overnight at room temperature. The mixture was then cen- trifuged at 4000 × g for 25 min to pellet the silica and the supernatant retained. The supernatant was then ﬁltered through a 0.2 µm ﬁlter to ensure sterility before storage at −20 °C for further use. Delipidated FBS was used in RPMI at a ﬁnal concentration of 10%. Cells were only kept in delipidated FBS for 3 days as any longer culture of cells in these conditions lead to large scale cell death in all conditions. Lentiviral transduction 293T-Pheonix cells were grown in DMEM (Gibco) containing 10% FBS and transfected with construct plasmid (NTshRNA and MPI shRNA1 Nature Communications\| (2023) 14:2132 12 15140122) at 37 °C with 5% COO2 unless otherwise stated. For experi- ments where delipidated FBS was used, this was done by the adding of 2 g of fumed silica to 100 ml of heat inactivated FBS (Sigma, F4135) and i i i h Th i h FBS was used in RPMI at a ﬁnal concentration of 10%. Cells wer kept in delipidated FBS for 3 days as any longer culture of cells in conditions lead to large scale cell death in all conditions. FBS was used in RPMI at a ﬁnal concentration of 10%. Cells were only kept in delipidated FBS for 3 days as any longer culture of cells in these conditions lead to large scale cell death in all conditions. FBS was used in RPMI at a ﬁnal concentration of 10%. Cell culture Cells were only kept in delipidated FBS for 3 days as any longer culture of cells in these conditions lead to large scale cell death in all conditions. 15140122) at 37 °C with 5% COO2 unless otherwise stated. For experi- ments where delipidated FBS was used, this was done by the adding of 2 g of fumed silica to 100 ml of heat inactivated FBS (Sigma, F4135) and stirring overnight at room temperature. The mixture was then cen- trifuged at 4000 × g for 25 min to pellet the silica and the supernatant retained. The supernatant was then ﬁltered through a 0.2 µm ﬁlter to ensure sterility before storage at −20 °C for further use. Delipidated https://doi.org/10.1038/s41467-023-37652-0 WT AML cells treated with both standard and FLT3 inhibitor therapies are able to escape cell death by adapting their metabolism, in this case by switching from glycolysis to fatty acid oxidation. Conversely treated AML cells with depleted MPI have preferential activation of the ATF6 arm of the unfolded proteins response and 2 TRIPZ inducible shRNA, Horizon Discovery, ATF6-YFP reporter ﬁg. S5f), packaging construct (psPAX) and glycoprotein (pMD2G) using lipofectamine (Gibco, 18324010) for transient transfection. After 24 h, medium was replaced by RPMI with 5% FBS before overnight incubation at 32 °C with 5% COO2. Media containing virus was har- vested and ﬁltered through a 0.45 µm ﬁlter to remove any cells 24 and 48 h post media change and either used immediately or stored at −80 °C in 1 ml aliquots for further use. and 2 TRIPZ inducible shRNA, Horizon Discovery, ATF6-YFP reporter ﬁg. S5f), packaging construct (psPAX) and glycoprotein (pMD2G) using lipofectamine (Gibco, 18324010) for transient transfection. After 24 h, medium was replaced by RPMI with 5% FBS before overnight incubation at 32 °C with 5% COO2. Media containing virus was har- vested and ﬁltered through a 0.45 µm ﬁlter to remove any cells 24 and 48 h post media change and either used immediately or stored at −80 °C in 1 ml aliquots for further use. volume of 10% of total culture volume. Samples were then cultured overnight before repeating. Virus was removed by washing three times with sterile PBS before continuing with culture as detailed below. For MPI and NTshRNA transduced samples, cells were cultured in normal media with the addition of puromycin (1 µg/ml ﬁnal con- centration, ThermoFisher, A1113803) for selection of transduced cells and doxycycline (1 µg/ml ﬁnal concentration, Sigma, D3072) for acti- vation of the shRNA construct. Activation of the construct and trans- duction efﬁciency was determined by the percentage of RFP + cells. Cells were then cultured as normal with the addition of 1 µg/ml dox- ycycline for 72 h before plating for experiments to induce knockdown of the target genes. For transduction of cell lines, 1 million cells were seeded in 1 ml RMPI containing virus with 4 µg/ml polybrene (Santa Cruz Bio- technology, TR1003) and spun for 90 min at 900 × g on 2 consecutive days. https://doi.org/10.1038/s41467-023-37652-0 8 \| Model: loss of MPI leads to cell death in AML through inhibition of FAO leading to PUFA accumulation and ferroptosis. A model of the proposed mechanism. WT AML cells treated with both standard and FLT3 inhibitor therapies are able to escape cell death by adapting their metabolism, in this case by switching from glycolysis to fatty acid oxidation. Conversely treated AML cells with depleted MPI have preferential activation of the ATF6 arm of the unfolded proteins response which inhibits fatty acid oxidation. This is paired with increased uptake of fatty acids, particularly polyunsaturated fatty acids, by MPI depleted cells. Both these effects lead to intracellular accumulation of PUFAs and PUFAs containing lipid species. These undergo lipid peroxidation leading to ferroptotic cell death in these cells. which inhibits fatty acid oxidation. This is paired with increased uptake of fatty acids, particularly polyunsaturated fatty acids, by MPI depleted cells. Both these effects lead to intracellular accumulation of PUFAs and PUFAs containing lipid species. These undergo lipid peroxidation leading to ferroptotic cell death in these cells. which inhibits fatty acid oxidation. This is paired with increased uptake of fatty acids, particularly polyunsaturated fatty acids, by MPI depleted cells. Both these effects lead to intracellular accumulation of PUFAs and PUFAs containing lipid species. These undergo lipid peroxidation leading to ferroptotic cell death in these cells. Fig. 8 \| Model: loss of MPI leads to cell death in AML through inhibition of FAO leading to PUFA accumulation and ferroptosis. A model of the proposed mechanism. WT AML cells treated with both standard and FLT3 inhibitor therapies are able to escape cell death by adapting their metabolism, in this case by switching from glycolysis to fatty acid oxidation. Conversely treated AML cells with depleted MPI have preferential activation of the ATF6 arm of the unfolded proteins response which inhibits fatty acid oxidation. This is paired with increased uptake of fatty acids, particularly polyunsaturated fatty acids, by MPI depleted cells. Both these effects lead to intracellular accumulation of PUFAs and PUFAs containing lipid species. These undergo lipid peroxidation leading to ferroptotic cell death in these cells. Fig. 8 \| Model: loss of MPI leads to cell death in AML through inhibition of FAO leading to PUFA accumulation and ferroptosis. A model of the proposed mechanism. https://doi.org/10.1038/s41467-023-37652-0 Cells were then placed in an incubator at 32 °C for 4 h before the addition of a further 3 ml of RPMI with 5% FBS. Cells were then placed in an incubator at 37 °C for 24 h before the virus was removed by washing three times with sterile PBS. For the ATF6 reporter, transduced cells were selected by treating cells with 1 µM tunicamycin (Tocris, 3516) to induce YFP expression in transduced cells before cell sorting for the YFP positive population. Cells were then cultured as normal. Primary samples were cultured in StemSpan II (STEMCELL- Technologies, 09605) supplemented with 150 ng/ml SCF (Biolegend, 573908), 150 ng/ml Flt-3 (Biolegend, 550602), 10 ng/ml IL-6 (Biole- gend, 570802), 25 ng/ml G-CSF (Biolegend, 578602), 20 ng/ml TPO (Biolegend, 763702), 1% HEPES (Gibco, 15630056) and 1% Penicillin/ Streptomycin. All samples were cultured at 37 °C with 5% COO2. Cells were not spun and instead viral supernatant was added to a ﬁnal Lentiviral transduction Lentiviral transduction 293T-Pheonix cells were grown in DMEM (Gibco) containing 10% FBS and transfected with construct plasmid (NTshRNA and MPI shRNA1 Nature Communications\| (2023) 14:2132 12 Article https://doi.org/10.1038/s41467-023-37652-0 https://doi.org/10.1038/s41467-023-37652-0 Fig. 7 \| MPI depletion in primary FLT3ITD AML samples causes ATF6 activation, lipid peroxidation and sensitization to FLT3-TKI therapy. a Percentage of live cells of NT shRNA and MPI shRNA1 primary FLT3ITD AML MNC treated with vehicle, AC220 (2.5 nM) or AC220 (2.5 nM) and mannose (100 µM), as indicated normalised to NT shRNA with vehicle for each sample. Treated for 3 days, N = 11, 1 way Anova with Tukey’s correction for multiple comparisons; (b) Percentage of live cells of NT shRNA and MPI shRNA1 normal CD34 + primary cells treated with vehicle, AC220 (2.5 nM) or AC220 (2.5 nM) and mannose (100 µM), as indicated normalised to NT shRNA with vehicle for each sample. Treated for 3 days, N = 3; (c) Mean ﬂuores- cence intensity at 510 nm of Bodipy 581/591 C11, which shows level of lipid perox- idation, in NT shRNA and MPI shRNA1 FLT3ITD primary AML MNC treated with vehicle, AC220 (2.5 nM) or AC220 (2.5 nM) and mannose (100 µM), as indicated normalised to NT shRNA with vehicle at 72 h after treatment. N = 4, 1 way Anova with Tukey’s correction for multiple comparisons (left). Representative plots of ﬂow cytometry ﬂuorescence intensity plot NT shRNA and MPI shRNA1 with AC220 (2.5 nM) and mannose (100 µM) as indicated at 510 nm (right); (d) Confocal microscopy images of FLT3ITD primary AML MNC stained for DAPI (blue, 1st col- umn) and ATF6 (green, 2nd column) with a merged image (3rd column), treated with vehicle, AC220 (2.5 nM) and mannose (100 µM) combinations as indicated 72 h after treatment with colocalisation analysis (right graph). N = 6 for all except MPIshRNA with AC220 where N = 7. 1 way Anova with Tukey’s correction; (e) Per- centage of live cells of NT shRNA and MPI shRNA1 primary FLT3ITD AML MNC treated with vehicle, AC220 (2.5 nM), Ferrostatin-1 (5 µM) or liproxstatin (1 µM) as indicated, normalised to NT shRNA with vehicle for each sample. Treated for 3 days, N = 3, 1 way Anova with Tukey’s correction for multiple comparisons. For all panels, ns = not signiﬁcant, =p < 0.05, =p < 0.01, =p < 0.005, ***=p < 0.001. Source data are provided as a Source Data ﬁle. All data presented as mean values ± SEM. Fig. Nature Communications\| (2023) 14:2132 Human samples Erastin (Sigma, E7781) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 4 µM, unless otherwise stated. Sulfosuccinimidyl Oleate (SSO, Cayman Chemicals, 11211) was dissolved in DMSO and cells treated at a ﬁnal concentration of 100 µM. Arachidonic acid (Sigma, 10931), palmitic acid (Acros organics, 129702500) and oleate (Sigma, O7501) were dissolved in isopropanol, with cells treated in concentrations as outlined below. AA147 (Tocris, 6759) was dissolved in DMSO and cells were treated at a ﬁnal con- centration of 5 µM. Tunicamycin (Tocris, 3516) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 1 µM. ZVAD (Sigma, V116) was dissolved in DMSO and cells were treated at a ﬁnal con- centration of 20 µM. Necrostatin-1 (Sigma, N9037) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 20 µM. GSK2656157 (SelleckChem, S7033) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 200 nM. MKC-3946 (Sell- eckChem, S8286) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 1 µM. Dimethyl alpha-ketoglutarate (Sigma, 349631) was used at a ﬁnal concentration of 4 mM. RSL3 (SelleckChem, S8155) was dissolved in DMS and used at a ﬁnal concentration of 100 nM unless otherwise stated. information was not available for the samples used. Bone marrow and peripheral blood samples containing >80% blasts were collected from adult patients with AML at diagnosis or relapse and cryopreserved after mononuclear cell (MNCs) isolation using Lymphoprep™(07851, STEMCELL Technologies) based density gradient centrifugation. Nor- mal CD34+ enriched samples were taken from myeloma patients at the time of peripheral blood stem cell collections. Samples were thawed and transduced via lentivirus with NTshRNA or MPIshRNA as described above. information was not available for the samples used. Bone marrow and peripheral blood samples containing >80% blasts were collected from adult patients with AML at diagnosis or relapse and cryopreserved after mononuclear cell (MNCs) isolation using Lymphoprep™(07851, STEMCELL Technologies) based density gradient centrifugation. Nor- mal CD34+ enriched samples were taken from myeloma patients at the time of peripheral blood stem cell collections. Samples were thawed and transduced via lentivirus with NTshRNA or MPIshRNA as described above. CRISPR KO gRNA sequences NTgRNA - ATTTTCGTACCCTGGGACGC MPIgRNA2 - AGGATCTTGGCATCCCCTC MPIgRNA5 – CAATCAGGAACTGAAACTC CRISPR KO gRNA sequences NTgRNA - ATTTTCGTACCCTGGGACGC MPIgRNA2 - AGGATCTTGGCATCCCCTC MPIgRNA5 – CAATCAGGAACTGAAACTC Generation of CRISPR knockout clones Generation of CRISPR knockout clones The knock-out of genes was accomplished using the CRISPR/ Cas9 system. For this, MOLM13, MV411 and THP-1 cell lines were transduced with Cas9 lentivirus (Addgene, lentiCas9-Blast, #52962), generating Cas9 expressing cell lines. Functional gRNA sequences were obtained from genome-wide gRNA library and ligated in the backbone, conjugating MPI gRNA with mCherry (Addgene, pKLV2-U6gRNA5(Bbsl)- PGKpuro2AmCherry-W, #67977). Virus for Cas9, MPI gRNA and NT gRNA were produced and transduced as described above. Obtaining single cell colonies was performed using methylcellulose media (H4531; STEMCELL Technologies, Cambridge, UK), where cells were cultured in semi-liquid conditions to minimize movement. Seeding cells in methylcellulose in a low density (~500 cells/mL) permitted separate colonies to form from a single cell. After picking the colonies from the assay and culturing them in small volumes, the generation of cell lines from a single parent all with the same construct was realized. Competition assays 125000 Cas9/KO-mCherry (NTgRNA or MPIgRNA) per ml were added along with 125000 Cas9 cells with no mCherry (parental) per ml and were cultured in the presence or absence of AC220 (1 nM). At days 3, 6 and 9 the ratio between mCherry positive and negative cells were measured by ﬂow cytometry using LSR Fortessa (BD Biosciences) instruments, with data normalised to the NT:parental ratio at day 3. Downstream analysis was performed using FlowJo version 10.6.1 (BD Biosciences). Example FACS gating strategies are available in the Supplementary Information. Viability assay Around 1 million cells per condition were harvested by centrifugation at 500xg, before washing twice with PBS at 4 °C. Cells were then resuspended in annexin V binding buffer (BioWorld, 21720002-1) and annexin V-FITC (BioLegend, 640945, 1:150) and 7-AAD (BD Bios- ciences, 559925, 1:150) were added. Positive control cells for single colour compensation of the FITC and 7-AAD were generated by heat- ing cell suspensions for 3 min at 70 °C. Cells were incubated for at least 15 min at 4 °C, protected from light. For analysis of live cells by DAPI exclusion cells were harvested as described above and resuspended in PBS. DAPI at a ﬁnal concentration of 0.5 µg/ml was added. Cells were then kept at 4 °C protected from light until analysis. Cells were then analysed using LSR Fortessa (BD Biosciences) instruments. Down- stream analysis was performed using FlowJo version 10.6.1 (BD Bios- ciences). Example FACS gating strategies are available in Supplementary Information. SiRNA transfection ATF6 (Ambion, AM16708) and non-silencing control (Ambion, 4390843) siRNAs were resuspended in nuclease free water and stored at −20 °C until further use. Molm13 cells were replated at a density of 200,000 cells/ml in normal RPMI with 10% FBS and 1% pen/strep 48 h before transfection. On day of transfection cells were pelleted, washed twice with PBS and ﬁnally resuspended in 100 µl buffer SF (Lonza, V4XC-2012). Cells were transferred to Lonza Nucelocuvettes and placed into the Lonza Nucelofector 4D X unit and transfected using program DJ-100. Cells were then removed and added to 2 ml of pre-warmed RPMI with 10% FBS and 1% pen/strep. Cells were taken for RNA extraction and qPCR analysis after 48 h and plated for annexin analysis at the same time. Cell counting C ll Cells were counted either using a CASY cell counter (Cambridge Biosciences) or a LUNA automated cell counter (Logos Biosystems). For LUNA cell counting, cell samples were mixed in a 1:1 ratio with 0.4% trypan blue solution. Human samples The use of human samples, collection and publication of individual- level clinical data was approved by the East of England - Cambridge Research Ethics Committee REC 21/EE/0123. The conduct of the study was fully compliant with the ethical approval and sex and gender Nature Communications\| (2023) 14:2132 13 https://doi.org/10.1038/s41467-023-37652-0 Article 1 µM, unless otherwise stated. Ceapin A7 (Sigma, SML2330) was dis- solved in DMSO and cells were treated at a ﬁnal concentration of 1 µM, unless otherwise stated. Fenoﬁbrate (Sigma, F6020) was dissolved in DMSO and cells were treated at a ﬁnal concentration at 10 µM, unless otherwise stated. Ferrostatin-1 (Sigma, SML0583) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 5 µM, unless otherwise stated. Erastin (Sigma, E7781) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 4 µM, unless otherwise stated. Sulfosuccinimidyl Oleate (SSO, Cayman Chemicals, 11211) was dissolved in DMSO and cells treated at a ﬁnal concentration of 100 µM. Arachidonic acid (Sigma, 10931), palmitic acid (Acros organics, 129702500) and oleate (Sigma, O7501) were dissolved in isopropanol, with cells treated in concentrations as outlined below. AA147 (Tocris, 6759) was dissolved in DMSO and cells were treated at a ﬁnal con- centration of 5 µM. Tunicamycin (Tocris, 3516) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 1 µM. ZVAD (Sigma, V116) was dissolved in DMSO and cells were treated at a ﬁnal con- centration of 20 µM. Necrostatin-1 (Sigma, N9037) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 20 µM. GSK2656157 (SelleckChem, S7033) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 200 nM. MKC-3946 (Sell- eckChem, S8286) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 1 µM. Dimethyl alpha-ketoglutarate (Sigma, 349631) was used at a ﬁnal concentration of 4 mM. RSL3 (SelleckChem, S8155) was dissolved in DMS and used at a ﬁnal concentration of 100 nM unless otherwise stated. 1 µM, unless otherwise stated. Ceapin A7 (Sigma, SML2330) was dis- solved in DMSO and cells were treated at a ﬁnal concentration of 1 µM, unless otherwise stated. Fenoﬁbrate (Sigma, F6020) was dissolved in DMSO and cells were treated at a ﬁnal concentration at 10 µM, unless otherwise stated. Ferrostatin-1 (Sigma, SML0583) was dissolved in DMSO and cells were treated at a ﬁnal concentration of 5 µM, unless otherwise stated. Treatments AC220 (Insight Biotechnology, HY13001) was dissolved in DMSO and cells were treated at 1 nM ﬁnal concentration, unless otherwise stated. Mannose (Sigma, 112585) was dissolved in sterile water and cells were treated at 100 µM ﬁnal concentration, unless otherwise stated. Eto- moxir (Cayman Chemicals, 11969) was dissolved in DMSO and cells were treated at 10 µM ﬁnal concentration for all apoptosis and lipid peroxidation assays and 50 µM for all SEAHORSE experiments, unless otherwise stated. MLS0315771 (Insight Biotechnology, HY112945) was dissolved in DMSO and cells were treated at a ﬁnal concentration of Human samples After transduction was conﬁrmed by the presence of RFP expres- sion by ﬂow cytometry, primary samples were cultured on a stroma of MS-5 mouse cells that had been irradiated in H5100 media (STEMCELL Technologies) supplemented with penicillin/streptomycin, interleukin- 3, G-CSF and TPO at 20 ng/ml ﬁnal. For all assays, AML cells were removed from the co-culture and cultured without stroma. Assays were performed as described in viability section, however less cells were used in each case as numbers of cells in the primary samples was limited. To ensure that only transduced primary cells were removed and used for assays, RFP-positive percentage was assessed to be above 95%. Nature Communications\| (2023) 14:2132 IC50 calculations 50000 NT gRNA, MPI gRNA2 and MPI gRNA5 MOLM13 cells were seeded and treated with increasing concentrations of AC220 in tripli- cate. After 3 days cell proliferation was measured using CellTiter 96 Nature Communications\| (2023) 14:2132 14 https://doi.org/10.1038/s41467-023-37652-0 Article AQueous Non-Radioactive Cell Proliferation Assay (Promega, G5421) and normalised to cells treated with vehicle only as 100% proliferation. AQueous Non-Radioactive Cell Proliferation Assay (Promega, G5421) and normalised to cells treated with vehicle only as 100% proliferation. (BD Biosciences) instruments. For data interpretation, cells of all conditions were grown in normal media with BSA only. These cells were harvested and stained as above and the mean ﬂuorescence intensity (MFI) was measured and subtracted from lipid supplemented conditions as a baseline of intracellular lipid content. Downstream analysis was performed using FlowJo version 10.6.1 (BD Biosciences). Example FACS gating strategies are available in Supplementary Information. Bodipy uptake assay py p y C1-Bodipy C12 500/510 (Thermo Fisher, D3823) was added to cells in normal culture conditions at a ﬁnal concentration of 1 µM, concurrent with other treatments before being placed in an incubator at 37 °C and 5% CO2 for 24 h. Cells were then pelleted and washed twice with PBS before resuspension in PBS and ﬂow cytometry analysis using LSR Fortessa (BD Biosciences) instruments. Downstream analysis was performed using FlowJo version 10.6.1 (BD Biosciences). Example FACS gating strategies are available in Supplementary Information. For liquid chromatography a ZIC-pHILIC guard column (SeQuant, 20 × 2.1 mm) and ZIC-pHILIC column (SeQuant, 150 × 2.1 mm, 5 µm, Merck KGaA) was used on a Thermo Scientiﬁc UltiMate 3000 HPLC system. The aqueous mobile-phase solvent consisted of a 0.1% ammonium hydroxide −20 mM ammonium carbonate solution and and the organic mobile phase was acetonitrile. A linear biphasic LC gradient was executed, staring from 80% organic phase to 80% aqu- eous phase. The gradient took 15 min for a total run time of 22 min. The ﬂow rate was set to 200 µl/min with column temperature being maintained at 45 °C. The mass spectrometer used was a qExactive Orbitrap Mass Spectrometer (Thermo Fisher Scientiﬁc). The MS operated in polarity switching mode. Pre-analysis calibration was car- ried out for both ionization mode using a custom CALMIX and a low m/z range tune ﬁle was used. Full scan (MS1) data was acquired during polarity switch in proﬁle mode. The resolution was 35,000 (at m/z range 75–1000). Automatic gain control was set to target of 1×106 with a max ﬁll time of 250 ms. The parameters for spray voltages are as follows: +4.5 kV (capillary +50 V, tube: +70 kV, skimmer: +20 V) and −3.5 kV (capillary −50 V, tube: −70 kV, skimmer: −20 V). The s-lens RF level was set to 50 for the front optics. The probe temperature was set at 50̊C and capillary temperature set at 375 ̊C The sheath gas ﬂow rate was 25 and auxiliary gas ﬂow rate was 15. Sweep gas ﬂow was set to rate of 1. The mass accuracy was sub 5 ppm and data was acquired using Thermo Xcalibur 4.3.73.11 software. Bodipy lipid peroxidation assay One million cells of each condition were harvested and the positive control was treated with 100 µM tetrabutyl hydroperoxide for the duration of the Bodipy labelling. Bodipy 581/591 lipid peroxidation sensor (Thermos Fisher, D3861) was added to a ﬁnal concentration of 4 µM and cells were incubated for 45 min at 37 °C with gentle shaking, protected from light. Cells were then removed, washed twice with PBS and analysed by ﬂow cytometry using LSR Fortessa (BD Biosciences) instruments. Downstream analysis was performed using FlowJo ver- sion 10.6.1 (BD Biosciences). Example FACS gating strategies are available in Supplementary Information. SEAHORSE MitoStress test The SEAHORSE MitoStress test was performed essentially per manu- facturer’s instructions. In short, cells were grown and treated as described above. The SEAHORSE cartridge was hydrated with sterile water overnight in a 37 °C incubator with no COO2, before the water being removed and replaced with SEAHORSE calibrant solution (Agi- lent, 100840-000) for at least 1 h before the cartridge is put into a SEAHORSE XF instrument. The cell plate was coated with Corning CellTak (Fisher, can no CB40240) adhesion solution at 22.4 µg/ml concentration in sodium bicarbonate at pH 8 for 15 min at room tem- perature before washing twice with sterile water. 105 cells were har- vested and washed with PBS before resuspension in 50 µl of pre- warmed SEAHORSE RPMI media (Agilent, 103576-100) and adding onto the cell plate. Cells were adhered by centrifugation at 100 × g for 1 min without braking, before incubation at 37 °C for 15 min in an incubator with no COO2. 130 µl of additional SEAHORSE media along with eto- moxir at a ﬁnal concentration of 50 µM (only to etomoxir treatment wells) was added to wells and incubated for a further 15 min. The drugs were added to the cartridge in the following manner: port A – oligo- mycin, 1 µM ﬁnal concentration, port B – FCCP, 0.3 µM ﬁnal con- centration, port C – rotenone/antimycin A, 0.5 µM ﬁnal concentration (Agilent, 103015-100), all of which were prepared in SEAHORSE media. The assay was set up on the SEAHORSE XF machine with a 2 minmixing, 2 min wait and 2 min measuring for each well at each timepoint. Metabolomic analysis Cells were treated with AC220 (1 nM) and mannose (100 µM) for 48 h before 30 million cells were harvested and washed. Pellets were snap frozen and sent to Metabolon (Morrisville, NC) for LC-MS analysis. After extraction of metabolites, samples were split into four aliquots to be analysed in acidic positive, acidic negative and basic negative eluted from a UPLC C18 column (Waters) with the fourth aliquot separated with negative ionisation on a UPLC HILIC column (Waters). All analysis was performed on a Q-Exactive mass spectrometer (Thermo Scientiﬁc) coupled to an ACQUITY UPLC system (Waters) coupled to a HESI-II electrospray ionisation source. MS operated at 35000 mass resolution with scan range between 70 and 1000 m/z. All extraction and analysis of RAW data was performed in house by Metabolon. Raw counts were normalised by Bradford protein concentration and then each bio- chemical is rescaled to the median levels of each biochemical across all 6 conditions which is set as reference. Z scores are then calculated as the difference between the observed value in one condition minus the mean, all divided by the standard deviation, i.e. x-µ/σ. Targeted metabolomic analysis following U-13C6-glucose labelling was per- formed as described previously9. For palmitate SEAHORSE assays it was performed essentially as above, but cells were cultured overnight in RPMI media lacking FBS and glutamine, supplemented with 50 µM palmitate-BSA or BSA (Sigma, A8806) only. SEAHORSE media was without glutamine and palmitate (50 µM) was added to the + palmitate conditions. The rest of the assay was run as described above. Palmitate labelling Cells were cultured in Plasmax media (Ximbio, 156371) supplemented with 5% dialysed FBS (Sigma). U-¹³C16-Palmitic acid (CK Isotopes, CLM409) was dissolved in isopropanol and added to cell culture concurrent with treatments at a ﬁnal concentration of 50 µM for 24 h. 500,000 cells were harvested washed twice with ice cold PBS before extraction in solvent containing 50% methanol (Fisher, 10675112), 30% acetonitrile (Fisher, 10407440) and 20% H2O (All HPLC grade, Fisher, 10777404). Samples were then centrifuged and the supernatant retained. Samples were kept at −80 °C until analysis. Nature Communications\| (2023) 14:2132 Western blot analysis Approximately 1 million cells for each condition were harvested and washed twice with PBS before ﬁxing with 4% paraformaldehyde in PBS for 10 min at room temperature. Cells were then washed before per- meabilisation with 0.3% triton x-100 in PBS for 15 min at room tem- perature, before a PBS wash and blocking with 0.2% ﬁsh skin gelatin for 1 h at room temperature with agitation. Cells were incubated with primary antibody diluted in blocking solution (ATF6, 1:200, Santa Cruz biotech, SC166659) for 1 h at room temperature with agitation before 5 washes with PBS. Samples were then incubated with secondary anti- body (AlexFluor 488, goat anti-rabbit, 1:1000, Thermo, A32731) pro- tected from light before 5 washes with PBS. Glass slides were coated with CellTak solution (22.4 µg/ml) at room temperature for 15 min before washing the slides twice with PBS. Cells were then pipetted onto the CellTak coated slides and left for 30 min at room temperature before the addition of DAPI Fluoromount (Southern Biotech, 0100-20) solution and covering with coverslips. Slides were then kept at 4 °C until imaging. All imaging was done on a Zeiss LSM 710 scanning confocal microscope at ×40 or ×63 magniﬁcation. Further image analysis and processing was performed using ImageJ, including use of the Coloc2 plugin for colocalisation analysis. For 4-HNE staining, the procedure was performed essentially as above, with anti-4-HNE anti- serum (Alpha Diagnostic International, HNE11-S) as the primary anti- body. Further analysis was performed with the FIJI software package for ImageJ, using the Coloc2 plugin for colocalisation. For enhancing contrast, saturated pixels were set to 0.3 or 0.5% and was performed on the whole image at once. For images where more than 0.3 or 0.5% of pixels were already saturated, this contrast enhancement had no visi- ble effect. In total, 106 Cells were lysed in Cell Signalling technololgies lysis buffer (Cell Signalling) supplemented with protease and phospha- tase inhibitors. DNA was sheared by passing the sample through a ﬁne gauge needle 8 times before centrifugation for 10 min at 14,000 x g. The supernatants were retained and stored at −20 °C before further use. NuPAGE sample buffer (Invitrogen) was added to samples before separation on 4-12% polyacrylamide gels (Invitrogen, NP0321). Quantitative RT-PCR (qPCR) Q (q ) RNA was extracted from ~1 million cells using the RNeasy mini kit (Qiagen, 74106) as per the manufacturer’s instructions. RNA con- centration was then measured using a nanodrop and immediately reverse transcribed using the High-Capacity cDNA Reverse Transcrip- tion Kit (ThermoFisher,. 4368814) as per the manufacturers intstruc- tions. cDNA was then stored at −20 °C until use. For qPCR 20 ng of cDNA was added to 5ul of PowerUp SYBR Green MasterMix (Ther- moFisher, A25742) with 250 nM of forward and reverse primers for the gene of interest. Samples in a 384 well plate were then analysed by BioRad CFX384 Real-Time System (BioRad) with the following proto- col: 95 °C for 2 min, 95 °C for 5 s, 60 °C for 30 s when the ﬂuorescence was measured before returning to step 2 and repeating 39 times, 95 °C for 5 s, 65 °C for 5 s, ﬂuorescence measurement, 95 °C for 30 s and holding at 16 °C forever. Proteostat staining Approximately 1 million cells per condition were collected and washed twice with PBS before ﬁxation for 10 min at room temperature by resuspension in 4% paraformaldehyde. Cells were then washed twice with PBS and permeabilised with 0.3% triton X-100 in PBS for 15 min before a further wash and resuspension in assay buffer from the pro- teostat protein aggregation kit (Enzo life sciences, ENZ51023) with proteostat stain (1:5000). Cells were then kept on ice before analysis. Example FACS gating strategies are available in Supplementary Information. qPCR primer sequences CPT1A - Forward: CAAACTGGACCGGGAGGAAA; Reverse: TGTGCTGG ATGGTGTCTGTC. PPARA - Forward: AGCTGTCACCACAGTAGCTT; Reverse: GGAAC TCTTCAGATAACGGGCT. ACOX1 - Forward: CGCCGAGAGATCGAGAACAT; Reverse: GCACT TTTCCTGACAGCCAC MPI - Forward: CTGCCGGGAAAGGCATACG; Reverse: AGCAATCC ACTGGCTTAGGG ERO1B - Forward: AGAGAACTGTTTCAAGCCTCG; Reverse: TCCAG ACACAAACCTTCTAGCC HERPUD1 - Forward: CGAGATTGGTTGGATTGGACC; Reverse: CA CCCAACGTGATGCAGGTA XBP1 spliced – Forward: TTGCTGAAGAGGAGGCGGAA; Reverse: CTGCACCTGCTGCGGACTCAG CPT1A - Forward: CAAACTGGACCGGGAGGAAA; Reverse: TGTGCTGG ATGGTGTCTGTC. PPARA - Forward: AGCTGTCACCACAGTAGCTT; Reverse: GGAAC TCTTCAGATAACGGGCT. Article Article Article https://doi.org/10.1038/s41467-023-37652-0 The peak areas of each metabolite and their respective iso- topologues were quantiﬁed using Thermo Traceﬁnder 4.1. Metabolites were identiﬁed by accurate mass of the singly charged ion and by retention times of authentic standards on the pHILIC column. The commercially standard compound mix (Merck: MSMLS-1EA) had been analysed previously on our LCMS system to determine accurate ion masses and retention times. Data were processed and corrected for natural abundance through Autoplotter. (Pietzke and Vazquez, 2020, Cancer Metab. doi: 10.1186/s40170-020-00220-x.). Western blot analysis Proteins were then transferred to PVDF membranes fol- lowed by blocking with 5% milk/Tris Buffered Saline with 0.1% tween (TBS-T), incubation with primary antibodies in 5% BSA/TBS-T over- night at 4 °C then corresponding secondary antibodies in 5% milk/ TBS-T for 1 h at room temperature before incubation of the mem- branes with ECL (BioRad,. 1705061) and visualisation using an Amersham Imager (GE Healthcare). Further analysis was performed in ImageJ. Primary antibodies were used at the following concentra- tions: ATF6 (Abcam, AB122897), 1:500, total Histone 3 (Cell Signal- ling, 44995), 1:5000, MPI (Santa Cruz Biotech, SC393477), 1:1000, PARP (Cell Signalling, 56255), 1:500, EIF2α (Cell Signalling, 9722), 1:1000, phosphor-EIF2α (Ser51, Cell Signalling, 9721), 1:1000. Uncropped and unprocessed scans of the blots are available in Supplementary Information. Bodipy neutral lipid stain Cells were grown as normal with the addition of fatty acids-BSA or BSA alone as vehicle at the following concentrations: palmitic acid: 50 µM, oleate: 50 µM and arachidonic acid 1 µM for 24 h. Around 1 million cells of each condition were then harvested and washed twice in PBS, before resuspension in PBS and incubating the samples with 2.5 µg/ml Bodipy 493/503 (ThermoFisher, D3922) for 20 min at 37 °C with agitation. Samples were then washed twice with PBS and resuspended in PBS and then kept on ice before analysis by ﬂow cytometry using LSR Fortessa Nature Communications\| (2023) 14:2132 15 Nature Communications\| (2023) 14:2132 Staining of surface proteins for ﬂow cytometry Approximately 1 million cells for each condition were harvested and washed twice with PBS before resuspension in PBS. FC block (Biole- gend, 422301, 1:200) was then added for 20 min at room temperature before the addition of primary antibody at a 1:100 dilution before incubation at 4 °C for 30 min. Cells were then washed 3 times with PBS before resuspension in PBS. Samples were then stored either on ice or at 4 °C protected from light until analysis by ﬂow cytometry using LSR Fortessa (BD Biosciences) instruments. The following antibodies were used: anti-human CD45-FITC (BioLegend, 304005), anti-mouse CD45-APC (BioLegend, 157605), anti-SLC7a11 (SantaCruz Biotech, SC98552). Downstream analysis was performed using FlowJo version 10.6.1 (BD Biosciences). Example FACS gating strategies are available in Supplementary Information. Lectin staining of surface glycoproteins Approximately 1 million cells for each condition were harvested and washed twice with PBS before resuspension in PBS. Lectin from Lyco- persicon esculentum (tomato) FITC conjugate (Sigma, L0401) was added in a 1:100 dilution before incubation for 1 h at 4 °C, protected from light. Cells were then washed twice with PBS, resuspended in PBS before ﬂow cytometry analysis using LSR Fortessa (BD Biosciences) instruments. Downstream analysis was performed using FlowJo ver- sion 10.6.1 (BD Biosciences). Example FACS gating strategies are available in Supplementary Information. Nature Communications\| (2023) 14:2132 16 Article https://doi.org/10.1038/s41467-023-37652-0 Reporting summary Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article. Gene-set enrichment analysis (GSEA) analysis Gene set enrichment analysis (GSEA) analysis RNA-sequencing data was analysed for GSEA using the Broad Institute software (http://software.broadinstitute.org/gsea/index.jsp). For GSEA analysis normalised read counts for all conditions were used and genes were ranked using the signal-to-noise metric and FDR and NES were calculated using 1000 gene-set permutation. All mice were housed in IVCs on a 12 h light/dark cycle at an ambient temperature of 20 °C and humidity of 27–35%. XBP1 total – Forward: TTCCGGAGCTGGGTATCTCA; Reverse: GAAAGGGAACCCCCGTATCC ATF6 – Forward: ATGAAGTTGTGTCAGAGAACC; Reverse: CTCTT TAGCAGAAAATCCTAG for 5 days. Survival time was measured from time of transplantation until time mice had to be culled due to overt clinical symptoms. IVIS bioluminescence imaging (PerkinElmer) was used to conﬁrm and track disease dissemination. In brief, D-luciferin (Cat#122799, Perkin Elmer) was administered by intraperitoneal (IP) injection as per the manu- facturer’s recommendation, followed by IVIS bioluminescence ima- ging under anaesthesia (Isoﬂurane). Bioluminescence as a surrogate for tumour burden, was quantiﬁed using Living Image Software (ver- sion 4.7.2, PerkinElmer). References References Data availability The RNA-sequencing data generated in this study have been deposited in the ArrayExpress database and are available at https://www.ebi.ac. uk/arrayexpress/experiments/E-MTAB-11750. Untargeted metabo- lomics analysis is available in Supplemental Data 1. All data are avail- able in the article, Supplementary Information and source data. Source data are provided with this paper. RNA sequencing Cells were treated with AC220 (1 nM) and mannose (100 µM) for 24 or 72 h as indicated. RNA was extracted and puriﬁed with the RNAeasy kit (Qiagen, 74106) as per manufacturer’s instructions. Concentration was measured by NanoDrop (ThermoFisher). Ribosomal depletion was performed using the QIAseq FastSelect™RNA Removal Kit—24sam- ples; HUMAN (Qiagen: THS-001Z-24). Library preparation was per- formed with the dUTP directional kit NEBNext® Ultra8482 II Directional RNA Library Prep Kit for Illumina®. Samples were analysed on a NovaSeq6000 S1 ﬂowcell as a paired end 150 bp (PE150) run. We followed recommended guidelines in the analysis of RNA-seq data for quality control, read mapping, quantiﬁcation of gene expression, assessment of reproducibility among biological replicates, and differ- ential gene expression (Conesa et al., 2016, Genome Biology, doi: 10.1186/s13059-016-0881-8.). First, TrimGalore 0.4.5 was used with default parameters and paired-end mode for quality-trimming and ﬁltering of read sequences. Then, STAR 2.6.1 (Dobin et al, 2013, Bioinformatics, doi: 10.1093/bioinformatics/bts635) was used to align the reads to the reference human genome assembly GRCh38 using the Ensembl release 95 annotation as reference transcriptome. Proportion of uniquely mapped reads was >91% in all samples. FeatureCounts 1.6.3 was run on paired-end reads to count fragments in annotated gene features, with parameters ‘-p -T 4 -t exon -g gene_id’ (Liao et al., 2014). The R/Bioconductor package DESeq2 was then used to perform dif- ferential gene expression analyses between samples and conditions (Love et al., 2014, Genome Biology, https://doi.org/10.1186/s13059- 014-0550-8). Raw data is available at https://www.ebi.ac.uk/ arrayexpress/experiments/E-MTAB-11750. A 8 week old male NBSGW (NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ ThomJ) mice (Jackson Laboratory) had either 5000 THP-1 NTgRNA or THP-1 MPIgRNA5 cells infused by intravenous injection via the tail vein. 1 week after this AML engraftment was measured by ﬂow cytometry based on percentage of human CD45 + cells in the peripheral blood. The next day mice were treated with 50 mg/mg cytarabine (Sigma, 251010) in PBS or PBS alone (vehicle) for 7 days via intraperitoneal injection. Survival time was measured from time of transplantation until time mice had to be culled due to overt clinical symptoms. y p Bone marrow tissue was harvested by dissection of the leg bones of the mouse before the bones were ﬂushed with PBS to obtain the cells. Public dataset analysis All data was visualised in Prism 8.4 (Graph Pad), with statistical tests outlined in ﬁgure legends for individual analyses. All data are shown as mean ± standard error of the mean, unless otherwise stated. GSE13159, GSE10358, GSE37642, GSE76009, GSE30377, GSE83533 and RNA-Seq from AML TCGA (data obtained from https://www. cbioportal.org/), BeatAML dataset (data obtained http://www. vizome.org/) and from manuscript 10.1056/NEJMoa1808777 (NEJM WUSM) were used to perform various analysis. For MPI expression, normalised expression was used and compared between different genotypes (FLT3ITD vs FLT3WT) or patient samples (diagnosis vs relapse). For analysis of MPI level based on AML karyotype pre nor- malised data from GEO GSE147515 was plotted based on AML kar- yotype and MPI level. For GSEA analysis, within relevant datasets, the expression proﬁle patients expressing high (i.e. above median) levels of MPI was compared to that of patients expressing low (i.e. below median) MPI levels. Genes were ranked using the signal-to-noise metric and FDR and NES were calculated using 1000 phenotype permutation. RNA sequencing After collection 2 ml of red blood cell lysis buffer (ThermoFisher, 00430054) was added and incubated for 5 min at room temperature before inactivation by the addition of 10 volumes of PBS. Cells were then pelleted for further ﬂow cytometry analysis as outlined previously above. 10 week old male NBSGW (NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ ThomJ) mice (Jackson Laboratory) had either 5000 THP-1 NTgRNA or THP-1 MPIgRNA5 cells infused by intravenous injection via the tail vein. 1 week after this AML engraftment was measured by ﬂow cytometry based on percentage of human CD45 + cells in the peripheral blood. The next day mice were treated with 50 mg/mg cytarabine (Sigma, 251010) in PBS for 7 days with or without liproxstatin (10 mg/kg) for 10 days via intraperitoneal injection. Survival time was measured from time of transplantation until time mice had to be culled due to overt clinical symptoms. For 4-HNE assessment a cohort of 3 mice in each treatment group was culled soon after treatment and bone marrow harvested. Thereafter human CD45 + leukaemic cells were sorted for immunocytochemistry as described above. Bone marrow tissue was harvested by dissection of the leg bones of the mouse before the bones were ﬂushed with PBS to obtain the cells. After collection 2 ml of red blood cell lysis buffer (ThermoFisher, 00430054) was added and incubated for 5 min at room temperature before inactivation by the addition of 10 volumes of PBS. Cells were then pelleted for further ﬂow cytometry analysis as outlined previously above. p y y y p y 10 week old male NBSGW (NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ ThomJ) mice (Jackson Laboratory) had either 5000 THP-1 NTgRNA or THP-1 MPIgRNA5 cells infused by intravenous injection via the tail vein. 1 week after this AML engraftment was measured by ﬂow cytometry based on percentage of human CD45 + cells in the peripheral blood. The next day mice were treated with 50 mg/mg cytarabine (Sigma, 251010) in PBS for 7 days with or without liproxstatin (10 mg/kg) for 10 days via intraperitoneal injection. Survival time was measured from time of transplantation until time mice had to be culled due to overt clinical symptoms. For 4-HNE assessment a cohort of 3 mice in each treatment group was culled soon after treatment and bone marrow harvested. Thereafter human CD45 + leukaemic cells were sorted for immunocytochemistry as described above. In vivo experiments Glutaminolysis is a metabolic dependency in FLT3(ITD) acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition. Blood 131, 1639–1653 (2018). 31. Lakhia, R. et al. PPARalpha agonist fenoﬁbrate enhances fatty acid beta-oxidation and attenuates polycystic kidney and liver disease in mice. Am. J. Physiol. Ren. Physiol. 314, F122–F131 (2018). 10. Lagadinou, E. D. et al. BCL-2 inhibition targets oxidative phosphor- ylation and selectively eradicates quiescent human leukemia stem cells. Cell Stem Cell 12, 329–341 (2013). 32. Hong, M. et al. Underglycosylation of ATF6 as a novel sensing mechanism for activation of the unfolded protein response. J. Biol. Chem. 279, 11354–11363 (2004). 11. Samudio, I. et al. Pharmacologic inhibition of fatty acid oxidation sensitizes human leukemia cells to apoptosis induction. J. Clin. Invest 120, 142–156 (2010). 33. Nolan, K. et al. Endoplasmic reticulum stress-mediated upregula- tion of miR-29a enhances sensitivity to neuronal apoptosis. Eur. J. Neurosci. 43, 640–652 (2016). 12. Raffel, S. et al. BCAT1 restricts alphaKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation. Nature 551, 384–388 (2017). 34. Gallagher, C. M. & Walter, P. Ceapins inhibit ATF6alpha signaling by selectively preventing transport of ATF6alpha to the Golgi appa- ratus during ER stress. Elife 5, e11880 (2016). 13. Farge, T. et al. Chemotherapy-resistant human acute myeloid leu- kemia cells are not enriched for leukemic stem cells but require oxidative metabolism. Cancer Disco. 7, 716–735 (2017). 35. Gallagher, C. M. et al. Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6alpha branch. Elife 5, e11878 (2016). 36. Plate, L. et al. Small molecule proteostasis regulators that repro- gram the ER to reduce extracellular protein aggregation. Elife 5, e15550 (2016). 14. Sharma, V., Ichikawa, M. & Freeze, H. H. Mannose metabolism: more than meets the eye. Biochem Biophys. Res Commun. 453, 220–228 (2014). 15. Ichikawa, M., Scott, D. A., Losfeld, M. E. & Freeze, H. H. The meta- bolic origins of mannose in glycoproteins. J. Biol. Chem. 289, 6751–6761 (2014). 37. Yoshida, H. et al. A time-dependent phase shift in the mammalian unfolded protein response. Dev. Cell 4, 265–271 (2003). 38. Yoshida, H., Matsui, T., Yamamoto, A., Okada, T. & Mori, K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891 (2001). 16. DeRossi, C. et al. In vivo experiments 1. Estey, E. H. Acute myeloid leukemia: 2021 update on risk-stratiﬁcation and management. Am. J. Hematol. 95, 1368–1398 (2020). In total, 3 × 106 luciferase expressing MOLM13 cells transduced with non-targeting CRISPR guide (NT-gRNA) or MPI KO CRISPR guide (MPI- gRNA) were transplanted into male 8-12 week old NSG (NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ) mice (Jackson Laboratory) which were sub- lethally irradiated (2 Gy). Mice were treated by oral gavage with AC220 (5 mg/kg) or vehicle (22% hydroxypropyl-β-cyclodextrin/0.3% DMSO) 2. Smith, C. C. et al. Validation of ITD mutations in FLT3 as a ther- apeutic target in human acute myeloid leukaemia. Nature 485, 260–263 (2012). 3. Intlekofer, A. M. et al. Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations. Nature 559, 125–129 (2018). 3. Intlekofer, A. M. et al. Acquired resistance to IDH inhibition through trans or cis dimer-interface mutations. Nature 559, 125–129 (2018). Nature Communications\| (2023) 14:2132 17 Article https://doi.org/10.1038/s41467-023-37652-0 26. Christopher, M. J. et al. Immune escape of relapsed AML cells after allogeneic transplantation. N. Engl. J. Med. 379, 2330–2341 (2018). 4. Marin-Bejar, O. et al. Evolutionary predictability of genetic versus nongenetic resistance to anticancer drugs in melanoma. Cancer Cell 39, 1135–1149 e1138 (2021). 27. Sharma, V. et al. Phosphomannose isomerase inhibitors improve N-glycosylation in selected phosphomannomutase-deﬁcient ﬁbro- blasts. J. Biol. Chem. 286, 39431–39438 (2011). 5. Fong, C. Y. et al. BET inhibitor resistance emerges from leukaemia stem cells. Nature 525, 538–542 (2015). 28. Perea, G. et al. Adverse prognostic impact of CD36 and CD2 expression in adult de novo acute myeloid leukemia patients. Leuk. Res 29, 1109–1116 (2005). 6. Boumahdi, S. & de Sauvage, F. J. The great escape: tumour cell plasticity in resistance to targeted therapy. Nat. Rev. Drug Disco. 19, 39–56 (2020). 7. Culp-Hill, R., D’Alessandro, A. & Pietras, E. M. Extinguishing the embers: targeting AML metabolism. Trends Mol. Med 27, 332–344 (2021). 29. Coort, S. L. et al. Sulfo-N-succinimidyl esters of long chain fatty acids speciﬁcally inhibit fatty acid translocase (FAT/CD36)-medi- ated cellular fatty acid uptake. Mol. Cell Biochem 239, 213–219 (2002). 8. Bjelosevic, S. et al. Serine biosynthesis is a metabolic vulnerability in FLT3-ITD-driven acute myeloid leukemia. Cancer Disco. 11, 1582–1599 (2021). 30. Guo, J. et al. Selective transport of long-chain fatty acids by FAT/ CD36 in skeletal muscle of broilers. Animal 7, 422–429 (2013). 9. Gallipoli, P. et al. In vivo experiments Ablation of mouse phosphomannose isomerase (Mpi) causes mannose 6-phosphate accumulation, toxicity, and embryonic lethality. J. Biol. Chem. 281, 5916–5927 (2006). 39. Wu, J. et al. ATF6alpha optimizes long-term endoplasmic reticulum function to protect cells from chronic stress. Dev. Cell 13, 351–364 (2007). 17. de Koning, T. J. et al. A novel disorder of N-glycosylation due to phosphomannose isomerase deﬁciency. Biochem Biophys. Res Commun. 245, 38–42 (1998). 40. Jao, T. M. et al. ATF6alpha downregulation of PPARalpha promotes lipotoxicity-induced tubulointerstitial ﬁbrosis. Kidney Int. 95, 577–589 (2019). 18. Jaeken, J. et al. Phosphomannose isomerase deﬁciency: a carbohydrate-deﬁcient glycoprotein syndrome with hepatic- intestinal presentation. Am. J. Hum. Genet 62, 1535–1539 (1998). 41. Miess, H. et al. The glutathione redox system is essential to prevent ferroptosis caused by impaired lipid metabolism in clear cell renal cell carcinoma. Oncogene 37, 5435–5450 (2018). 19. Niehues, R. et al. Carbohydrate-deﬁcient glycoprotein syndrome type Ib. Phosphomannose isomerase deﬁciency and mannose therapy. J. Clin. Invest 101, 1414–1420 (1998). 42. Zou, Y. et al. A GPX4-dependent cancer cell state underlies the clear-cell morphology and confers sensitivity to ferroptosis. Nat. Commun. 10, 1617 (2019). 20. Sharma, V. et al. Mannose supplements induce embryonic lethality and blindness in phosphomannose isomerase hypomorphic mice. FASEB J. 28, 1854–1869 (2014). 43. Soga, T. et al. Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione con- sumption. J. Biol. Chem. 281, 16768–16776 (2006). 21. Shtraizent, N. et al. MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect. Elife 6, e22477 (2017). 22. Cazet, A., Charest, J., Bennett, D. C., Sambrooks, C. L. & Contessa, J. N. Mannose phosphate isomerase regulates ﬁbroblast growth fac- tor receptor family signaling and glioma radiosensitivity. PLoS One 9, e110345 (2014). 44. Dalleau, S., Baradat, M., Gueraud, F. & Huc, L. Cell death and dis- eases related to oxidative stress: 4-hydroxynonenal (HNE) in the balance. Cell Death Differ. 20, 1615–1630 (2013). 45. Dixon, S. J. et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife 3, e02523 (2014). 23. Gonzalez, P. S. et al. Mannose impairs tumour growth and enhances chemotherapy. Nature 563, 719–723 (2018). 46. Zilka, O. et al. On the Mechanism of Cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death. ACS Cent. Sci. 3, 232–243 (2017). 24. Pacharne, S. et al. Reprints and permissions information is available at http://www.nature.com/reprints 63. Ma, X. et al. CD36-mediated ferroptosis dampens intratumoral CD8(+) T cell effector function and impairs their antitumor ability. Cell Metab. 33, 1001–1012 e1005 (2021). Publisher’s note Springer Nature remains neutral with regard to jur- isdictional claims in published maps and institutional afﬁliations. 64. Xu, S. et al. Uptake of oxidized lipids by the scavenger receptor CD36 promotes lipid peroxidation and dysfunction in CD8(+) T cells in tumors. Immunity 54, 1561–1577 e1567 (2021). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons 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/. 65. Broadﬁeld, L. A., Pane, A. A., Talebi, A., Swinnen, J. V. & Fendt, S. M. Lipid metabolism in cancer: New perspectives and emerging mechanisms. Dev. Cell 56, 1363–1393 (2021). 66. DeLany, J. P., Windhauser, M. M., Champagne, C. M. & Bray, G. A. Differential oxidation of individual dietary fatty acids in humans. Am. J. Clin. Nutr. 72, 905–911 (2000). Author contributions P.G. and K.W. designed the experiments. K.W. and L.S.D. performed laboratory assays. K.M.R. G.V.H. designed and performed the palmitate labelling assay. A.M.S.M., C.P., V.D. and S.C.J. helped with experiments. L.N.L. and P.G. performed computational analysis of published datasets. P.M. performed RNA sequencing analysis. K.W., G.G., C.M., K.R.K. and R.A. designed, performed or helped in vivo studies. J.H.M.P. provided reagents and input for UPR analysis. C.P. and K.R-P. provided input on methodology, reagents, experimental design and analysis of UPR. K.T. and G.S.V. designed and performed original CRISPR screen. K.W. and P.G. wrote and edited the manuscript. P.G. designed the study and supervised the project with B.J.P.H. mentorship/support at the start. All authors critically reviewed the manuscript and approved the ﬁnal version. 51. Torretta, S. et al. D-mannose suppresses macrophage IL-1beta production. Nat. Commun. 11, 6343 (2020). 52. Zhang, D. et al. D-mannose induces regulatory T cells and sup- presses immunopathology. Nat. Med. 23, 1036–1045 (2017). 53. Larrue, C. et al. Antileukemic Activity of 2-Deoxy-d-Glucose through Inhibition of N-Linked Glycosylation in Acute Myeloid Leukemia with FLT3-ITD or c-KIT Mutations. Mol. Cancer Ther. 14, 2364–2373 (2015). 54. Williams, A. B. et al. Fluvastatin inhibits FLT3 glycosylation in human and murine cells and prolongs survival of mice with FLT3/ITD leu- kemia. Blood 120, 3069–3079 (2012). 55. Tcheng, M. et al. Very long chain fatty acid metabolism is required in acute myeloid leukemia. Blood 137, 3518–3532 (2021). 56. Stevens, B. M. et al. Fatty acid metabolism underlies venetoclax resistance in acute myeloid leukemia stem cells. Nat. Cancer 1, 1176–1187 (2020). https://doi.org/10.1038/s41467-023-37652-0 (SBF004\1099) J.H.M.P. was supported by a research grant from Sci- ence Foundation Ireland (SFI) under Grant Number 16/RC/3948 and co- funded under the European Regional Development Fund and by FutureNeuro industry partners. K.T. was funded by Wellcome Trust (Grant References: RG94424, RG83195, G106133), UKRI Medical Research Council (RG83195) and Leukaemia UK (G108148). 48. Saito, Y. et al. Mannose and phosphomannose isomerase regulate energy metabolism under glucose starvation in leukemia. Cancer Sci. 112, 4944–4956 (2021). 49. Schneider, A. et al. Successful prenatal mannose treatment for congenital disorder of glycosylation-Ia in mice. Nat. Med. 18, 71–73 (2011). 50. Minikel, E. V. et al. Evaluating drug targets through human loss-of- function genetic variation. Nature 581, 459–464 (2020). Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-37652-0. 58. Joshi, S. K. et al. The AML microenvironment catalyzes a stepwise evolution to gilteritinib resistance. Cancer Cell 39, 999–1014 e1018 (2021). Correspondence and requests for materials should be addressed to Paolo Gallipoli. Correspondence and requests for materials should be addressed to Paolo Gallipoli. 59. Dixon, S. J. et al. Ferroptosis: an iron-dependent form of non- apoptotic cell death. Cell 149, 1060–1072 (2012). 60. Stockwell, B. R. et al. Ferroptosis: A Regulated. Cell Death Nexus Link. Metab., Redox Biol., Dis. Cell 171, 273–285 (2017). Peer review information : Nature Communications thanks Eric Chevet, Courtney Jones and Silvia von Karstedt for their contribution to the peer review of this work. Peer reviewer reports are available. 61. Hangauer, M. J. et al. Drug-tolerant persister cancer cells are vul- nerable to GPX4 inhibition. Nature 551, 247–250 (2017). 62. Lin, Z. et al. The lipid ﬂippase SLC47A1 blocks metabolic vulner- ability to ferroptosis. Nat. Commun. 13, 7965 (2022). Reprints and permissions information is available at http://www.nature.com/reprints Competing interests The authors declare no competing interests. 57. Bosc, C. et al. Mitochondrial inhibitors circumvent adaptive resis- tance to venetoclax and cytarabine combination therapy in acute myeloid leukemia. Nat. Cancer 2, 1204–1223 (2021). Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-37652-0. Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41467-023-37652-0. In vivo experiments SETBP1 overexpression acts in the place of class- deﬁning mutations to drive FLT3-ITD-mutant AML. Blood Adv. 5, 2412–2425 (2021). 47. Yuk, H., Abdullah, M., Kim, D. H., Lee, H. & Lee, S. J. Necrostatin-1 prevents ferroptosis in a RIPK1- and IDO-independent manner in hepatocellular carcinoma. Antioxid. (Basel) 10, 1347 (2021). 25. Li, S. et al. Distinct evolution and dynamics of epigenetic and genetic heterogeneity in acute myeloid leukemia. Nat. Med. 22, 792–799 (2016). Nature Communications\| (2023) 14:2132 18 Article https://doi.org/10.1038/s41467-023-37652-0 Acknowledgements We wish to thank the Barts Cancer Institute tissue bank for sample col- lection and processing. This research was supported by the BCI Flow cytometry facility (CRUK Core Award C16420/A18066). This work was supported by the Wellcome Trust (PG, 109967/Z/15/Z), the American Society of Haematology (PG, Global Research Award) and Cancer Research UK (PG, Advanced Clinician Scientist fellowship, C57799/ A27964). K.R-P. was supported by the Academy of Medical Sciences © The Author(s) 2023 19 Nature Communications\| (2023) 14:2132
W3019357706.txt	null	en		Killer Archaea: Virus-Mediated Antagonism to CRISPR-Immune Populations Results in Emergent Virus-Host Mutualism	MBio	2,020	cc-by	6,993	RESEARCH ARTICLE Ecological and Evolutionary Science crossm Killer Archaea: Virus-Mediated Antagonism to CRISPR-Immune Populations Results in Emergent Virus-Host Mutualism Samantha J. DeWerff,a Maria A. Bautista,a* Matthew Pauly,a* Changyi Zhang,a Rachel J. Whitakera,b a Department of Microbiology, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA b Infection Genomics for One Health Theme, Carl R. Woese Institute for Genomic Biology, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA Theory, simulation, and experimental evolution demonstrate that diversiﬁed CRISPR-Cas immunity to lytic viruses can lead to stochastic virus extinction due to a limited number of susceptible hosts available to each potential new protospacer escape mutation. Under such conditions, theory predicts that to evade extinction, viruses evolve toward decreased virulence and promote vertical transmission and persistence in infected hosts. To better understand the evolution of hostvirus interactions in microbial populations with active CRISPR-Cas immunity, we studied the interaction between CRISPR-immune Sulfolobus islandicus cells and immune-deﬁcient strains that are infected by the chronic virus SSV9. We demonstrate that Sulfolobus islandicus cells infected with SSV9, and with other related SSVs, kill uninfected, immune strains through an antagonistic mechanism that is a protein and is independent of infectious virus. Cells that are infected with SSV9 are protected from killing and persist in the population. We hypothesize that this infection acts as a form of mutualism between the host and the virus by removing competitors in the population and ensuring continued vertical transmission of the virus within populations with diversiﬁed CRISPR-Cas immunity. ABSTRACT IMPORTANCE Multiple studies, especially those focusing on the role of lytic viruses in key model systems, have shown the importance of viruses in shaping microbial populations. However, it has become increasingly clear that viruses with a long hostvirus interaction, such as those with a chronic lifestyle, can be important drivers of evolution and have large impacts on host ecology. In this work, we describe one such interaction with the acidic crenarchaeon Sulfolobus islandicus and its chronic virus Sulfolobus spindle-shaped virus 9. Our work expands the view in which this symbiosis between host and virus evolved, describing a killing phenotype which we hypothesize has evolved in part due to the high prevalence and diversity of CRISPRCas immunity seen in natural populations. We explore the implications of this phenotype in population dynamics and host ecology, as well as the implications of mutualism between this virus-host pair. KEYWORDS archaea, CRISPR-Cas, chronic viruses, coevolution, mutualism, symbiosis, transmission mode, virus-host interactions Citation DeWerff SJ, Bautista MA, Pauly M, Zhang C, Whitaker RJ. 2020. Killer archaea: virus-mediated antagonism to CRISPR-immune populations results in emergent virus-host mutualism. mBio 11:e00404-20. https://doi.org/ 10.1128/mBio.00404-20. Editor Jennifer B. H. Martiny, University of California, Irvine Copyright © 2020 DeWerff et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Rachel J. Whitaker, rwhitaker@life.illinois.edu. V iruses can play inﬂuential roles in population dynamics, ecology, and evolution in all three domains of life. Most studies of viruses of microbes have focused on the negative effects of viral infection, in part due to the study of primarily lytic viruses which must kill their host in order to reproduce successfully. However, there has recently been a greater appreciation for the beneﬁcial, mutualistic interactions between virus and host and the coevolutionary forces that may shift this symbiosis from antagonistic to mutualistic (1–4). Chronic viruses are transmitted vertically from mother to daughter cell. Unlike their lytic and temperate counterparts, chronic viruses can also be transmitted horizontally by producing new virions without killing their hosts (often March/April 2020 Volume 11 Issue 2 e00404-20 * Present address: Maria A. Bautista, Department of Biological Sciences, University of Calgary, Calgary, Canada; Matthew Pauly, Division of Viral Hepatitis, National Center for HIV, Hepatitis, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, Georgia, USA. Received 26 February 2020 Accepted 2 April 2020 Published 28 April 2020 ® mbio.asm.org 1 ® DeWerff et al. 105 1 OD600nm 103 0.1 102 101 Virus (pfu/m L) 104 100 0.01 0 1 Uninfected Infected 2 Days 3 4 Limit SSV9 FIG 1 Challenge with SSV9 yields isolated chronically infected S. islandicus strains. Growth of an isolated, chronically infected strain (Δcas6:SSV9.1, solid blue line) and an uninfected isogenic strain (Δcas6 strain, gold line), as monitored by optical density. Throughout the growth curves, SSV9 production (blue dashed line) was monitored in the chronically infected strain by plaque assay. Error bars show mean results ⫾ standard errors of the means (SEM) (n ⫽ 3). through budding) (1, 4). Because of this mixed mode of transmission, selection can favor the vertical or horizontal mode depending on factors such as the availability of susceptible hosts and costs associated with viral carriage (1, 5, 6). We have shown that highly active and diverse CRISPR-Cas immunity can create conditions of low-susceptibility host density in which lytic viruses can go extinct (7). These conditions are deﬁned as distributed immunity, where CRISPR-immune hosts have a diversity of spacers targeting a virus, thus leading to a decreased probability of a single escape mutation by the virus leading to the emergence of a viral epidemic of the population (8, 9). Under these conditions, nonproductive infection of CRISPRimmune hosts leads to degradation of viral genomes and therefore comes at a signiﬁcant cost to the virus’s ﬁtness (7, 10–13). We predict that conditions of highly distributed immunity would result in a shift toward vertical transmission of chronic viruses. We have identiﬁed the structure of distributed immunity that is maintained over time in the natural geographically isolated population of the archaeon Sulfolobus islandicus from Kamchatka, Russia (14). We used viruses isolated from both Kamchatka and Yellowstone National Park, USA, populations to investigate their interactions with immune hosts and whether they evolve mechanisms to persist in host populations with highly diversiﬁed CRISPR-Cas immunity (14–16). Through plaque assays with enrichment supernatants and a diversity of 11 wild S. islandicus hosts from Yellowstone and Kamchatka, the primary viruses that we isolated from the Kamchatka population are Sulfolobus spindle-shaped viruses (SSVs) from the Fuselloviridae family (17). Schleper et al. ﬁrst discovered SSVs as chronic viruses that have a circular double-stranded DNA genome that can be maintained in a population through integration into the host genome or be kept as an episome, both of which are transmitted vertically (18). SSVs continuously bud from host cells without killing them at a rate of approximately 1 PFU for every 10,000 cells and can be transmitted horizontally to a new host (Fig. 1) (19). We have demonstrated experimentally that the CRISPR-Cas system can actively prevent productive SSV9 infection and that in this system, exposure of viral supernatants to the host causes dormancy (17). However, while this study suggests a possible infection-independent response of the challenged host, it lacks the context of population dynamics and a chronically infected host. Within the population context, it has been shown that Kamchatka population genomes show a high level of population-distributed immunity to SSVs (15). This results in very few susceptible hosts and a probability of evolutionary emergence of a successful viral escape variant on the scale of 10⫺4 (see Fig. S1 in the supplemental material) (8, 9, 14, 15). Here, we test the virus-host interaction and competitive host ﬁtness when the virus competes against cells that are CRISPR immune and susceptible. In doing so, we uncover a novel antagonistic interaction between chronically infected strains and uninfected counterparts that we hypothesize drives a mutualism between viruses and their archaeal hosts in populations with distributed immunity. March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 2 ® Virus-Mediated Antagonism by Chronically Infected Strains 1:1 10:1 100 Abundance (%) 50 10 6 10 5 0 6 4 2 0 Day 6 4 2 0 6 10 7 6 4 10 8 Day 10 11 10 10 10 9 50 10 8 10 7 10 6 10 5 0 6 106 0 10 9 10 12 100 4 107 50 10 10 Abundance (%) 8 10 11 100 Abundance (%) 10 9 SSV9 SSV9 (genomes/mL) 10 Infected SSV9 (genomes/mL) 10 10 50 Day Uninfected SSV9 (genomes/mL) 100 2 4 Day Immune 0 2 Day B 0 0 6 4 2 0 0 50 2 50 0 Abundance (%) 100:1 100 Abundance (%) Abundance (%) 100 0 A Day FIG 2 The chronically infected strain outcompetes the immune strain and can invade when chronically infected cells are rare. Relative abundances in competitions between a CRISPR-immune strain (RJW003, black) and an uninfected strain (Δcas6 strain, gold) (A) or between a CRISPR-immune strain and a chronically infected strain (Δcas6:SSV9.1, blue) (B) at initial frequencies of 1:1, 10:1, and 100:1. Strains grown to mid-log phase were mixed and monitored for 6 days by qPCR for host cell type. Days 2 and 4 show relative abundances before a 1:5 dilution into fresh medium. Error bars show mean results ⫾ SEM (n ⫽ 3). Presence of virus (SSV9, white circles) in competitions of 1:1, 10:1, and 100:1 dilutions of chronically infected strains as monitored by qPCR of whole culture aliquots. Error bars show mean results ⫾ SEM (n ⫽ 3). RESULTS AND DISCUSSION Viral challenge with SSV9 leads to chronic infection in immune-deﬁcient strains. The CRISPR-immune strain RJW003 is derived from type strain M.16.4 (isolated from Kamchatka, Russia) and has a perfect spacer match to that of SSV9 (also from Kamchatka), which is sufﬁcient for CRISPR-Cas immunity and prevention of viral infection (see Table S1 in the supplemental material) (17). This strain is marked with a deletion at the lacS locus, which is neutral in this context but allows identiﬁcation in competition with other strains through colony staining or quantitative PCR (qPCR) techniques (Fig. S3). We created a CRISPR immunity-deﬁcient strain of our Sulfolobus islandicus Δcas6 type strain (RJW002 Δcas6) and infected it with SSV9 (17). Chronically infected cells can grow while new viral particles are being shed but have a slight cost in ﬁnal optical density compared to those of uninfected isogenic strains in stationary phase. The peak of viral production occurs during exponential phase (Fig. 1). The chronically infected strain was sequenced to determine integration of the SSV9 genome; however, read mapping analysis supported the ﬁnding that the virus was being maintained as a nonintegrated episome at a 6-fold copy number relative to the number on the host chromosome. Chronically infected strains outcompete immune strains. To quantify the cost of viral infection, we tested the relative ﬁtnesses of the chronically infected and uninfected isogenic strains in competition with RJW003, the CRISPR-Cas-immune ΔlacS genetic derivative. We predicted that the immune strain would outcompete the chronically infected strain due to its ability to prevent viral infection and the cost of infection. We found that the immune strain had a relative ﬁtness above that of an uninfected CRISPR-Cas knockout strain in competition, suggesting that CRISPR-Cas is beneﬁcial and not costly in this genetic background (Fig. S2). In contrast to our prediction, the chronically infected strain outcompeted the immune strain (Fig. 2) and had a higher selection rate coefﬁcient for immune cells than the immune-deﬁcient, uninfected isogenic control strain (0.594 ⫾ 0.064/day and ⫺0.374 ⫾ 0.154/day for infected and March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 3 ® DeWerff et al. B 100 Abundance (%) 50 0 50 Immune SSV13 Infected 4 3 2 Day SSV14 Infected SSV17 Infected D 100 Abundance (%) 100 50 50 4 4 3 2 1 0 Day 3 0 0 2 Abundance (%) C SSV11 Infected 1 0 4 3 2 1 0 0 Day 1 Abundance (%) 100 0 A Day FIG 3 Competitive advantage is not speciﬁc to SSV9. Relative abundances in competitions between the immune strain (RJW003, black) and immune-deﬁcient (Δcas6 background) strains infected with related SSVs: SSV11 (A), SSV13 (B), SSV14 (C), and SSV17 (D). Chronically infected strains were added to mid-log phase of an immune culture and monitored by qPCR for 4 days without dilutions with fresh medium. Error bars show mean results ⫾ SEM (n ⫽ 3). uninfected strains, respectively; P value ⫽ 0.001). This ﬁtness advantage is independent of which strain carries the lacS deletion (Fig. S4). Additionally, this competitive beneﬁt still occurs when chronically infected cells are initially as rare as 1:100 (Fig. 2). SSVs are a common type of virus found among the Kamchatka population; therefore, we isolated new strains chronically infected with related viruses to determine if this competitive advantage is speciﬁc to SSV9 or a broad phenotype. We isolated chronically infected strains that were infected with other SSVs isolated from both the Kamchatka region (SSV13, SSV14, and SSV17) and Yellowstone National Park (SSV11) (Table S1). Competition between the immune strain and these chronically infected strains shows that this competitive beneﬁt is not speciﬁc to SSV9-infected cells but can be seen with viruses from both locations (Fig. 3). Shared open reading frames between these SSVs and SSV9 were compared with tblastn, revealing 1 gene sharing the same presence-absence pattern of the killing phenotype; however, competition with a chronically infected strain containing a deletion of this gene still showed a killing phenotype (Fig. S5) (20). To test whether ﬁtness advantage resulted from the faster growth of infected strains, we quantiﬁed the CFU of competing strains. As shown in Fig. 4, there was a decrease in the immune population after 24 h of coincubation at a 10:1 initial ratio, despite the fact that the population was initially in exponential growth, with an average doubling time of 14.2 h, suggesting that immune cells die and do not simply grow more slowly than infected cells. When looking at serial dilutions of the competing colonies, there was a mix of immune and chronically infected colonies seen on the initial day, but by day 1, there was evidence of only the infected strain, although it had not yet reached carrying capacity (Fig. S3). This killing effect would also explain the anomalous plaque formation observed with chronic viruses that do not kill their hosts (18). We hypothesize that the direct killing of uninfectable cells is a beneﬁt for the virus, as it removes potentially nonpermissive CRISPR immune hosts and prevents particle loss to CRISPR-Cas targeting, while the chronically infected host remains unaffected. This phenotype may explain some of the cost of infection (Fig. 1), as segregants are killed when they lose the virus. In this emergent mutualism, infection also beneﬁts the host cell in competition with immune cells. This is an advantage in a metapopulation March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 4 ® Virus-Mediated Antagonism by Chronically Infected Strains Abundance (copies/mL) A 1011 1010 10 9 10 8 0 1 2 3 4 Day Chronically Infected SSV9 CRISPR Immune Uninfected 10 11 10 12 10 11 10 10 10 10 10 9 10 9 10 8 10 8 0 1 2 3 4 SSV9 (genomes/mL) Abundance (copies/mL) B 10 7 Day FIG 4 A chronically infected strain is antagonistic to an immune strain. Relative abundances in competitions between a CRISPR-immune strain (RJW003, black) and an uninfected strain (Δcas6 strain, gold) (A) or between a CRISPR-immune strain and a chronically infected strain (Δcas6:SSV9.1, blue) (B). Uninfected or chronically infected strains were added to the mid-log phase of an immune culture and monitored by qPCR for 4 days without dilutions with fresh medium. In a representative experiment of three biological replicates, error bars show mean results ⫾ SEM (3 technical replicates). Virus abundance was measured by qPCR (dashed line). like that found in Sulfolobus islandicus strains from Yellowstone National Park (21), in which invasion of local environments that have immunity can increase host ﬁtness. Virus-mediated antagonism is mediated by a proteinaceous killing factor. To determine the mechanism of antagonism, we tested whether killing is due to active infection of immune cells by SSV9. Cell-free supernatants were collected by ﬁltration and boiled for 30 min; after this time, we determined the titers of untreated and boiled samples, and boiled samples were shown to have infectious units below the limit of detection (Fig. S4). As shown in Fig. 5, at 24 h, there was a severe loss of viability of the cells exposed to PFU-free supernatants from the chronically infected strain, in contrast to that of controls, and this viability remained low throughout the experiment. This demonstrates that infectious particles and active infection are not needed for the infected-cell antagonism on uninfected cells, making this phenotype clearly distinct from some dormancy phenotypes which may produce the similar result of immune cell death by activation of CRISPR targeting of host transcripts seen with Cas13 targeting in Listeria ivanovii (22). However, this may explain previous results in the Sulfolobus islandicus and SSV9 system which suggested that the presence of virus, even if inactivated by UV, can cause a stall in host growth. Here, we show that this phenotype in the context of the mixed population is the basis of a beneﬁt for both the virus and the chronically infected strain. This extracellular killing mechanism is also different from those of abortive infection systems that can also utilize toxin-antitoxin systems due to the killing activity not requiring active infection (23). Our data show that killing is not contact mediated, that it can occur without the presence of infected cells, and that a killing factor would be extremely stable in an acidic hot spring environment compared to in a viral particle. To test the nature of the killing factor, we treated cell-free supernatants from an uninfected and a chronically infected strain with the protease pepsin overnight. After 3 doses, it was found that the immune strain was still viable if challenged with the March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 5 DeWerff et al. ® FIG 5 Infection-mediated antagonism does not require active infection. (A) Representative images of CRISPR-immune strain RJW002 growth after incubation with boiled cell-free supernatants from an uninfected (Δcas6 strain) or a chronically infected (Δcas6:SSV9.1) strain. Immune cells were dosed with PFU-free boiled supernatants on days 0, 1, and 2. (B) Representative images of CRISPR-immune strain RJW002 growth on days 0 and 3 after incubation with boiled cell-free supernatants from an uninfected (Δcas6 strain) or a chronically infected (Δcas6:SSV9.1) strain with and without pepsin treatment. Immune cells were dosed with PFU-free boiled treated or untreated supernatants on days 0, 1, and 2. pepsin-treated supernatants or the supernatants from the uninfected strain (Fig. 5B). From this, we suggest that the mechanism of action is a toxin secreted into the medium similar in mechanism to that of killer yeast strains, where infection with an RNA virus causes the production and secretion of a protein toxin (24). Addictive plasmids evolved a similar method of utilizing a toxin for ensuring vertical transmission; however, it is not exported from the cell (25, 26). The identity of this toxin or whether it is virus or host encoded is unknown at this time. The proteinaceous toxins (sulfolobicins) speciﬁc to this genus that have been isolated from vesicles from Sulfolobus species were not encoded by the virus or the M.16.4 background (27, 28). Chronically infected supernatants concentrated to a 4⫻ relative concentration by tangential-ﬂow ﬁltration through a 30,000 molecular-weight-cutoff (MWCO) ﬁlter and boiled still show killing activity and growth inhibition (Fig. S8). The activity of the concentrated supernatants suggests that the killing factor is larger than a 30,000 MWCO, as it is not lost in the ﬁltration process. The killing phenotype selects for successful horizontal transmission. Since chronically infected cells continue to grow while producing the extracellular killing factor, we hypothesize that the virus encodes a rescue factor or antitoxin that makes chronically infected cells resistant to its effects. This would provide a screening mechanism to promote new horizontal infections established in nonimmune cells while limiting the loss of particles to CRISPR-immune cells. To test this, we competed the chronically infected and uninfected strains with the CRISPR-Cas-immune strain or March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 6 ® Virus-Mediated Antagonism by Chronically Infected Strains 10 4 8 10 3 10 7 10 2 10 6 10 5 10 1 0 5 10 15 10 10 10 5 10 9 10 4 10 8 10 3 10 7 10 2 10 6 10 5 10 1 0 20 5 10 4 10 8 10 3 10 7 10 2 10 6 10 5 10 1 0 5 10 15 20 20 10 10 10 5 10 9 10 4 10 8 10 3 10 7 10 2 10 6 10 1 10 5 10 0 0 5 10 15 20 Day Day Immune 15 Virus (pfu/mL) 10 9 Abundance (copies/mL) D 10 5 Virus (pfu/mL) Abundance (copies/mL) 10 10 10 Day Day C Virus (pfu/mL) 10 9 10 B 10 5 Abundance (copies/mL) 10 10 Virus (pfu/mL) Abundance (copies/mL) A Immune Deficient Infected SSV9 FIG 6 Persistence of immune and immune-deﬁcient strains in competition. Relative abundances in competitions between the CRISPR-immune strain (A and C) or an immune-deﬁcient strain (B and D), with the chronically infected strain at a ratio of 1:1 (A and B, circles) or 1:100 (C and D, triangles). Strains were grown to mid-log phase and mixed before being sampled for host abundance by qPCR. Supernatants were saved, and PFU were quantiﬁed by plaque assay to determine the amount of the virus (right axis). Every 3 days, the culture was sampled and then diluted 1:5 in fresh medium. Shown are the results of a representative experiment of three biological replicates; error bars show mean results ⫾ SEM (3 technical replicates). Virus abundance was measured by qPCR (dashed line). immune-deﬁcient strain. If the killer phenotype selects for successful horizontal transmission, we predicted that the CRISPR-immune strain would not persist in a long-term competition with the chronically infected strain and that the immune-deﬁcient strains would persist, as they could chronically infect cells through horizontal transmission (Fig. S6). However, we found instead that both the immune and the immune-deﬁcient strains can persist throughout the competition and at similar levels, contrary to our hypothesis (Fig. 6). Samples throughout the competition were plated for colonies and stained with X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) to determine the genetic background. While obtaining well-isolated colonies of the tested population (initially immune or immune deﬁcient) was rare, all colonies tested positive for the presence of SSV9 through colony PCR, indicating successful horizontal transmission. While this was expected for the immune-deﬁcient population, this was surprising for the immune population. We hypothesize that this may be due to a CRISPR-Cas failure rate, evolution of viral escape mutants, or some combination of the two. Though surprising, this result supports the supposition that, while it seems that there is a strong vertical transmission component in this system, the possibility of a horizontal component should not be excluded, even for immune populations. A virus-resistant strain is outcompeted by a chronically infected strain. To further test our hypothesis that active infection is not needed for killing but needed for survival, a virus-resistant strain was competed with a chronically infected strain. This virus-resistant strain was isolated from a viral challenge and found to have deletions of two surface pilin proteins needed to establish infection (20). When this strain is competed with a chronically infected strain, it is driven to almost extinction by day 6 (Fig. 7). Without the ability to establish chronic infection by horizontal transmission, the chronically infected strain has a larger competitive advantage over the resistant strain than the CRISPR-immune strain, with selection rates of 0.883 ⫾ 0.015/day against the resistant strain and 0.594 ⫾ 0.064/day against the CRISPR-immune strain, as stated March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 7 DeWerff et al. ® FIG 7 A viral-infection-resistant strain is outcompeted in a competition with a chronically infected strain. Shown are relative abundances in competitions between a virus-resistant strain (Δ6068) and an uninfected strain (RJW003 Δcas6) (A) and between Δ6068 and a chronically infected strain (RJW003 Δcas6: SSV9.3). Strains grown to mid-log phase were mixed and monitored for 6 days by qPCR for host cell type. Days 2 and 4 show relative abundances before a 1:5 dilution into fresh medium. Error bars show mean results ⫾ SEM (n ⫽ 3). earlier. Additionally, because the resistant strain is unable to be infected, this supports our earlier ﬁnding that active infection is not required for killing but instead for protection against toxin produced by other infected stains. Chronically infected supernatants show killing activity on contemporary Sulfolobus islandicus strains. Work until this point has been performed with a variety of strains within the same M.16.4 genetic background. However, if this is an emergent mutualism, it should not affect just one strain in a population that is known to be diverse. Therefore, killing activity was assessed on S. islandicus strains isolated from the same hot spring at the same time, as well as on two distantly related organisms, Sulfolobus acidocaldarius and Sulfolobus solfataricus P2 (29–33). Supernatants were collected from uninfected and chronically infected strains by ﬁltration, and aliquots we boiled for 30 min to remove infectious particles. Sulfolobus strains were then dosed with media, uninfected supernatants, or chronically infected supernatants for 3 days and then spotted for viability. While one distantly related strain showed complete resistance to the virus and killing activity (S. acidocaldarius), there was a mix of phenotypes seen, suggesting that some strains may be susceptible to the killing activity and not to viral infection or, conversely, to viral infection but not the killing activity (Fig. 8). Additionally, strains were grown on lawns and spotted with 3 doses of relative 4⫻-concentrated samples as described earlier at 0, 18, and 24 h (Fig. S8). Supporting the cell viability spots, almost all strains show some growth inhibition from the chronically infected supernatants; however, this is less pronounced in some of the boiled supernatants, which supports the idea that the inhibition is due to virus infection and not killing by a toxin. Overall though, the killing activity of contemporary strains suggests that this phenotype may be a driving force in the ecology of acidic hot springs and that it promotes a strong mutualism between SSV9 and its hosts. Concluding remarks. The population structure of susceptible hosts can drive the evolution of their viruses. While in some cases, this is theoretically predicted to select for decreases in virulence and increased vertical transmission, in Sulfolobus islandicus and SSV9, this interaction in the context of highly distributed immunity may select for a unique way of ensuring viral persistence by removing competing nonpermissive hosts. We have shown that strains with CRISPR-Cas immunity and viral resistance are subjected to this killing phenotype, suggesting that while active infection is not needed for killing, it may be needed to prevent killing. Our hypothesis is that the chronically infected cells produce a toxin that can be secreted and produce an antitoxin that remains within the infected cell. This would allow for speciﬁc targeting of the nonpermissive host that either degrades the viral genome through CRISPR-Cas immunity or prevents infection, as with potentially virus-resistant strains. This phenotype seems March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 8 ® Virus-Mediated Antagonism by Chronically Infected Strains M.16.46 M.16.47 ed ec ted bo ile d bo il Inf ted Inf ec ted ted ec ec inf Un di a inf Me M.16.40 M.16.30 M.16.23 M.16.22 M.16.13 Un ted Inf bo ec ile ted d Inf ec ted bo ile d ec ec inf inf Un Un M.16.4 M.16.12 M.16.2 S. solfataricus S. acidocaldarius Δcas6 RJW002 Me dia ted Supernatants FIG 8 Treatment of chronically infected supernatants on related Sulfolobaceae strains shows decreased viability. S. islandicus (RJW002, Δcas6, and M.16 strains), Sulfolobus acidocaldarius, and Sulfolobus solfataricus were grown in liquid media dosed with uninfected or chronically infected supernatants with and without prior boiling. Strains were incubated for 3 days and then serially diluted and spotted onto a solid medium to determine viability. Representative images from two biological replicates are shown. especially beneﬁcial for a chronic virus with a mixed mode of transmission, as the continued production of viral particles would allow an uninfected, permissive host to be selected in the population, as well as remove competing strains to ensure vertical transmission. This is especially important in populations with high levels of distributed CRISPR-Cas immunity, where new viruses would most often be degraded, leading to viral extinction. Together with the beneﬁts to the infected host as shown in this study, we believe that this interaction may shift toward a mutualistic interaction between the virus and its host, and future studies into the mechanism and ecology of this interaction may further support the changing evolution of this symbiosis. MATERIALS AND METHODS Microbial strains. All Sulfolobus islandicus strains (see Table S1 in the supplemental material) were grown in dextrin-tryptone medium supplemented with uracil (DTU), as needed (29, 34). Cultures were grown at 75°C without shaking. Viral stocks and supernatants were collected at exponential growth of March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 9 DeWerff et al. ® a chronically infected strain, ﬁltered through a 0.2-␮m polyethersulfone (PES) bottle-top ﬁlter (Nalgene catalog number 595-4520), and then stored at 4°C. Infections with SSV viruses. An S. islandicus immune-deﬁcient ΔCas6 deletion strain was challenged with viruses as described previously (17). Following the 5-h incubation with virus ﬁltrate, cells were resuspended in 70 ml DTU in a 75-cm2 culture ﬂask and incubated at 75°C. Host growth was monitored, and after 8 days or when an optical density at 600 nm (OD600) of 0.10 was reached, cultures were plated in DTU-Gelrite overlays for colony isolation. To isolate a chronically infected strain after 10 to 14 days of incubation at 75°C, colonies were picked and resuspended in DTU and tested for infection using 16S rRNA- and SSV-speciﬁc primers. Colonies that tested positive for infection were then grown, supernatants were collected, and 10 ␮l was spotted on a lawn of the strain S. islandicus Y08.82.36 for production of PFU. Library prep and sequencing. Genomic libraries were prepared for all viruses using the Nextera XT kit (Illumina) according to the manufacturer’s instructions. Libraries were pooled and sequenced using paired-end MiSeq version 2.5 by the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois at Urbana-Champaign. Reads were quality ﬁltered using FASTX-Toolkit, and adapters were trimmed using Cutadapt (35). Host genome assemblies were compared to those of host strain M.16.4 using breseq (36). Determining SSV integration status. Quality ﬁltered reads from a chronically infected strain were mapped to the SSV9 integrase by using bwa (37). Read names were extracted, and the mate pairs of mapped reads were then pulled into a separate ﬁle. Read mates were mapped back to the S. islandicus M.16.4 genome using bwa, and the alignment was visualized using Geneious. Areas of read coverage of the mate pairs were analyzed, and we found no evidence of reads mapping to the host genome above our map quality score threshold. Growth curves. To compare levels of growth of the uninfected and infected strains, cells were collected by centrifugation at 4,000 ⫻ g for 20 min. Cells were resuspended in fresh DTU and then diluted to a ﬁnal volume of 20 ml at an initial OD600 of 0.04 and incubated without shaking at 75C. Cell growth was determined by measuring optical density every 24 h. At the time of sampling, 500-␮l aliquots were taken and culture supernatants were collected by centrifugation at 15,000 ⫻ g in a microcentrifuge. Collected supernatants were then used for a plaque-forming assay. One hundred microliters of the supernatant was mixed with 500 ␮l of a 10⫻-concentrated strain, S. islandicus Y08.82.36. This mixture was incubated at 75°C for 30 min, and then 5 ml of an overlay sucrose-yeast (SY) medium was poured on SY plates as previously described (18). Plates were incubated at 75°C for 48 h. Transfer competitions. Strains used in competitions were grown to mid-log phase (OD600 of 0.10 to 0.18). Cells were collected by centrifugation at 4,000 ⫻ g for 20 min and resuspended in fresh media. Cells were then inoculated at the respective ratio of each type so that there was approximately 3 ⫻ 109 combined total cells. Cultures were incubated for 2 days at 75°C without shaking; after 2 days, the culture was diluted 1:5 into fresh medium. This was repeated 2 more times, for a total competition duration of 6 days. At the start of the competition and before and after dilution, 200 ␮l of culture was collected and frozen for analysis of cell abundance by qPCR and 200 ␮l of supernatant was collected for analysis of viral abundance (see Table S3 for primers). Primers targeting a 150-bp portion within the lacS gene or the residues left after lacS deletion were used to distinguish cell types using a previously described qPCR protocol. Standard curves were created using extracted genomic DNA from puriﬁed strains with and without the lacS gene. In the long-term transfer competitions, the experiment was conducted in a similar manner, but cells were transferred every 3 days. Nontransfer competitions. Strains used in competitions were grown to mid-log phase (OD600, 0.10 to 0.18). Cells were collected by centrifugation at 4,000 ⫻ g for 20 min and resuspended in fresh media. Cells were then inoculated at the respective ratio of each type so that there were approximately 3 ⫻ 109 total combined cells. Cultures were incubated at 75°C for 4 days, and 200 ␮l of culture was collected every day and frozen for analysis of cell abundance by qPCR as described above. Heat-killed supernatant assays. Cultures of the chronically infected strain or the isogenic uninfected strain were grown to an optical density of 0.15 to 0.20 and then ﬁltered through a 0.22-␮m PES ﬁlter. Supernatants were then placed in boiling water for 30 min. Supernatant samples from before and after boiling were tested for infectious particles by plaque assay as described above. When pepsin was used, the treatment of the supernatants was completed with immobilized pepsin (Thermo Scientiﬁc catalog number 20343) according to the manufacturer’s instructions and incubated at 37°C overnight in a rotator. Strains tested were grown to mid-log phase and then diluted to an OD600 of 0.03 in fresh medium, the boiled supernatants, or the unboiled supernatants. At 24, 48, and 72 h, cultures were centrifuged, and cells were resuspended in fresh doses of medium or the supernatants. Every 24 h, cell viability was tested by spotting 10-␮l cell dilutions onto DTU plates. SUPPLEMENTAL MATERIAL Supplemental material is available online only. FIG S1, EPS ﬁle, 1.6 MB. FIG S2, EPS ﬁle, 1.5 MB. FIG S3, EPS ﬁle, 1.7 MB. FIG S4, EPS ﬁle, 1.5 MB. FIG S5, EPS ﬁle, 2.2 MB. FIG S6, TIF ﬁle, 4.9 MB. March/April 2020 Volume 11 Issue 2 e00404-20 mbio.asm.org 10 ® Virus-Mediated Antagonism by Chronically Infected Strains FIG S7, EPS ﬁle, 0.8 MB. FIG S8, EPS ﬁle, 1.9 MB. TABLE S1, DOCX ﬁle, 0.02 MB. TABLE S2, DOCX ﬁle, 0.01 MB. ACKNOWLEDGMENTS This work was supported by National Science Foundation grants DOB 1342876 and IOS 16-505. We thank M. Young and J. Weitz for thoughtful discussions, E. Rowland and D. Campbell for thoughtful feedback on experimental troubleshooting, W. England for help with bioinformatics, and D. Krause for help with oligonucleotide design and bioinformatics and thoughtful discussion. We thank the W. M. Keck Center for Comparative and Functional Genomics at the University of Illinois Urbana-Champaign for the Illumina sequencing done for this work. Conceptualization, S.J.D. and R.J.W.; Methodology, S.J.D., M.A.B., M.P., and R.J.W.; Investigation, S.J.D., M.A.B., and M.P.; Resources C.Z.; Writing – Original Draft, S.J.D.; Writing – Review & Editing, S.J.D. and R.J.W.; Funding Acquisition, R.J.W. REFERENCES 1. Bull JJ, Molineux IJ, Rice WR. 1991. Selection of benevolence in a host-parasite system. Evolution 45:875– 882. https://doi.org/10.1111/j .1558-5646.1991.tb04356.x. 2. Poisot T, Bever JD, Thrall PH, Hochberg ME. 2014. Dispersal and spatial heterogeneity allow coexistence between enemies and protective mutualists. Ecol Evol 4:3841–3850. https://doi.org/10.1002/ece3.1151. 3. Roossinck MJ. 2011. The good viruses: viral mutualistic symbioses. Nat Rev Microbiol 9:99 –108. https://doi.org/10.1038/nrmicro2491. 4. Shapiro JW, Williams ESCP, Turner PE. 2016. Evolution of parasitism and mutualism between ﬁlamentous phage M13 and Escherichia coli. PeerJ 4:e2060. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC 4888304/. https://doi.org/10.7717/peerj.2060. 5. Turner PE, Cooper VS, Lenski RE. 1998. Tradeoff between horizontal and vertical modes of transmission in bacterial plasmids. Evolution 52: 315–329. https://doi.org/10.2307/2411070. 6. Bull JJ. 1994. Virulence. Evolution 48:1423–1437. https://doi.org/10.1111/ j.1558-5646.1994.tb02185.x. 7. Childs LM, England WE, Young MJ, Weitz JS, Whitaker RJ. 2014. CRISPRinduced distributed immunity in microbial populations. PLoS One 9:e101710. https://doi.org/10.1371/journal.pone.0101710. 8. Chabas H, Lion S, Nicot A, Meaden S, van Houte S, Moineau S, Wahl LM, Westra ER, Gandon S. 2018. Evolutionary emergence of infectious diseases in heterogeneous host populations. PLoS Biol 16:e2006738. https://doi.org/10.1371/journal.pbio.2006738. 9. Pilosof S, Alcala-Corona SA, Wang T, Kim T, Maslov S, Whitaker RJ, Pascual M. 2020. The network structure and eco-evolutionary dynamics of CRISPR-induced immune diversiﬁcation. bioRxiv 850800. https://www .biorxiv.org/content/10.1101/850800v1. 10. van Houte S, Ekroth AKE, Broniewski JM, Chabas H, Ashby B, BondyDenomy J, Gandon S, Boots M, Paterson S, Buckling A, Westra ER. 2016. The diversity-generating beneﬁts of a prokaryotic adaptive immune system. Nature 532:385–388. https://doi.org/10.1038/nature17436. 11. Westra ER, van Houte S, Oyesiku-Blakemore S, Makin B, Broniewski JM, Best A, Bondy-Denomy J, Davidson A, Boots M, Buckling A. 2015. Parasite exposure drives selective evolution of constitutive versus inducible defense. Curr Biol 25:1043–1049. https://doi.org/10.1016/j.cub.2015.01.065. 12. Chabas H, Nicot A, Meaden S, Westra ER, Tremblay DM, Pradier L, Lion S, Moineau S, Gandon S. 2019. Variability in the durability of CRISPR-Cas immunity. Philos Trans R Soc Lond B Biol Sci 374:20180097. https://doi .org/10.1098/rstb.2018.0097. 13. Common J, Morley D, Westra ER, van Houte S. 2019. CRISPR-Cas immunity leads to a coevolutionary arms race between Streptococcus thermophilus and lytic phage. Philos Trans R Soc Lond B Biol Sci 374: 20180098. https://doi.org/10.1098/rstb.2018.0098. 14. Held NL, Herrera A, Cadillo-Quiroz H, Whitaker RJ. 2010. CRISPR associated diversity within a population of Sulfolobus islandicus. PLoS One 5:e12988. https://doi.org/10.1371/journal.pone.0012988. 15. Pauly MD, Bautista MA, Black JA, Whitaker RJ. 2019. Diversiﬁed local March/April 2020 Volume 11 Issue 2 e00404-20 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. CRISPR-Cas immunity to viruses of Sulfolobus islandicus. Philos Trans R Soc Lond B Biol Sci 374:20180093. https://doi.org/10.1098/rstb.2018 .0093. Held NL, Whitaker RJ. 2009. Viral biogeography revealed by signatures in Sulfolobus islandicus genomes. Environ Microbiol 11:457– 466. https:// doi.org/10.1111/j.1462-2920.2008.01784.x. Bautista MA, Zhang C, Whitaker RJ. 2015. Virus-induced dormancy in the archaeon Sulfolobus islandicus. mBio 6:e02565-14. https://doi.org/10 .1128/mBio.02565-14. Schleper C, Kubo K, Zillig W. 1992. The particle SSV1 from the extremely thermophilic archaeon Sulfolobus is a virus: demonstration of infectivity and of transfection with viral DNA. Proc Natl Acad Sci U S A 89:7645–7649. https://doi.org/10.1073/pnas.89.16.7645. Quemin ERJ, Chlanda P, Sachse M, Forterre P, Prangishvili D, Krupovic M. 2016. Eukaryotic-like virus budding in archaea. mBio 7:e01439-16. https://doi.org/10.1128/mBio.01439-16. Rowland EF, Bautista MA, Zhang C, Whitaker RJ. 2020. Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus. Mol Microbiol https://onlinelibrary.wiley.com/doi/abs/10.1111/mmi.14435. Accessed 10 January 2020. Redder P, Peng X, Brügger K, Shah SA, Roesch F, Greve B, She Q, Schleper C, Forterre P, Garrett RA, Prangishvili D. 2009. Four newly isolated fuselloviruses from extreme geothermal environments reveal unusual morphologies and a possible interviral recombination mechanism. Environ Microbiol 11:2849 –2862. https://doi.org/10.1111/j.1462-2920.2009 .02009.x. Meeske AJ, Nakandakari-Higa S, Marrafﬁni LA. 2019. Cas13-induced cellular dormancy prevents the rise of CRISPR-resistant bacteriophage. Nature 570:241–245. https://doi.org/10.1038/s41586-019-1257-5. Koonin EV, Makarova KS, Wolf YI. 2017. Evolutionary genomics of defense systems in archaea and bacteria. Annu Rev Microbiol 71:233–261. https://doi.org/10.1146/annurev-micro-090816-093830. Bevan EA, Herring AJ, Mitchell DJ. 1973. Preliminary characterization of two species of dsRNA in yeast and their relationship to the “killer” character. Nature 245:81– 86. https://doi.org/10.1038/245081b0. Lehnherr H, Maguin E, Jafri S, Yarmolinsky MB. 1993. Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained. J Mol Biol 233:414 – 428. https://doi.org/10.1006/jmbi.1993 .1521. Gerdes K, Bech FW, Jørgensen ST, Løbner-Olesen A, Rasmussen PB, Atlung T, Boe L, Karlstrom O, Molin S, von Meyenburg K. 1986. Mechanism of postsegregational killing by the hok gene product of the parB system of plasmid R1 and its homology with the relF gene product of the E. coli relB operon. EMBO J 5:2023–2029. https://doi.org/10.1002/j .1460-2075.1986.tb04459.x. Ellen AF, Rohulya OV, Fusetti F, Wagner M, Albers S-V, Driessen AJM. 2011. The sulfolobicin genes of Sulfolobus acidocaldarius encode novel mbio.asm.org 11 ® DeWerff et al. 28. 29. 30. 31. 32. 33. antimicrobial proteins. J Bacteriol 193:4380 – 4387. https://doi.org/10 .1128/JB.05028-11. Prangishvili D, Holz I, Stieger E, Nickell S, Kristjansson JK, Zillig W. 2000. Sulfolobicins, speciﬁc proteinaceous toxins produced by strains of the extremely thermophilic archaeal genus Sulfolobus. J Bacteriol 182: 2985–2988. https://doi.org/10.1128/jb.182.10.2985-2988.2000. Whitaker RJ, Grogan DW, Taylor JW. 2003. Geographic barriers isolate endemic populations of hyperthermophilic archaea. Science 301: 976 –978. https://doi.org/10.1126/science.1086909. Cadillo-Quiroz H, Didelot X, Held NL, Herrera A, Darling A, Reno ML, Krause DJ, Whitaker RJ. 2012. Patterns of gene ﬂow deﬁne species of thermophilic archaea. PLoS Biol 10:e1001265. https://doi.org/10.1371/ journal.pbio.1001265. Brock TD, Brock KM, Belly RT, Weiss RL. 1972. Sulfolobus: a new genus of sulfur-oxidizing bacteria living at low pH and high temperature. Arch Mikrobiol 84:54 – 68. https://doi.org/10.1007/bf00408082. Zillig W, Stetter KO, Wunderl S, Schulz W, Priess H, Scholz I. 1980. The Sulfolobus-“Caldariella” group: taxonomy on the basis of the structure of DNA-dependent RNA polymerases. Arch Microbiol 125:259 –269. https:// doi.org/10.1007/BF00446886. Sakai HD, Kurosawa N. 2018. Saccharolobus caldissimus gen. nov., sp. March/April 2020 Volume 11 Issue 2 e00404-20 34. 35. 36. 37. nov., a facultatively anaerobic iron-reducing hyperthermophilic archaeon isolated from an acidic terrestrial hot spring, and reclassiﬁcation of Sulfolobus solfataricus as Saccharolobus solfataricus comb. nov. and Sulfolobus shibatae as Saccharolobus shibatae comb. nov. Int J Syst Evol Microbiol 68:1271–1278. https://doi.org/10.1099/ijsem.0.002665. Zhang C, Whitaker RJ. 2012. A broadly applicable gene knockout system for the thermoacidophilic archaeon Sulfolobus islandicus based on simvastatin selection. Microbiology 158:1513–1522. https://doi.org/10.1099/ mic.0.058289-0. Martin M. 2011. Cutadapt removes adapter sequences from highthroughput sequencing reads. EMBnet J 17:10. https://doi.org/10 .14806/ej.17.1.200. Barrick JE, Colburn G, Deatherage DE, Traverse CC, Strand MD, Borges JJ, Knoester DB, Reba A, Meyer AG. 2014. Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq. BMC Genomics 15:1039. https://doi.org/10.1186/1471-2164-15 -1039. Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25:1754 –1760. https://doi .org/10.1093/bioinformatics/btp324. mbio.asm.org 12
https://openalex.org/W3083581329	https://hal.science/hal-03086685/document	English	null	Inferring number of populations and changes in connectivity under the n-island model	Heredity	2,021	cc-by	15,924	Inferring number of populations and changes in connectivity under the n-island model Arredondo, Beatriz Mourato, Khoa Nguyen, Simon Boitard, Willy Rodríguez, Olivier Mazet, Lounès Chikhi To cite this version: Armando Arredondo, Beatriz Mourato, Khoa Nguyen, Simon Boitard, Willy Rodríguez, et al.. Infer- ring number of populations and changes in connectivity under the n-island model. Heredity, 2021, 126 (6), pp.896-912. 10.1101/2020.09.03.282251. hal-03086685 Distributed under a Creative Commons Attribution 4.0 International License Inferring number of populations and changes in connectivity under the n-island model Armando Arredondo, Beatriz Mourato, Khoa Nguyen, Simon Boitard, Willy Rodríguez, Olivier Mazet, Lounès Chikhi To cite this version: Armando Arredondo, Beatriz Mourato, Khoa Nguyen, Simon Boitard, Willy Rodríguez, et al.. Infer- ring number of populations and changes in connectivity under the n-island model. Heredity, 2021, 126 (6) 896 912 10 1101/2020 09 03 282251 h l 03086685 Inferring number of populations and changes in connectivity under the n-island model Armando Arredondo, Beatriz Mourato, Khoa Nguyen, Simon Boitard, Willy Rodríguez, Olivier Mazet, Lounès Chikhi 3 Instituto Gulbenkian de Ciência, Oeiras, Portugal 4 CBGP, Université de Montpellier, CIRAD, INRAE, Institut Agro, IRD, Montpellier, France 5 ENAC - Ecole Nationale de l’Aviation Civile, Université de Toulouse, Toulouse, France 6 Laboratoire Cogitamus, Toulouse, France 7 Laboratoire Évolution & Diversité Biologique (EDB UMR 5174), CNRS, IRD, UPS, Université de Toulouse Midi-Pyrénées, Toulouse, France Abstract Inferring the demographic history of species is one of the greatest challenges in populations genetics. This history is often represented as a history of size changes, ignoring population structure. Alternatively, when structure is assumed, it is deﬁned a priori as a population tree and not inferred. Here we propose a framework based on the IICR (Inverse Instantaneous Coalescence Rate). The IICR can be estimated for a single diploid individual using the PSMC method of Li and Durbin (2011). For an isolated panmictic population, the IICR matches the population size history, and this is how the PSMC outputs are generally interpreted. However, it is increasingly acknowledged that the IICR is a function of the demographic model and sampling scheme with limited connection to population size changes. Our method ﬁts observed IICR curves of diploid individuals with IICR curves obtained under piecewise stationary symmetrical island models. In our models we assume a ﬁxed number of time periods during which gene ﬂow is constant, but gene ﬂow is allowed to change between time periods. We infer the number of islands, their sizes, the periods at which connectivity changes and the corresponding rates of connectivity. Validation with simulated data showed that the method can accurately recover most of the scenario parameters. Our application to a set of ﬁve human PSMCs yielded demographic histories that are in agreement with previous studies using similar methods and with recent research suggesting ancient human structure. They are in contrast with the view of human evolution consisting of one ancestral population branching into three large continental and panmictic populations with varying degrees of connectivity and no population structure within each continent. Inferring number of populations and changes in connectivity under the n-island model Received: 30 August 2020 / Revised: 11 March 2021 / Accepted: 12 March 2021 / Published online: 12 April 2021 © The Author(s) 2021. This article is published with open access HAL Id: hal-03086685 https://hal.science/hal-03086685v1 Submitted on 17 Mar 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. 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 * Armando Arredondo arredond@insa-toulouse.fr Heredity (2021) 126:896–912 https://doi.org/10.1038/s41437-021-00426-9 ARTICLE ARTICLE Introduction Such models thus assume panmixia over large geographic regions and long periods (Gutenkunst et al., 2009, Noskova et al., 2019). Panmictic and tree models are useful approximations, and in the last decades they have proven their utility in building stories of human expansions and population splits (Guten- kunst et al., 2009, Li and Durbin, 2011). However, the meaning of such stories is questionable (Mazet et al., 2016, Scerri et al., 2019, Wakeley, 1999). Also, most tree models assume the existence of clear splitting events similar to those separating species, and some tree models assume, as in most species trees, that there is no gene ﬂow between branches (even when they represent populations or continents). The latter assumption may then require the inference of admixture events (e.g., Kuhlwilm et al., 2016). The approach presented in the present study differs from the approaches mentioned above in several ways. First, we aim at inferring the number of populations rather than set- ting it a priori. Second, we ask whether it is possible to date and quantify changes in connectivity (i.e., gene ﬂow) rather than changes in population size. For that, we use the pie- cewise stationary n-island model in which continuous gene ﬂow happens between populations at a constant rate during speciﬁc periods (called components) but is allowed to change between periods (see below and Rodríguez et al. (2018)). This model differs from tree models in that we do not estimate parameters such as splitting times which may or may not be meaningful or appropriate for various species (Scerri et al., 2019) depending on their actual (unknown) demographic history. We acknowledge the limitations of the n-island model as it ignores spatial distances and other complexities of real species (Chikhi et al., 2018), but our choice for the current study is also guided by simplicity and computational considerations. We focus on changing pat- terns of connectivity since they may have been crucial in the recent evolutionary history of many species (Fenderson et al., 2020, Goldstein and Chikhi, 2002, Mazet et al., 2016, Quéméré et al., 2012, Salmona et al., 2017, Scerri et al., 2018, Steinrücken et al., 2019), particularly in the context of Pleistocene climate change and habitat fragmentation. Also, it is important to clarify whether the IICR contains useful information for parameter estimation and model choice (Mazet et al., 2016). Introduction challenges of population geneticists (Beaumont, 2004, Goldstein and Chikhi, 2002, Hey and Machado, 2003, Johri et al., 2020, Li and Durbin, 2011). It is an important and challenging statistical problem, but it is also central to our understanding of the evolutionary history of species. Indeed, the demographic history that we assume or infer for a particular population or species implicitly or explicitly provides the null model against which selected loci could in theory be identiﬁed (Beaumont and Nichols, 1996, Cavalli- Sforza, 1966, Goldstein and Chikhi, 2002, Johri et al., Reconstructing the demographic history of populations from the analysis of genomic data is one of the greatest Associate editor: Giorgio Bertorelle * Armando Arredondo arredond@insa-toulouse.fr * Lounès Chikhi lounes.chikhi@univ-tlse3.fr 1 Université de Toulouse, Institut National des Sciences Appliquées, Institut de Mathématiques de Toulouse, Toulouse, France 2 Institut de Mathématiques de Toulouse; UMR5219. Université de Toulouse, Toulouse, France Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41437- 021-00426-9. Associate editor: Giorgio Bertorelle * Armando Arredondo arredond@insa-toulouse.fr * Lounès Chikhi lounes.chikhi@univ-tlse3.fr 1 Université de Toulouse, Institut National des Sciences Appliquées, Institut de Mathématiques de Toulouse, Toulouse, France 2 Institut de Mathématiques de Toulouse; UMR5219. Université de Toulouse, Toulouse, France Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41437- 021-00426-9. Associate editor: Giorgio Bertorelle Associate editor: Giorgio Bertorelle Associate editor: Giorgio Bertorelle Associate editor: Giorgio Bertorelle Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41437- 021-00426-9. * Armando Arredondo arredond@insa-toulouse.fr * Lounès Chikhi lounes.chikhi@univ-tlse3.fr * Lounès Chikhi lounes.chikhi@univ-tlse3.fr 1 Université de Toulouse, Institut National des Sciences Appliquées, Institut de Mathématiques de Toulouse, Toulouse, France 2 Institut de Mathématiques de Toulouse; UMR5219. Université de Toulouse, Toulouse, France Inferring number of populations and changes in connectivity under the n-island model 897 still limited number of species (Lapierre et al., 2017). We stress though that this research ﬁeld is very active and new methods are regularly proposed that go beyond the simpli- ﬁed classiﬁcations proposed here. For instance, recent methods allow to infer complex demographic histories from full genomes (Steinrücken et al., 2019, Wang et al., 2020). 2020). Introduction In a period of global environmental change, the reconstructed demographic history should allow evolu- tionary biologists to correlate changes in population size or connectivity with past environmental changes or species association and interactions (Goossens et al., 2006, Hecht et al., 2018, 2020, Mona et al., 2014, Quéméré et al., 2012, Salmona et al., 2017). Here, we propose to use a different strategy based on the IICR (Inverse Instantaneous Coalescence Rate) introduced by Mazet et al. (2016). We propose to perform demographic inference under the piecewise stationary n-island model (Rodríguez et al., 2018), based on the symmetrical n-island model (Wright, 1931), using the IICR or estimates obtained from sequence data. The IICR, as deﬁned by Mazet et al. (2016) for a sample of size two, is equivalent to the full distribution of coalescence times for that sample (i.e., the distribution of T2). Simulations by Chikhi et al. (2018) and Rodríguez et al. (2018) under various models of population structure suggest that the IICR is sensitive to population structure or ﬂuctuations in migration rates (i.e., changes in connectivity). In other words, by addressing these challenges we expect to increase our understanding of the environmental (including species interactions) and anthropogenic factors that have inﬂuenced genomic diversity in various species. Also, understanding how past climatic events or human activities have inﬂuenced genomic diversity today may become particularly illuminating for conservation biologists regarding the likely long-term consequences of ongoing climate change and human actions (Poelstra et al., 2021). g However, to understand how the past inﬂuenced the pre- sent patterns of genomic diversity one major question is whether our conclusions or inferences may fundamentally change depending on the family of models assumed and the questions asked (Beaumont, 2004, Chikhi et al., 2018, 2010, Mazet et al., 2016, Pouyet et al., 2018, Rodríguez et al., 2018, Wakeley, 1999). Currently, the solutions to this com- plex inferential problem have been to assume that differ- entiation between geographic locations can be neglected and local panmixia assumed, and then infer population size changes (Li and Durbin, 2011, Liu and Fu, 2015). Alter- natively, other studies have assumed simpliﬁed tree models with a priori ﬁxed numbers of populations (i.e., the popula- tion trees are not inferred). Additionally, in the case of human evolutionary history, the branches of the assumed tree may represent predeﬁned continental populations. Introduction The work presented here may thus represent an interesting endeavor, particularly given that there is an increasing recognition of ancient human structure by researchers of different ﬁelds (Scerri et al., 2019, 2018). h i f i l h d i i l d i Methods can also be classiﬁed by the type of data used. Currently, most genomic methods use the allele frequency spectrum (AFS) (Excofﬁer et al., 2013, Gutenkunst et al., 2009, Liu and Fu, 2015) or the AFS combined with other summary statistics (Boitard et al., 2016). The AFS can be computed from RAD-Seq data for many non-model species (Poelstra et al., 2021) or from full genome sequences for a The inferential method is implemented in a program called SNIF (Structured Non-stationary Inferential A. Arredondo et al. 898 demes in any given generation, such that mi = ∑i≠jqjisj/si. She also showed that measuring time in units of 2N gen- erations and making N go to inﬁnity in such a way that the number of migrants stays bounded, the model converges to a continuous-time Markov process. It is possible to con- struct a transition rate matrix Q that provides a good approximation of the gene genealogies of the discrete time model. In this transformation, qij goes to zero in such a way that the product 2Nqjisj/si converges and has limit Mij/2. Thus we can express the transition rates in Q in terms of n, si, and Mij. In the case of the symmetrical island model (Wright, 1931), all the migration rates Mij between any pair of islands i and j are equal, so we use the notation M = (n − 1)Mij to denote the migration rate received by any given island. In addition to this base model, we use an extension, the NSSC, presented in Rodríguez et al. (2018) which allows to introduce demographic events that change the rate M or relative deme size s at certain points in time (see section “The piecewise stationary n-island coalescent”). We note however that throughout the manuscript we will only focus on symmetrical models with constant size (see section “Discussion” for extensions to symmetrical models with population size changes). Framework). We validated the method with data simulated under piecewise stationary n-island models and inferred connectivity graphs which are a visual representation of the times at which gene ﬂow changed and of the magnitude of these changes. Introduction We then applied SNIF to human genomic data using ﬁve published PSMC curves (Prado-Martinez et al., 2013), allowing in each case the number of compo- nents to vary between analyses, and compared the inferred histories and connectivity graphs between individuals and with previously inferred scenarios by Rodríguez et al. (2018) and Noskova et al. (2019). Beyond human data we ﬁnd that a crucial issue is the estimation of the IICR from genomic data. Indeed, the stochasticity generated during the estimation of the IICR in very ancient times, and possibly recent times, with humps that are difﬁcult to interpret, may lead to the inference of events that may never have taken place. Methods To use the IICR as a summary of genomic information we ﬁrst assume that an IICR curve can be obtained, which we will use as the target for demographic inference. With simulated data (sequences or T2 values) this target curve can be obtained under any predeﬁned coalescent model that could be expressed with a simulation tool (e.g., the ms program (Hudson, 2002)). With real genome-wide sequence data, the curve can be estimated with the PSMC method of Li and Durbin (2011). We then try to identify a piecewise stationary n-island model that generates an IICR that is identical or similar to the target IICR (or PSMC curve). The similarity between the two IICR curves is quantiﬁed with a distance metric deﬁned below. We use a genetic algorithm to explore the parameter space (number of populations, migration rate within a time component, and timing of these changes assuming a ﬁxed number of components for each independent analysis) and minimize that distance. We compute the IICR under the non-stationary structured coa- lescent (NSSC) of Rodríguez et al. (2018). For the demographic histories under these models, we study the IICR of a sample of size 2 (see section S1.1 of the Supplementary Materials), and we use it as a statistic for demographic inference. We do this by comparing the IICR of many hypothetical demographic scenarios to a target IICR curve. This target IICR may be simulated, or it may be obtained from diploid individuals via full genome studies (Prado-Martinez et al., 2013). In such cases, these target IICRs are themselves inferred demographies under the assumptions of a particular model. For example, the PSMC method (Li and Durbin, 2011) uses the population size change model, where a single panmictic population varies in size according to a function N(t) = N(0)λ(t) (see Tavaré (2004)). It was shown by Mazet et al. (2016) that the IICR of a sample of size 2 under this model is exactly the λ(t) relative size changing function, and it relates to the dis- tribution of the time to coalescence T2 as: PðT2>tÞ ¼ exp R t 0 dx λðxÞ ; IICRðtÞ ¼ λðtÞ ¼ PðT2>tÞ f T2ðtÞ : ð1Þ ^ϕγ;B ¼ ^φ ¼ ðN; φÞ 2 R ϕγ;B such that 0<Nmin ⩽N ⩽Nmax : In section S1.2 of the Supplementary Materials, we present the piecewise-continuous version of this parameter space for both the scaled and unscaled IICRs, as well as the relationship between them. Given a ﬁxed integer γ of demographic events to con- sider (γ ⩾0) and a collection of bounds B in the form of: B ¼ ½nmin; nmax; ½t1 min; t1 max ¼ ½tγ min; tγ max; ½M0 min; M0 max ¼ ½Mγ min; Mγ max; ½s0 min; s0 max ¼ ½sγ min; sγ max ; ð2Þ The structured coalescent and the IICR ð1Þ The theoretical framework we use for modeling structure is based on the ﬁnite Herbots’s model of the structured coa- lescent (Herbots, 1994), which is a backwards-in-time view of the gene genealogies. We have n populations or demes that are assumed to behave as haploid Wright–Fisher models of size Ni = 2siN genes, where si is the relative deme size and N is large. Migration occurs between demes as in each generation a proportion qij of lineages migrates from deme i to deme j. Herbots denoted by mi the proportion of the population of deme i that was received from other The IICR is not tied to any particular model, structured or otherwise. It is deﬁned using the distribution of the coalescent times of a sample of size two. It can be approximated to arbitrary numerical precision under the assumptions of the NSSC (Rodríguez et al., 2018); it can also be computed empirically by simulating a sample of coalescent times (Chikhi et al., 2018); or it can be read from full sequence genomic data using the appropriate methods (e.g., Li and Durbin (2011)). Inferring number of populations and changes in connectivity under the n-island model 899 we deﬁne the parameter space Φγ,B as: ϕγ;B ¼ φ ¼ ðn; t1 ¼ tγ; M0 ¼ Mγ; s0 ¼ sγÞ 2 N R3γþ3; s:t: 8i : 2 ⩽nmin ⩽n ⩽nmax; 0<ti min ⩽ti ⩽ti max; 0<Mi min ⩽Mi ⩽Mi max; 0<si min ⩽si ⩽si maxg: ð3Þ Given a target IICR0 and a parameter space Φγ,B, we want to ﬁnd a parameter tuple φ in Φγ,B such that the exact IICR curve corresponding to the model deﬁned by φ (denoted by IICRφ) approximates IICR0. We thus want to ﬁnd the minimal distance: ð3Þ We deﬁne bounds for each variable because we use a constrained optimization algorithm, for which all para- meters must be bounded (see section “Optimization frame- work: search algorithm and optimality criteria”). Also, since we focus on the case where there are no deme size changes, we enforce this by using B, as making si min ¼ si max ¼ 1 for all 0 ⩽i ⩽γ effectively ﬁxes all deme sizes to 1, and reduces the number of parameters to 2γ + 2. min φ2Φγ;B d IICR0; IICRφ : ð4Þ ð4Þ Regarding the distance d, a straightforward deﬁnition would be: d IICR0; IICRφ ¼ Z 1 0 IICR0ðtÞ IICRφðtÞ wðtÞdt; ð5Þ d IICR0; IICRφ ¼ Z 1 0 IICR0ðtÞ IICRφðtÞ wðtÞdt; The parameter space We ﬁrst deﬁne the parameter space, as this directly determines the family of demographic histories that we can explore and infer from. The piecewise stationarity refers to the fact that, although migration rate is constant and identical between any pair of islands, this rate may be different between consecutive time periods (components), and there is a ﬁxed number γ that represents the number of demographic events. To say that there are γ ⩾0 changes of gene ﬂow thus means that there are c = γ + 1 components or periods of constant gene ﬂow. Likewise, the deme size, which is the same for all islands, may in theory change through time in the general model presented in Rodríguez et al. (2018). In the present study we focus on models with constant population size but we present a more general model where deme sizes can change between com- ponents. In this more general case, the parameter space includes the number of islands n, the times ti for the demo- graphic events, and the values of both the migration rate Mi and the local deme size si at each new demographic period. Note that n is inferred but it does not change through time. We thus assume no extinction, no population split, and no creation of new populations. The computation of the IICR uses functions that receive the time t in units of 2N generations, and return values in units of N generations per coalescence, so these IICR functions are dimensionless in the sense that they operate in a relative frame of reference. In order to compare the IICR with PSMC inferences, we need to re-scale both the time and the IICR values by a reference deme of size N which speciﬁes how many haploid genes correspond to a local deme size of 1 as follows: sIICRðgÞ ¼ N IICRðg=2NÞ; In order to compare the IICR with PSMC inferences, we need to re-scale both the time and the IICR values by a reference deme of size N which speciﬁes how many haploid genes correspond to a local deme size of 1 as follows: where sIICR(g) refers to the scaled IICR, and IICR(t) to the unscaled (dimensionless) one. Note that we use g for generations as the variable name for sIICR to further stress the difference. The piecewise stationary n-island coalescent scenarios is the IICR. In the Supplementary Materials we provide a brief overview of how to perform its computation for any given φ ∈Φγ,B based on the work of Rodríguez et al. (2018). Optimization framework: search algorithm and optimality criteria ð2Þ The search algorithm explores the parameter space and uses an optimality criterion to select the structured scenario that best explains a given target IICR curve, either scaled or unscaled. We also assume that the underlying coalescence times for these target IICRs have cumulative distribution F0 and density f0. we deﬁne the parameter space Φγ,B as: we deﬁne the parameter space Φγ,B as: The parameter space The parameter space for the sIICR can be thought of as a simple one-dimensional addition to Φγ,B: ^ϕγ;B ¼ ^φ ¼ ðN; φÞ 2 R ϕγ;B such that 0<Nmin ⩽N ⩽Nmax : Computing and scaling the IICR Z 0 ð5Þ ð5Þ Given any demographic scenario from Φγ,B, the associated coalescent process is an instance of the NSSC of Rodríguez et al. (2018). Our main object of interest regarding these where w(t) is a weight function that should take into account the natural distribution of the information in an IICR. One where w(t) is a weight function that should take into account the natural distribution of the information in an IICR. One 900 A. Arredondo et al. An alternative option for the deﬁnition of d in (4) could be one that takes into account the ultimate visual nature of the curve ﬁtting task. Assuming that the points {τj} are log- distributed and that they will be used for visualization purposes in a horizontally log-scaled plot like Fig. S37, then the deﬁnition: reasonable solution for w is to take a quantity proportional to the density f0 of the coalescence times because it ensures that the integral in (5) is ﬁnite, and also because it assigns more weight to the temporal periods where the target IICR0 is expected to be more accurate and reliable since more coalescences are likely to have happened. We thus consider the family of weight functions: dðIICR0; IICRφÞ ¼ X I j¼1 yj IICRφðτjÞ ; ð8Þ ð8Þ wðtÞ ¼ f ω 0 ðtÞ k f ω 0 k1 ; ð6Þ wðtÞ ¼ f ω 0 ðtÞ k f ω 0 k1 ; ð6Þ where ∥⋅∥1 is the L1-norm and ω > 0 is a weight-shifting parameter, with the purpose of dampening (if ω < 1) or exaggerating (if ω > 1) the effect of the weight f0. Unless otherwise noted, we take ω = 1, which corresponds in practice to giving more weight to recent periods of the IICR in direct proportion to the density f0 in an n-island model. captures the perceived visual difference between the plots of the two curves. We distinguish distance (5) from (8) by denoting them dω and dvisual respectively. We keep both deﬁnitions because we found that the weighted family of distances generally performs better than the visual distance under certain validation tests, but also that the dvisual distance can be used to choose the optimal weight parameter in dω (see Fig. S46). Computing and scaling the IICR In practice, we need to consider that all we know about IICR0 is a stepwise discretization over a bounded interval of time, so a numerical approximation of the distance (5) is required. This includes approximating the density f0 of the underlying T2 distribution. Given a division of time into I intervals ½τj1; τjÞ for 1 ⩽j ⩽I, where τ0 = 0 and τI < 1, we can consider a discrete representation of IICR0 in the form of a collection of I values {yj} such that: Regarding the optimization problem (4) itself, we use the Differential Evolution method (Storn and Price, 1997). As a genetic meta-heuristics, this algorithm maintains and evolves (using mutation and recombination parameters) a population of solutions iteratively. As a global optimization algorithm, it features mechanisms for escaping local optima of the parameter space. In section S2.3 of the Supplementary Materials, we explore the potential effects on the inference results of tuning some of the parameters provided by this algorithms implementation. For our validations, the method runs by using multiple steps of optimization which we refer to as rounds. In addition, we stress that the method should be used multiple times on real data sets. We set a maximum number of allowed rounds, as well as a tolerance ε for the distance which controls the minimum number of rounds. IICR0ðtÞ ¼ yj; 8t 2 ½τj1; τjÞ; 1 ⩽j ⩽I: ð7Þ ð7Þ We can use this form to compute a numerical approximation for the integral in (5). For instance, a ﬁrst degree approximation would yield: d IICR0; IICRφ ¼ X I j¼1 yj IICRφðτjÞ wðτj1Þ ðτj τj1Þ: As for the values of w(τj), notice that from (1) we have the identity: As for the values of w(τj), notice that from (1) we have the identity: Validation framework We applied the SNIF inferential method to target IICRs generated under piecewise stationary n-island models of increasing complexity (i.e., number of components) and with known parameter values (N, n, ti, Mi) and then compared the inferred parameter values to those actually used (see Fig. 1). In what follows we present various ways of generating random demographic scenarios and then computing appropriate IICR curves from them for use in validation. We applied the SNIF inferential method to target IICRs generated under piecewise stationary n-island models of increasing complexity (i.e., number of components) and with known parameter values (N, n, ti, Mi) and then compared the inferred parameter values to those actually used (see Fig. 1). f 0ðtÞ ¼ exp Z t 0 dτ IICR0ðτÞ =IICR0ðtÞ; which, using the representation (7), can be discretized into: which, using the representation (7), can be discretized into: In what follows we present various ways of generating random demographic scenarios and then computing appropriate IICR curves from them for use in validation. f 0ð0Þ ¼ 1=y0; f 0ðτjÞ ¼ exp P j k¼1 τk1τk yk =yj; 1 ⩽j ⩽I: The three types of target IICRs After sorting the times ti, we can deﬁne the L sampled scenarios by constructing, for 1 ⩽j ⩽L, the tuple nj; tj 1 ¼ tj γ; Mj 0 ¼ Mj γ; sj 0 ¼ sj γ . This sampling strategy makes it very unlikely to sample exactly the same parameter values twice or to sample exactly the same Mi values in two consecutive components. However, it sometimes produces demographic scenarios in which consecutive ti and/or Mi values may be close to each other, and thus difﬁcult to distinguish. This makes it thus harder on our inferential framework compared to cases where we would choose contrasted scenarios with clearly separated events with major changes in migration rates. In other words, our inferential method was sometimes inferring parameters in the case of extremely difﬁcult scenarios as we show below. We explored three different types of target IICRs (see Fig. S3) that could be obtained given a scenario φ ∈Φγ,B. All IICRs were discretized so as to be comparable to PSMC plots (see eq. (7)). Validating SNIF on PSMC plots across the parameter space described above would be extremely time consuming as it would require simulating genomes and then running the PSMC method (or other related methods) on these genomes before applying our approach. We thus only ran the PSMC method in the case of the scenarios inferred for the human data so as to integrate the uncertainty due to the PSMC inferential process. The issue of uncertainty is cru- cial but our aim is not to test the PSMC or other inferential methods. To clarify this we explain below the different types of IICR that could be computed given a scenario φ ∈ Φγ,B (see also Fig. 1). In section “Validation” we show the results obtained using this sampling method with L = 400 scenarios. The bounds for sampling and inferring are shown in Table S1. In particular, we note that in practice we disallow deme size changes by ﬁxing the bounds of the si to 1, which consequently reduces the parameter space to just 2γ + 2 parameters. Exact IICR We can compute the IICR for any n-island model at any time value t, but to generate input data we need a discretization as in (7), so considering that we take a log-distributed sample of size I in the interval ½tmin; tmax, we end up with a suitable IICR0. Sampling the parameter space We then have that for any given ω, w(τj) can be expressed as: Given a parameter space Φγ,B (we only discuss the unscaled case here for brevity, but the same principles apply to a scaled parameter space), we sample demographic scenarios from which we compute the corresponding IICRs. We used two sampling strategies which we call continuous and dis- crete sampling. as: wðτjÞ ¼ f ω 0 ðτjÞ= X I k¼1 f ω 0 ðτkÞ: 901 Inferring number of populations and changes in connectivity under the n-island model Continuous sampling Assuming that we want to realize L independent tests, this sampling strategy consists in using uniform or log-uniform distributions for each of the 3γ + 3 random variables: inference process always allowed n to take any value between 2 and 50. The choice of the L independent simulated data sets was done using the following procedure. We ﬁrst considered the following cartesian product of dimension 2γ + 2: n Ufnmin; nmin þ 1; ¼ ; nmaxg; n Ufnmin; nmin þ 1; ¼ ; nmaxg; n Ufnmin; nmin þ 1; ¼ ; nmaxg; t1 LU10½t1 min; t1 max; ¼ ; tγ LU10½tγ min; tγ max; M0 U½M0 min; M0 max; ¼ ; Mγ U½Mγ min; Mγ max; s0 U½s0 min; s0 max; ¼ ; sγ U½sγ min; Mγ max; ð9Þ K ¼ n tγ Mγþ1 f1g: t1 LU10½t1 min; t1 max; ¼ ; tγ LU10½tγ min; tγ max; t1 LU10½t1 min; t1 max; ¼ ; tγ LU10½tγ min; tγ max; t1 LU10½t1 min; t1 max; ¼ ; tγ LU10½tγ min; tγ max; M0 U½M0 min; M0 max; ¼ ; Mγ U½Mγ min; Mγ max; M0 U½M0 min; M0 max; ¼ ; Mγ U½Mγ min; Mγ max; and then uniformly drew L tuples from K without replacement. We then sorted the sampled event times obtaining thus a set of L demographic scenarios. We drew randomly (without replacement) from the set K, rejecting scenarios with identical Mi values in two consecutive components, until we reached L accepted scenarios. s0 U½s0 min; s0 max; ¼ ; sγ U½sγ min; Mγ max; ð9Þ ð9Þ where U denotes a uniform distribution (discrete in the case of n and continuous for the rest) and LU10 denotes a log- uniform distribution of base 10. Sampling the parameter space This distribution is used for sampling the times of changes in a logarithmic space in order to take into account the natural distribution of information in an IICR. The three types of target IICRs After that, we run the inference algorithm on this target IICR curve (using wider bounds than those in B) to obtain an estimated (or inferred) demographic history ^φ0 ¼ ðN0; n0; t0 i ; M0 i Þ, which we then compare to the known ^φ in order to assess the accuracy of the infer- ence methodology (section “Results”). Fig. 1 Flowchart of the validation procedures. Starting from a parameter space ^Φγ;B we use one of two sampling methods (section “Sampling the parameter space”) to generate a demographic history ^φ deﬁned (for the scaled case) by the parameters (N, n, ti, Mi). We then compute the IICR of that demographic history using one of three methods (section “The three types of target IICRs”) to obtain the target IICR. After that, we run the inference algorithm on this target IICR curve (using wider bounds than those in B) to obtain an estimated (or inferred) demographic history ^φ0 ¼ ðN0; n0; t0 i ; M0 i Þ, which we then compare to the known ^φ in order to assess the accuracy of the infer- ence methodology (section “Results”). T-sim IICR The T-sim IICR is obtained by simulating a ﬁnite collection of T2 realizations using ms and then building an empirical IICR as in Mazet et al. (2016), using the Kaplan–Meier estimator (Kaplan and Meier, 1958), with log-distributed times. We stress that ms scales time in units of 4N generations whereas our models use a scale of 2N generations (see the example in Fig. S3), so this must be kept in mind when writing ms commands. The bounds for the ti are speciﬁed in generations, so given a generation time of 25 years, we effectively allowed for the inference of demographic events between 10 thou- sand and 10 million years ago. Regarding the number of components, we choose c ∈{2, 3, 4, 5}; i.e., between one and four demographic events in agreement with Mazet et al. (2016) who suggested that a minimum of three events were necessary to explain the two humps, and in agreement with our validation simulations which suggest that inference above ﬁve components is difﬁcult. Seq-sim IICR We simulate genomic sequences with ms and then apply the PSMC method for obtaining the IICR0 to be used by the inference method. The three types of target IICRs Since simulating genomes and performing PSMC analyses is signiﬁcantly more time consuming than the other two methods, we limited our- selves to validating the Seq-sim IICR for the human PSMC based scenarios that we obtain after performing the demo- graphic inference described in section “Application to humans”. The results of this step are shown in section S5.1 of the Supplementary Materials. Some of the analyzed PSMC curves exhibit an increase in effective size in the recent past, which could be due to a genuine population growth as noted by Mazet et al. (2016). Given that we choose to speciﬁcally rule out changes in deme sizes, we account for this fact by running every inference a second time, ignoring this period of possible recent expansion. This is accomplished using an option that allows to limit the interval where the distance function is computed. In this case, we restricted both this range and the bounds for the ti to be between 50 thousand and 10 million years ago, thus ignoring any population size change that may have happened in the last 50,000 years. Note that this option is also useful to ignore very ancient sections of the PSMC plots which may be difﬁcult to trust. The three types of target IICRs Note that even though this IICR has been discretized, its values are exact within machine precision, so it is still an artiﬁcial product com- pared to real data. Discrete sampling Here we sampled L = 100 independent scenarios from the same parameter space, but using the following set of predeﬁned values: For the validations using the exact IICR in section “Validation using exact target IICRs” we chose for the distance tolerance between a target and an inferred IICR a value of ε = 10−10 for the unscaled IICRs and an equivalent value of ε = 10−7 for the scaled IICR (since the simulated N was always 1000). It should be noted that this value of ε is quite small even for double-precision ﬂoating-point arith- metic, and thus is only a reasonable choice for validation using exact IICRs (i.e., those where the distance could theoretically be zero). n 2 n ¼ f2; 5; 10; 15; 20g; ti 2 t ¼ f1=10; 1=2; 1; 2; 5; 10; 20; 50g 8i; Mi 2 M ¼ f1=10; 1=5; 1=2; 1; 2; 5; 10; 20; 50g 8i; si 2 s ¼ f1g 8i; n 2 n ¼ f2; 5; 10; 15; 20g; ti 2 t ¼ f1=10; 1=2; 1; 2; 5; 10; 20; 50g 8i; Mi 2 M ¼ f1=10; 1=5; 1=2; 1; 2; 5; 10; 20; 50g 8i; si 2 s ¼ f1g 8i; n 2 n ¼ f2; 5; 10; 15; 20g; ti 2 t ¼ f1=10; 1=2; 1; 2; 5; 10; 20; 50g 8i; Mi 2 M ¼ f1=10; 1=5; 1=2; 1; 2; 5; 10; 20; 50g 8i; si 2 s ¼ f1g 8i; The inference process was, however, done within the continuous space. For instance, under this validation scheme (see section “Validation using T-sim IICRs”) we only simulated data with 2, 5, 10, 15, 20 islands but the 902 A. Arredondo et al. Fig. 1 Flowchart of the validation procedures. Starting from a parameter space ^Φγ;B we use one of two sampling methods (section “Sampling the parameter space”) to generate a demographic history ^φ deﬁned (for the scaled case) by the parameters (N, n, ti, Mi). We then compute the IICR of that demographic history using one of three methods (section “The three types of target IICRs”) to obtain the target IICR. Validation using exact target IICRs populations from this analysis because their corresponding inferred histories are similar to the other three (see Figs. S40 through S46). For each one of the selected scenarios we simulated nreps = 30 chromosomes of length L = 108 base pairs, using the effective size N inferred by SNIF, a per-base per-generation mutation rate of μ = 1.25 × 10−8 (see (Scally and Durbin, 2012) and references therein). We kept for consistency the scaled recombination rate of ρ = θ/ 5 as in Li and Durbin (2011), and we ran the ms command with θ = 4μLN using: A ﬁrst set of ﬁgures (Fig. 2, Figs. S6–S11 and S13–S18) represents the simulated and inferred parameter values on the horizontal and vertical axes, respectively, using the continuous sampling strategy. As explained in section “Sampling the parameter space”, the range of possible values in the inference process was always wider than the range used for the simulated values (see Table S1 for the exact values). We quantiﬁed the inference error for each parameter by computing the Normalized Root-Mean-Square Deviation (nRMSD). This value is displayed in the lower- right corner of each panel of the previously mentioned ﬁgures, and summarized for all parameters in Fig. S20. For reference, we also highlight the y = x line, indicating what would be a perfect inference, and the region corresponding to 10% of relative error around this line (50% of relative error in the case of the ti parameters). A summary of how many tests fall within this margin of error (and others) is displayed in Figs. S12 and S19. Altogether, these ﬁgures always show the data points near the y = x, hence sug- gesting that the inferred parameter was identical or very close to the simulated parameters. This is particularly obvious for all the parameters corresponding to scenarios with up to four components, where the nRMSD values stayed below 0.5 (the case of the ti parameters is excep- tional, since the exponential distribution of its range dis- proportionately affects the error measures). For instance, Fig. 2a shows the results for a model with three compo- nents, in which there is a nearly perfect match (nRMSD close to or below 0.1 for the non-t parameters) between the simulated and inferred values for the model parameters. Application to humans We applied our method to the human genomes published in the great apes study by Prado-Martinez et al. (2013). Namely, we used the PSMC ﬁles of ﬁve sampled humans identiﬁed as Dai, French, Karitiana, Sardinian, and Yoruba (see Fig. S37). For each human PSMC curve we performed demographic inference independently within the following bounds: Since real human PSMCs are unlikely to have been generated by an n-island model, the default value of ω used for simulated data may not be the most appropriate, and we thus performed inferences with ω ∈{1, 0.5, 0.2}. Decreasing values of ω give increasing weight to the most ancient part of the PSMC (see the weighted distances (6)). The resulting inferred demographic scenarios are shown in section “Application to humans”. n 2 f2; 3; ¼ ; 100g; ti 2 ½4 102; 4 105 8i; Mi 2 ½1=20; 20 8i; si ¼ 1 8i; N 2 ½102; 104: ð10Þ ti 2 ½4 102; 4 105 8i; ð10Þ Mi 2 ½1=20; 20 8i; To validate the inference process using PSMC outputs, we generated 10 Seq-sim IICRs corresponding to the inferred demographic scenarios for the French, Karitiana, and Yoruba individuals. We exclude the Dai and Sardinian si ¼ 1 8i; N 2 ½102; 104: N 2 ½102; 104: Inferring number of populations and changes in connectivity under the n-island model 903 Validation using exact target IICRs For ﬁve- and six-component scenarios the results are still nearly perfect for most of the simulated scenarios but we observe an increasing number of cases (i.e., simulated scenarios) where the parameters are poorly estimated, with the exception of n, M0 (and N for scenarios with scaled IICRs) which are almost always well estimated also in such cases (nRMSD values consistently close to or below 0.1). In particular, we can appreciate a gradual degradation of the correspondence between simulated and inferred migration values when the number of components c increases, as the nRMSD monotonically increases to over 0.5 for c = 6. These cases can be identiﬁed by the dots scattered in the different panels. They start to appear in scenarios with three components, but their number grows with the number of components. ms 2 nreps -t θ -r L -p 8 -I … where the rest of the command follows according to the inferred demography (see Fig. S3 for a reference). After that we prepared a .psmcfa ﬁle as input for PSMC, always choosing a bin size of s = 100. Then we ran the PSMC with the command: psmc -N25 -t15 -r5 -p "4+25*2+4+6" … following Li and Durbin (2011) on human data. We then scaled the resulting curve using the information in the generated .psmc ﬁle and used these PSMC curves as targets to determine whether we could indeed infer the parameters used for such complex scenarios. We also applied SNIF to genomic data simulated under the scenarios used to describe recent human evolutionary history by Gutenkunst et al. (2009) and Noskova et al. (2019). Here, we thus ask the following two questions: if human evolution were indeed closer to such splitting models, would our method infer again an n-island model with similar parameters to those inferred from the humans PSMCs? additionally, do these models generate IICR plots that are similar to the human PSMCs? The results of these validations are shown in section S5.1 of the Supplementary Materials. Results In this section we show the results of validating SNIF using target IICRs from known demographic histories; the application of the method to real human data; and the comparison of the obtained results with previously pub- lished demographic histories for humans. These poorly estimated parameters are surprising given the near perfect estimation obtained for most parameter combinations. This is particularly striking because these dots do not seem to be distributed in any clear area of the parameter space. We see at least two possible and non- exclusive interpretations for this result. One is that the The results of the validations are presented in Figs. 2–4 in the main manuscript and Figs. S6–S36 in the Supple- mentary Materials. Another set of ﬁgures (Figs. 5–8 and Figs. S40–S46) present the results of the application to human data. 904 A. Arredondo et al. Fig. 2 Scatter plots of simulated and inferred parameters. a Cor- responds to scenarios with c = 3 components, and b to scenarios with c = 6 components. The different sub-panels represent the simulated (horizontal axis) versus inferred (vertical axis) parameter values for all the parameters (or a representative selection of parameters in the case of b) of L = 400 unscaled simulated scenarios. (horizontal axis) versus inferred (vertical axis) parameter values for all the parameters (or a representative selection of parameters in the case of b) of L = 400 unscaled simulated scenarios. Fig. 2 Scatter plots of simulated and inferred parameters. a Cor- responds to scenarios with c = 3 components, and b to scenarios with c = 6 components. The different sub-panels represent the simulated (horizontal axis) versus inferred (vertical axis) parameter values for all the parameters (or a representative selection of parameters in the case of b) of L = 400 unscaled simulated scenarios. search algorithm had not yet converged when the maximum number of rounds was reached. value consistently decreases with more rounds (see Fig. S5), but the inferred parameter values may not converge to the simulated ones because with more components there is a higher probability that two consecutive simulated Mi values are very close, thus making the corresponding time of the event challenging to infer. The maximum number of rounds was set to 500 in all simulations because we had found that less than 50 rounds were more than enough in the ﬁrst tests carried out with one or two components. Results 3 Connectivity graphs of 100 independently inferred histories obtained by sampling for each scenario from the values indicated by the dotted lines. a Scenarios with c = 3 components. b Scenarios with c = 4 components. The right sub- panels show a side histogram with only the inferred migration rates for those components with a speciﬁc simulated migration rate (10 for (a) and 1 for (b)). s of d pling e otted 3 ith c sub- am ation with ion b)). Fig. 3 Connectivity graphs of 100 independently inferred histories obtained by sampling for each scenario from the values indicated by the dotted lines. a Scenarios with c = 3 components. b Scenarios with c = 4 components. The right sub- panels show a side histogram with only the inferred migration rates for those components with a speciﬁc simulated migration rate (10 for (a) and 1 for (b)). Fig. 3 Connectivity graphs of 100 independently inferred histories obtained by sampling for each scenario from the values indicated by the dotted lines. a Scenarios with c = 3 components. b Scenarios with c = 4 components. The right sub- panels show a side histogram with only the inferred migration rates for those components with a speciﬁc simulated migration rate (10 for (a) and 1 for (b)). here that the incorrectly inferred migration values are clustered around other simulated values, indicating a mis- match in a particular component assignment which does not affect the rest of the inferred demographic history (we present a quantiﬁcation of this effect for a particular case in Fig. S21). quantifying it for the case of c = 5 in Fig. S21. One way to mitigate the effect of this misassignment issue in the ana- lysis of the results is to visualize the simulated and inferred scenarios using what we call a connectivity graph. This connectivity graph represents the times at which migration changes against the values of the migration rates. Such connectivity graphs are featured in the next section. For example, consider the right sub-panel of (a). We see that most repetitions correctly inferred a value close to M = 10 for the components with that simulated migration rate. Results The search algorithm might however need more than 500 rounds to reach the optimal solution for scenarios of increasing complexity. We thus asked whether the maximum the number of rounds had been reached in the scenarios analysed and whether the proportion of scenarios with 500 rounds increased with the number of components. We found indeed that the proportion of simulations for which that maximum was reached increased with the number of components. For instance, all ﬁve- and six- component scenarios stopped their parameter search at 500 rounds, hence suggesting that at least some had not yet reached an optimum solution. For the cases with one- and two-component scenarios, all 800 independent simulations reached convergence in less than 150 rounds (see Fig. S4). Again, the choice of the tolerance ε plays a role in these results, and selecting larger tolerances will tend to produce earlier convergence in general, but not necessarily better results. The second possible reason for the poorly estimated parameters in Fig. 2 may be related to the fact that some simulated components may have a short duration that do not leave a signiﬁcant mark on the IICR curve, thus leading them to be “skipped”. We refer to this issue as component misidentiﬁcation or misassignment, which could lead to a particular estimated parameter to be plotted in the wrong panel. For instance, the method may miss the ﬁrst change in migration rate at t1 and identify the second change in migration at t2. In such a case the method will assign the inferred t2 value to the set of inferred t1 values and plot it in the t1 panel. This wrongly assigned t2 value will thus appear away from the diagonal in the t1 panel even if it was well- estimated. Such misassignment cases for one parameter will also have consequences for the Mi plots, and thus will generate several misassignments across panels. They are also expected to increase in frequency as the number of components increases and as the ti values become closer to each other. This phenomenon can be observed clearly in the right panels of Fig. 3. We also present an attempt at As a test we randomly identiﬁed a couple of scenarios with six components that had bad estimates and re-ran the algorithm with 5000 rounds. We found that the distance Inferring number of populations and changes in connectivity under the n-island model 905 Fig. Results However, there were cases where a given component i was simulated with a migration rate of Mi = 10, but it was missed entirely (maybe because it did not generate a very different IICR or because it had a short duration), and thus the inferred migration value for component i ultimately reﬂected either Mi−1 or Mi+1. In panel (b) we can observe the same effect with higher intensity because with more components it is more likely for them to be misassigned or misidentiﬁed during inference. See Fig. S21 for a quantiﬁ- cation of this effect on scenarios of c = 5 components. Fig. 3 Connectivity graphs of 100 independently inferred histories obtained by sampling for each scenario from the values indicated by the dotted lines. a Scenarios with c = 3 components. b Scenarios with c = 4 components. The right sub- panels show a side histogram with only the inferred migration rates for those components with a speciﬁc simulated migration rate (10 for (a) and 1 for (b)). Validation using T-sim IICRs The simulated n and N values are represented by black circles whereas the inferred values for the corresponding parameters are represented by red and green full circles and by gray histogram bars. generations due to the stochasticity of the IICR plot. This appears to generate some variance in the estimates of N and n but the connectivity graph shows the same trend (increasing connectivity) as in the simulations. In the case of panel (d) we can see that the method had some difﬁ- culty in estimating several of the changes in Mi values. This is not surprising as some of the randomly simulated changes do not seem to lead to major changes in the IICR curves. This generates again some variance in the N and n estimates. We also observe a signiﬁcant variance in the connectivity graph even if several runs overlap nearly perfectly with the simulated connectivity graph. to estimate properly, at least in the recent times. These graphs however summarize extremely different scenarios, including scenarios in which consecutive Mi values may be similar. We thus stress that the quality of the inference is dependent on the timing of the changes in migration rates and on the size of the change in Mi values. Figure 4 shows the results for four different scenarios. In each of the four panels, we represented the inferred and target IICR plots, connectivity graphs, N (the size of each the islands) and n (the number of islands) for the corre- sponding model. Panels (a) and (b) correspond to three- and four-component scenarios, whereas panels (c) and (d) show the results for two ﬁve-component scenarios, one for which we obtained very good estimates and one for which the estimates were poorer. In panels (b) and (c) the inferred and simulated Mi and ti values are on top of each other as can be seen in the connectivity graphs. Similarly, N and n are also well estimated. Here, the IICR plots also overlap, although this does not always guarantee perfect parameter estimation, as is the case in panels (a) and (d). Validation using T-sim IICRs The connectivity graphs and IICR plots obtained from simulated T2 values show that again the scenarios are gen- erally very well reconstructed (Fig. 3 and Figs. S22–S36). y y ( g g ) In Fig. 3 the connectivity graphs obtained for all the scenarios simulated with three and four components show that the inferred times at which migration rates changed (green vertical lines) and the inferred migration rates (green horizontal lines) are generally overlapping close to the simulated values (dotted vertical and horizontal gray lines). In the right panels of this ﬁgure, we show a subset of the inferred migration histogram (in red). Namely, we show the distributions of the migration values that were inferred for components with a simulated migration value of Mi = 10 for panel (a) and Mi = 1 for panel (b). This allows us to better appreciate the variance of the inferred migration values in the context of the simulated ones, as well as the component misassignment effect mentioned earlier. Indeed, we note These connectivity graphs (and the one obtained for ﬁve components shown in Fig. S34) also show that there are regions of the parameter space where the green lines are more widely distributed. For instance, in the recent past of Fig. 3b (ti < 10−3 generations) when the simulated Mi value was 0.1 or 0.2 the inferred values seem to vary between 0.05 and 0.3, suggesting that the method identiﬁes periods with low migration rates but that the exact value is difﬁcult 906 A. Arredondo et al. Fig. 4 Simulated and inferred IICR plots, connectivity graphs, N and n. The four panels correspond to four different scenarios. a A c = 3 components scenario. b A c = 4 components scenario. c A c = 5 components scenario. d A c = 5 components scenario. The left part of each panel represents the target and inferred IICRs (top), and the connectivity graphs (down). The right half of each panel shows the simulated and inferred values for n (top) and N (down). In each IICR graph, the ragged blue line represents the target IICR whereas the red lines represent 10 independently inferred IICRs. The vertical blue and red lines are located at the simulated and inferred values of the event times ti, respectively. In the connectivity graphs, the black and green lines represent the simulated and inferred connectivity scenarios, respectively. Validation using T-sim IICRs Interestingly, in panel (a) the ﬁrst change in migration rate (at t1 = 200 generations) is estimated at around 900 Altogether the validation tests and ﬁgures above suggest that our framework is able to infer changes in connectivity under the n-island model, and that some scenarios can be extremely well inferred whereas others may be more difﬁ- cult depending on their effect on the IICR plots. We also observe that for real data it may be helpful to run the ana- lyses for a varying number of rounds, since too few rounds may negatively affect the quality of the ﬁt. Also, once a scenario has been inferred, it is advisable, as an additional validation step, to simulate data under the inferred scenario Inferring number of populations and changes in connectivity under the n-island model 907 Fig. 5 Results of performing demographic inference on the French PSMC curve. a Shows the IICR plot inferred for c = 5 components and a weight parameter of ω = 0.2. The vertical lines represent the inferred times of the demographic events. b Shows the connectivity Fig. 5 Results of performing demographic inference on the French PSMC curve. a Shows the IICR plot inferred for c = 5 components and a weight parameter of ω = 0.2. The vertical lines represent the inferred times of the demographic events. b Shows the connectivity graph for the same inferred scenario. As a reference point, the con- nectivity graph of the scenario proposed in Rodríguez et al. (2018) is also shown. The vertical axis in b represent migration rates (M). graph for the same inferred scenario. As a reference point, the con- nectivity graph of the scenario proposed in Rodríguez et al. (2018) is also shown. The vertical axis in b represent migration rates (M). graph for the same inferred scenario. As a reference point, the con- nectivity graph of the scenario proposed in Rodríguez et al. (2018) is also shown. The vertical axis in b represent migration rates (M). Fig. 5 Results of performing demographic inference on the French PSMC curve. a Shows the IICR plot inferred for c = 5 components and a weight parameter of ω = 0.2. The vertical lines represent the inferred times of the demographic events. b Shows the connectivity graph for the same inferred scenario. As a reference point, the con- nectivity graph of the scenario proposed in Rodríguez et al. Validation using T-sim IICRs This is what we do with the real human data in the next section. Validation using T-sim IICRs a Shows the inferred number of islands n and b the inferred reference sizes N for each human population and each used combination of the weight parameter ω and number of components c (only 4 and 5 are shown here). The bars with the darker color, marked ’restricted’ in the legend, correspond to inferences realized with the option of ignoring recent population expansion. and use SNIF to re-infer the parameters. This is what we do with the real human data in the next section. The full set of results related to the inference of human demographies can be found in Figs. S40–S46, which were placed in the Supplementary Materials for the sake of brevity. The most striking feature of this extended set of plots is the sensitivity of the ﬁt to the value of the weight- shifting parameter ω. Smaller values allow the optimizer to distribute more of the demographic events towards the ancient past and thus allows this region to be better ﬁtted by the inferred IICR. This functionality (together with being able to ignore certain parts of the plots for the computation of the distance function) can be used to make explicit the knowledge (or beliefs) of the researcher regarding the accuracy of the PSMC curve. We notice that the Yoruba individual cannot be well ﬁtted in the recent past for any value of ω, even outside of the designated period of recent population expansion. The full set of results related to the inference of human demographies can be found in Figs. S40–S46, which were placed in the Supplementary Materials for the sake of brevity. The most striking feature of this extended set of plots is the sensitivity of the ﬁt to the value of the weight- shifting parameter ω. Smaller values allow the optimizer to distribute more of the demographic events towards the ancient past and thus allows this region to be better ﬁtted by the inferred IICR. This functionality (together with being able to ignore certain parts of the plots for the computation of the distance function) can be used to make explicit the knowledge (or beliefs) of the researcher regarding the accuracy of the PSMC curve. We notice that the Yoruba individual cannot be well ﬁtted in the recent past for any value of ω, even outside of the designated period of recent population expansion. and use SNIF to re-infer the parameters. Validation using T-sim IICRs The most striking feature of this extended set of plots is the sensitivity of the ﬁt to the value of the weight- shifting parameter ω. Smaller values allow the optimizer to distribute more of the demographic events towards the ancient past and thus allows this region to be better ﬁtted by the inferred IICR. This functionality (together with being able to ignore certain parts of the plots for the computation of the distance function) can be used to make explicit the knowledge (or beliefs) of the researcher regarding the accuracy of the PSMC curve. We notice that the Yoruba individual cannot be well ﬁtted in the recent past for any value of ω, even outside of the designated period of recent population expansion. Figure 6 shows in panel (a) the number of demes n and in panel (b) the reference size N that were inferred from each of the ﬁve ﬁtted human PSMCs. Of note here is that all individuals except the Yoruba show a consistent value for these inferred parameters across both number of Fig. 6 Results of performing demographic inference on the human PSMC curves. a Shows the inferred number of islands n and b the inferred reference sizes N for each human population and each used combination of the weight parameter ω and number of components c (only 4 and 5 are shown here). The bars with the darker color, marked ’restricted’ in the legend, correspond to inferences realized with the option of ignoring recent population expansion. and use SNIF to re-infer the parameters. This is what we do with the real human data in the next section. The full set of results related to the inference of human demographies can be found in Figs. S40–S46, which were l d i th S l t M t i l f th k f Fig. 6 Results of performing demographic inference on the human PSMC curves. a Shows the inferred number of islands n and b the inferred reference sizes N for each human population and each used combination of the weight parameter ω and number of components c (only 4 and 5 are shown here). The bars with the darker color, marked ’restricted’ in the legend, correspond to inferences realized with the option of ignoring recent population expansion. Fig. 6 Results of performing demographic inference on the human PSMC curves. Fig. 6 Results of performing demographic inference on the human PSMC curves. a Shows the inferred number of islands n and b the inferred reference sizes N for each human population and each used combination of the weight parameter ω and number of components c (only 4 and 5 are shown here). The bars with the darker color, marked ’restricted’ in the legend, correspond to inferences realized with the option of ignoring recent population expansion. Validation using T-sim IICRs (2018) is also shown. The vertical axis in b represent migration rates (M). e do the the μ = her d ω the the vely Mya t al. μ = The full set of results related to the inference of human demographies can be found in Figs. S40–S46, which were placed in the Supplementary Materials for the sake of brevity. The most striking feature of this extended set of plots is the sensitivity of the ﬁt to the value of the weight- shifting parameter ω. Smaller values allow the optimizer to distribute more of the demographic events towards the ancient past and thus allows this region to be better ﬁtted by the inferred IICR. This functionality (together with being able to ignore certain parts of the plots for the computation of the distance function) can be used to make explicit the knowledge (or beliefs) of the researcher regarding the accuracy of the PSMC curve. We notice that the Yoruba individual cannot be well ﬁtted in the recent past for any value of ω, even outside of the designated period of recent population expansion. Figure 6 shows in panel (a) the number of demes n and in and use SNIF to re-infer the parameters. This is what we do with the real human data in the next section. Application to humans Figures 5 and 6 show the results of using SNIF on the human data. In Fig. 5, panel (a) shows the PSMC curve of the French individual (scaled with a mutation rate of μ = 1.25 × 10−8 and a generation time of 25 years) together with the best ﬁtting IICR for the model with c = 5 and ω = 0.2. Panel (b) shows the connectivity graphs of the same inferred demographic scenario. We note that the connectivity pattern consisting of a period of relatively high connectivity between roughly 500 kya and 2 Mya agrees with previous results published in Rodríguez et al. (2018). Note that this study used a mutation rate of μ = 2.5 × 10−8 and not 1.25 × 10−8 as we do here and as originally stated. The absolute timing of events and deme sizes are thus different (see corrections in Rodríguez et al., 2021). The full set of results related to the inference of human demographies can be found in Figs. S40–S46, which were placed in the Supplementary Materials for the sake of brevity. Application to humans For reference purposes, we also show the real PSMC curve of the French individual. The gray vertical lines indicate the inferred event times in the C3PO model, and the colored vertical lines the inferred event times by SNIF. b Connectivity graph of the inferred scenario. For reference, we show the inferred event times in the C3PO model as gray vertical lines. Fig. 8 Application of our inference method to a generally accepted tree-like human demographic scenario with three modern populations. a Inferred number of islands for each modern population. b Inferred local size of each island. Shown here are the scenarios with 4 and 5 components c, and all three values of the weight-shifting parameter ω. The bars with the darker color, marked ‘restricted' in the legend, correspond to inferences realized with the option of ignoring recent population expansion. o er ze e e d' Fig. 8 Application of our inference method to a generally accepted tree-like human demographic scenario with three modern populations. a Inferred number of islands for each modern population. b Inferred local size of each island. Shown here are the scenarios with 4 and 5 components c, and all three values of the weight-shifting parameter ω. The bars with the darker color, marked ‘restricted' in the legend, correspond to inferences realized with the option of ignoring recent population expansion. Fig. 8 Application of our inference method to a generally accepted tree-like human demographic scenario with three modern a smaller one ancestral to the modern Eurasian population, whatever this terminology may mean. This ancestral lineage split about 22 Kya into the precursors of the European and Asian populations, which at this point began an exponential increase in size that continued to present day. During this period, all three lineages continued to exchange gene ﬂow asymmetrically. The times for these resize and splitting events are represented as dotted vertical gray lines in Fig. 7. a smaller one ancestral to the modern Eurasian population, whatever this terminology may mean. This ancestral lineage split about 22 Kya into the precursors of the European and Asian populations, which at this point began an exponential increase in size that continued to present day. During this period, all three lineages continued to exchange gene ﬂow asymmetrically. The times for these resize and splitting events are represented as dotted vertical gray lines in Fig. 7. components and value of ω. Application to humans Figures 5 and 6 show the results of using SNIF on the human data. In Fig. 5, panel (a) shows the PSMC curve of the French individual (scaled with a mutation rate of μ = 1.25 × 10−8 and a generation time of 25 years) together with the best ﬁtting IICR for the model with c = 5 and ω = 0.2. Panel (b) shows the connectivity graphs of the same inferred demographic scenario. We note that the connectivity pattern consisting of a period of relatively high connectivity between roughly 500 kya and 2 Mya agrees with previous results published in Rodríguez et al. (2018). Note that this study used a mutation rate of μ = 2.5 × 10−8 and not 1.25 × 10−8 as we do here and as originally stated. The absolute timing of events and deme sizes are thus different (see corrections in Rodríguez et al., 2021). Figure 6 shows in panel (a) the number of demes n and in panel (b) the reference size N that were inferred from each of the ﬁve ﬁtted human PSMCs. Of note here is that all individuals except the Yoruba show a consistent value for these inferred parameters across both number of 908 A. Arredondo et al. Fig. 7 Application of our inference method to a tree-like human demographic scenario with three modern populations. a IICR plots showing the resulting IICR curve of the European population under this model and the inferred IICR curve obtained with our method (where the recent period of human expansion was ignored) for c = 5 components and a weight parameter of ω = 0.25. For reference purposes, we also show the real PSMC curve of the French individual. The gray vertical lines indicate the inferred event times in the C3PO model, and the colored vertical lines the inferred event times by SNIF. b Connectivity graph of the inferred scenario. For reference, we show the inferred event times in the C3PO model as gray vertical lines. Fig. 7 Application of our inference method to a tree-like human demographic scenario with three modern populations. a IICR plots showing the resulting IICR curve of the European population under this model and the inferred IICR curve obtained with our method (where the recent period of human expansion was ignored) for c = 5 components and a weight parameter of ω = 0.25. Fig. 8 Application of our inference method to a generally accepted tree-like human demographic scenario with three modern populations. a Inferred number of islands for each modern population. b Inferred local size of each island. Shown here are the scenarios with 4 and 5 components c, and all three values of the weight-shifting parameter ω. The bars with the darker color, marked ‘restricted' in the legend, correspond to inferences realized with the option of ignoring recent population expansion. Application to humans The larger variance of the estimated values for the Yoruba individual suggests that a symmetrical island model may not be enough to explain the patterns of diversity in all ﬁve sampled human IICRs. Figures 7 and 8, and S38 show the results of applying our method to the IICR curves associated with the demographic model for human expansion published by Noskova et al. (2019), which we will refer to as the Classical 3-Populations model—C3PO for short. The C3PO model is a tree-like model with three modern populations that exchange gene ﬂow asymmetrically. It is based on the model of Gutenkunst et al. (2009) and has the same structure but with a higher likelihood and thus can be seen as an improved model with a better ﬁt to the data. The model stipulates the existence of an ancestral population that experienced an increase in size around 275 thousand years ago (Kya), and then a splitting event at about 150 Kya. This split resulted in two popula- tions that exchanged gene ﬂow asymmetrically: a large one that eventually became the modern African population, and It is clear that the nature of this model does not lend itself to be exactly modeled by a symmetrical n-island model, but the piecewise stationarity of our family of models should still be able to pick up the main demographic events. For example, from an n-island perspective, a merger or joining of two populations (going backwards in time) may be represented by an increase in gene ﬂow, although this effect may be confounded by the actual changes in both the sizes of the populations and migration rates taking place during these events. Also of note is the fact that the ﬁrst merger event is not visible to our method because it marks the start Inferring number of populations and changes in connectivity under the n-island model 909 of the recent population expansion and is thus excluded from the distance computation. stationary scenarios (c = 1), and increases linearly with the number of components. For the M parameters it reaches a value of about 0.5 at six components, and we see that the ﬁrst and last components are better estimated than the middle ones. It is likely that the component misidentiﬁca- tion phenomenon is contributing to this effect. Application to humans The number of islands n and the reference effective size N are con- sistently well estimated, reaching an nRMSD of about 0.1 in the worst cases. The ti parameters exhibit the worst nRMSD values, varying between 1 and 2 in the worst cases. Although in these cases, the fact that time is log-spaced and spans several orders of magnitude causes outliers to have a disproportionate contribution to this statistic. As can be seen in panel (a) of Fig. 7, these IICRs do not exhibit any major features past the 300 Kya mark, so they do not agree with the human PSMCs of Fig. S37 (of which the representative ones are again shown in Fig. 7 in dashed trace for reference), and they also do not generate sig- niﬁcant events in the inferred demographic histories. Par- ticularly, varying the value of the weight-shifting parameter ω did not make a great effect in this set of inferences (which is in contrast with the results shown in Fig. 5). This inferred demographic history can be roughly summarized from panel (b) as having a period of relative high gene ﬂow followed by a sharp decrease near the 300 Kya mark, which can be very clearly attributed to the size increase of the ancestral population in the C3PO model. Human evolution This is expected because of how the two models have fundamen- tally incompatible structures, not only regarding the island versus tree aspect, but also due to the size changes in the C3PO model that affect the IICR potentially as much as gene ﬂow does. However, we do identify the approximate timings of the two visible demographic events when using c = 5 components and the more recent-weighted value of ω These ﬁndings regarding changes in connectivity and number of islands are in agreement with the results of Rodríguez et al. (2018), in which a hand-ﬁtting approach of the IICRs was used to arrive at an estimate of 10 islands with a similar value of N and a comparable period featuring a signiﬁcant increased of gene ﬂow between 600 Kya and 2 Mya. Note that the timing in years and the deme sizes in Rodríguez et al. (2018) differ due to the change in mutation rate (Rodríguez et al., 2021). We also compared our results with the tree model for human evolution published by Noskova et al. (2019) (the C3PO model), which is a revision of the model from Gutenkunst et al. (2009) and represents a simpliﬁed model of human evolution (Jouganous et al., 2017, Kamm et al., 2019). The C3PO model proposes an ancestral human population that experiences two splits: an old one that resulted in the current African “population” and another more recent one that resulted in the current European and Asian “populations”. The parameters of this model include the times of these events, the population size history of these populations and their ancestral branches, and the migration rates between them. The summary statistic tar- geted by these methods is the AFS, and we see that a ﬁtting AFS does not guarantee a ﬁtting IICR and vice versa (Beichman et al., 2017, Chikhi et al., 2018). Indeed, the Human evolution An application of our method to ﬁve publicly available human PSMCs suggests that the backwards long term his- tory of the sampled individuals, when accounting for pos- sible recent expansions and the noise introduced by the PSMC method, can be accurately modeled in the framework of a symmetrical island model of ~10–12 demes with varying levels of connectivity through time. Only one of the ﬁve samples (Yoruba) displayed less consistent evidence of this ﬁnding, which may indicate that more complex models (possibly including asymmetric gene ﬂow, spatial modeling of the environment, or changes in deme sizes) could be needed to explain the full complexity of the data. The inferred number of demes and their relatives sizes for each population can be observed in Fig. 8. The numbers for the African population is in sharp contrast with the other two populations. We can also observe that for the three populations there is more variance (compared to the results from Fig. 6) in the inferred values of n and N across the different values of c and ω. This may indicate a weaker link to an underlying n-island model. In general, there is little agreement between the demo- graphic histories inferred by our method from the PSMC data and the simulated IICRs from the C3PO model. This is expected because of how the two models have fundamen- tally incompatible structures, not only regarding the island versus tree aspect, but also due to the size changes in the C3PO model that affect the IICR potentially as much as gene ﬂow does. However, we do identify the approximate timings of the two visible demographic events when using c = 5 components and the more recent-weighted value of ω = 1. These results also serve as additional validation that our method will not return the same parameter values regardless of the source of the data. They also suggest that the C3PO model is unlikely to be a good model to under- stand questions about ancient human structure and evolution. In general, there is little agreement between the demo- graphic histories inferred by our method from the PSMC data and the simulated IICRs from the C3PO model. Conclusion One novel aspect of our approach is that the number of demes gets inferred as one of the model parameters, and it is in fact the best estimated parameter, which is in agreement with Mazet et al. (2015) that used information from the distribution of T2 values and a likelihood approach. These authors how- ever, only analysed stationary models. Here we found that other parameters were also well estimated when the number of components was low, but we also observed that the estimated value of n scaled well with increasing model complexity. A similar consistency can be observed with the deme size parameter N (see Fig. S20). We give up some ﬂexibility in the model by keeping the number of demes constant throughout the history of the population, so the timed demographic events cannot represent splits or joining of populations even though such events are likely to have taken place in the history of species. Additionally, in the n-island model we do not account for possible asymmetrical gene ﬂow or different deme sizes, even when the theoretical framework does allow for such representations. However, it is a more challenging problem to validate due to the fact that during any given component, changing both the migration rate and the deme size have confounding effects on the IICR curve which can be hard to separate. This requires a dedicated study with a different methodology which we will explore in a future work. In summary, we have presented here an inference method for automatically estimating demographic parameters under a piecewise stationary symmetrical island model that uses the IICR as its summary statistic. The underlying metho- dology consists in quantifying the discrepancy between a target IICR and many simulated IICR curves for a large number of candidate scenarios, and using this metric to drive a global optimization process. With a large number of validations we have shown that the method works accu- rately and consistently for a diverse range of parameter values, and we additionally showed an application to human data that agrees with and improves upon previously pub- lished results using similar approaches. Discussion Likewise, when we use the C3PO model to generate IICR curves, and infer the corresponding demographic history using SNIF, we ﬁnd results that do not resemble those obtained from the human IICRs, and are less consistent across different runs than when inferring directly from the human IICRs. g y These ﬁndings suggest that tree models ﬁtted with the AFS like those considered here do not offer a deﬁnitive answer to the question of human evolution and other families of models should be explored (Goldstein and Chi- khi, 2002, Scerri et al., 2019, 2018). It remains to be seen however how well models inferred with our method ﬁt the real AFS of their respective human populations. A general treatment of this question is beyond the work presented here. However, in section S5.2 of the Supplementary Materials we compare the AFS of a sample of 216 humans from the Yoruba population (Lapierre et al., 2017) to the one inferred by the GADMA method from Noskova et al. (2019) and the one corresponding to three variations of the inferred demo- graphic model by our method (see Fig. S47). These simu- lations suggest that existing AFSs could be easily ﬁtted with a structured model similar to those inferred by SNIF, but in which we would allow for a recent population size change. Conclusion We believe that despite its current scope, our method can be of great value during the initial exploration of the parameter space for simple models, and thus can also pro- vide a starting point for manually ﬁtting the IICR with models that could express spatial structure and varying N (Rodríguez et al., 2018). Discussion Our validations show that the inference framework pre- sented here is able to accurately infer structure parameters (number of islands and their sizes) within a symmetrical island model given an IICR estimate like the PSMC. It is also able to date up to ﬁve events of changes in migration rate (i.e., six components) with good precision and con- sistency, as long as the underlying model is compatible with a symmetrical island model. The nRMSD (Fig. S20) of the simulated vs. inferred scenario parameters is zero for 910 A. Arredondo et al. IICRs may come in the form of more than one IICR sam- pled from an asymmetrical demographic model (for which the initial sampling deme does result in different curves (Chikhi et al., 2018), as opposed to the n-island model where demes are by deﬁnition indistinguishable). They may also be in the form of multiple IICRk curves where k is the number of sampled haploid genomes. Indeed, the IICR of Mazet et al. (2016) was deﬁned for k = 2, and this is the IICR that we have been studying in our previous works. However, the concept can be extended to more haploid genomes in the same way that the MSMC method (Schiffels and Durbin, 2013) is an extension of the PSMC to multiple genomes, which takes into consideration the distribution of the coalescent time Tk. The precise concept of the IICRk is currently being developed in a separate study. These approaches may prove beneﬁcial in choosing between structured and non-structured models. Indeed, Grusea et al. (2018) shows that using more than one IICR curve can help discriminate between structured and nonstructured scenarios in the n-island model. Finally, the incorporation of larger samples not only enables exploring more complex scenar- ios, but it also allows using other summary statistics to complement the IICR, most notably among them the AFS, which is widely used for the purposes of demographic inference. IICRs of the populations from the C3PO model do not resemble those of the real humans. Likewise, when we use the C3PO model to generate IICR curves, and infer the corresponding demographic history using SNIF, we ﬁnd results that do not resemble those obtained from the human IICRs, and are less consistent across different runs than when inferring directly from the human IICRs. IICRs of the populations from the C3PO model do not resemble those of the real humans. Data availability The implementation of SNIF its documentation, and the data and scripts required to reproduce our results can be found in github.com/arredondos/snif. Another potential direction is to use multiple IICR curves simultaneously during the inference process. These multiple Inferring number of populations and changes in connectivity under the n-island model 911 Acknowledgements We would like to thank Simona Grusea, Josué Corujo, Pierre Lacoste and Rémi Tournebize, for their input and suggestions over many lengthy and productive discussions. We also thank the three reviewers for their positive and constructive comments that allowed us to signiﬁcantly improve the manuscript. Armando Arredondo was funded by the Université Fédérale Toulouse Midi Pyrénées (UFTMiP) and the Région Occitanie (formerly Midi-Pyr- énées) with PhD grant No. 31I2017M248. Lounès Chikhi was funded by Fundação para a Ciência e Tecnologia (ref. PTDC-BIA-EVL/ 30815/2017). Olivier Mazet and Lounès Chikhi were funded by the 2015–2016 BiodivERsA COFUND call for research proposals, with the national funders ANR (ANR-16-EBI3-0014) and the Fundação para a Ciência e Tecnologia ref. Biodiversa/0003/2015 and PT-DLR (01LC1617A). This work was also supported by the LABEX entitled TULIP (ANR-10-LABX-41 and ANR-11-IDEX-0002-02) as well as the LIA BEEG-B (Laboratoire International Associé-Bioinformatics, Ecology, Evolution, Genomics and Behaviour). We acknowledge an Investissement d’Avenir grant of the Agence Nationale de la Recherche (CEBA: ANR-10-LABX-25-01). We are grateful to the GenOuest bioinformatics platform for providing computing and sto- rage resources. Chikhi L, Sousa VC, Luisi P, Goossens B, Beaumont MA (2010) The confounding effects of population structure, genetic diversity and the sampling scheme on the detection and quantiﬁcation of population size changes. Genetics 186(3):983–995 Excofﬁer L, Dupanloup I, Huerta-Sánchez E, Sousa VC, Foll M (2013) Robust demographic inference from genomic and snp data. PLoS Genet 9(10):e1003905 Fenderson LE, Kovach AI, Llamas B (2020) Spatiotemporal landscape genetics: investigating ecology and evolution through space and time. Mol Ecol 29(2):218–246 Goldstein DB, Chikhi L (2002) Human migrations and population structure: what we know and why it matters. Ann Rev Genom Human Genet 3(1):129–152 Goossens B, Chikhi L, Ancrenaz M, Lackman-Ancrenaz I, Andau P, Bruford MW et al. (2006) Genetic signature of anthropogenic population collapse in orang-utans. PLoS Biol4(2):285 Grusea S, Rodríguez W, Pinchon D, Chikhi L, Boitard S, Mazet O (2018) Coalescence times for three genes provide sufﬁcient information to distinguish population structure from population size changes. Data availability J Math Biol 78(1–2):189–224 Gutenkunst RN, Hernandez RD, Williamson SH, Bustamante CD (2009) Inferring the joint demographic history of multiple populations from multidimensional SNP frequency data. PLoS Genet 5(10):e1000695 Conﬂict of interest The authors declare no competing interests. Conﬂict of interest The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Hecht LB, Thompson PC, Rosenthal BM (2020) Assessing the evo- lutionary persistence of ecological relationships: a review and preview. Infect Geneti Evol. 84:104441 Herbots HMJD (1994) Stochastic models in population genetics: genealogy and genetic differentiation in structured populations. PhD thesis University of London 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/. Hey J, Machado CA (2003) The study of structured populations-new hope for a difﬁcult and divided science. Nat Rev Genet 4(7):535 Hudson RR (2002) Generating samples under a Wright–Fisher neutral model of genetic variation. Bioinformatics 18(2):337-338 Johri P, Riall K, Jensen JD (2020) The impact of purifying and background selection on the inference of population history: problems and prospects.Molecular Biology and Evolution, msab050, https://doi.org/10.1093/molbev/msab050 Jouganous J, Long W, Ragsdale AP, Gravel S (2017) Inferring the joint demographic history of multiple populations: beyond the diffusion approximation. Genetics 206(3):1549–1567 Kamm J, Terhorst J, Durbin R, Song YS (2019) Efﬁciently inferring the demographic history of many populations with allele count data. J Am Stat Assoc 115(531):1472-1487 Compliance with ethical standards Hecht LB, Thompson PC, Rosenthal BM (2018) Comparative demo- graphy elucidates the longevity of parasitic and symbiotic rela- tionships. Proc R Soc B 285(1888):20181032 References Nat Rev Genet 13 (10):745–753 Noskova E, Ulyantsev V, Koepﬂi K-P, O’Brien SJ, Dobrynin P (2019) GADMA: Genetic algorithm for inferring demographic history of multiple populations from allele frequency spectrum data. Giga- Science 9(3):giaa005 Scerri EM, Chikhi L, Thomas MG (2019) Beyond multiregional and simple out-of-Africa models of human evolution. Nat Ecol Evol 3 (10):1370–1372 Scerri EML, Thomas MG, Manica A, Gunz P, Stock JT, Stringer C, Grove M, Groucutt HS, Timmermann A, Rightmire GP, d’Errico F, Tryon CA, Drake NA, Brooks AS, Dennell RW, Durbin R, Henn BM, Lee-Thorp J, deMenocal P, Petraglia MD, Thompson JC, Scally A, Chikhi L (2018) Did our species evolve in sub- divided populations across Africa and why does it matter? Trends Ecol Evol 33(8):582-594 Poelstra J, Salmona J, Tiley GP, Schußler D, Blanco MB, Andriam- beloson JB, Manzi S, Campbell CR, Bouchez O, Etter PD, et al. (2021) Cryptic patterns of speciation in cryptic primates: micro- endemic mouse lemurs and the multispecies coalescent. Syst Biol 70(2):203-218 Pouyet F, Aeschbacher S, Thiéry A, Excofﬁer L (2018) Background selection and biased gene conversion affect more than 95% of the human genome and bias demographic inferences. Elife 7: e36317 Schiffels S, Durbin R (2013) Inferring human population size and separation history from multiple genome sequences. Nat Genet 8 (46):919–925 Prado-Martinez J, Sudmant PH, Kidd JM, Li H, Kelley JL, Lorente- Galdos B, Veeramah KR, Woerner AE, O’Connor TD, Santpere G et al. (2013) Great ape genetic diversity and population history. Nature 499(7459):471–475 Steinrücken M, Kamm J, Spence JP, Song YS (2019) Inference of complex population histories using whole-genome sequences from multiple populations. Proc Natl Acad Sci116(14):1–6 Quéméré E, Amelot X, Pierson J, Crouau-Roy B, Chikhi L (2012) Genetic data suggest a natural prehuman origin of open habitats in northern Madagascar and question the deforesta- tion narrative in this region. Proc Natl Acad Sci 109(32): 13028–13033 Storn R, Price K (1997) Differential evolution-a simple and efﬁcient heuristic for global optimization over continuous spaces. J Glob Optim 11(4):341–359 Tavaré S (2004) Part I: Ancestral inference in population genetics. In: Picard J. (eds) Lectures on Probability Theory and Statistics. Lecture Notes in Mathematics (Ecole d’Eté de Probabilités de Saint-Flour XXXI - 2001), vol 1837. Springer, Berlin, Heidel- berg. References Beaumont M (2004) Recent developments in genetic data analysis: what can they tell us about human demographic history? Heredity 92(5):365–379 Kaplan EL, Meier P (1958) Nonparametric estimation from incomplete observations. J Am Stat Assoc 53(282):457–481 Kuhlwilm M, Gronau I, Hubisz MJ, de Filippo C, Prado-Martinez J, Kircher M, Fu Q, Burbano HA, Lalueza-Fox C, de La Rasilla M et al. (2016) Ancient gene ﬂow from early modern humans into eastern neanderthals. Nature 530(7591):429–433 Beaumont MA, Nichols RA (1996) Evaluating loci for use in the genetic analysis of population structure. Proc R SocLond 263 (1377):1619–1626 Lapierre M, Lambert A, Achaz G (2017) Accuracy of demographic inferences from the site frequency spectrum: the case of the yoruba population. Genetics 206(1):439–449 Beichman AC, Phung TN, Lohmueller KE (2017) Comparison of single genome and allele frequency data reveals discordant demographic histories. G37(11):3605–3620 Li H, Durbin R (2011) Inference of human population history from individual whole-genome sequences. Nature 475(7357):493–496 Boitard S, Rodríguez W, Jay F, Mona S, Austerlitz F (2016) Inferring population size history from large samples of genome-wide molecular data-an approximate bayesian computation approach. PLoS Genet 12(3):e1005877 Liu X, Fu Y-X (2015) Exploring population size changes using SNP frequency spectra. Nat Genet 47(5):555-559 Mazet O, Rodríguez W, Chikhi L (2015) Demographic inference using genetic data from a single individual: separating popula- tion size variation from population structure. Theor Popul Biol 104:46–58 Cavalli-Sforza LL (1966) Population structure and human evolution. Proc R Soc Lond 164(995):362–379 Chikhi L, Rodríguez W, Grusea S, Santos P, Boitard S, Mazet O (2018) The IICR (inverse instantaneous coalescence rate) as a summary of genomic diversity: insights into demographic infer- ence and model choice. Heredity 120:13–24 Mazet O, Rodríguez W, Grusea S, Boitard S, Chikhi L (2016) On the importance of being structured: instantaneous coalescence rates 912 A. Arredondo et al. and human evolution—lessons for ancestral population size inference? Heredity 116(4):362 Salmona J, Heller R, Quéméré E, Chikhi L (2017) Climate change and human colonization triggered habitat loss and fragmentation in madagascar. Mol Ecol 26(19):5203–5222 Mona S, Ray N, Arenas M, Excofﬁer L (2014) Genetic consequences of habitat fragmentation during a range expansion. Heredity 112 (3):291–299 Scally A, Durbin R (2012) Revising the human mutation rate: impli- cations for understanding human evolution. References https://doi.org/10.1007/978-3-540-39874-5_1 Rodríguez W, Mazet O, Grusea S, Arredondo A, Corujo JM, Boitard S, Chikhi L (2018) The IICR and the non-stationary structured coalescent: towards demographic inference with arbitrary changes in population structure. Heredity 121 (6):663–678 Wakeley J (1999) Nonequilibrium migration in human history. Genetics 153(4):1863–1871 Rodríguez W, Mazet O, Grusea S, Arredondo A, Corujo JM, Boitard S, Chikhi L (2021) The IICR and the non-stationary structured coalescent: towards demographic inference with arbitrary chan- ges in population structure. (Correction to Heredity 2018, vol. 121(6), p. 663–678). Heredity 126(3):xxx–xxx Wang K, Mathieson I, O’Connell J, Schiffels S (2020) Tracking human population structure through time from whole genome sequences. PLoS Genet 16(3):e1008552 Wright S (1931) Evolution in Mendelian populations. Genetics 16 (2):97
https://openalex.org/W3044919154	https://threatenedtaxa.org/index.php/JoTT/article/download/4552/6883	English	null	Prevalence and morphotype diversity of Trichuris species and other soil-transmitted helminths in captive non-human primates in northern Nigeria	Journal of threatened taxa	2,020	cc-by	6,532	The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication. www.threatenedtaxa.org ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) Building evidence for conservation globally Journal of Threatened Taxa For Focus, Scope, Aims, Policies, and Guidelines visit https://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-0 For Article Submission Guidelines, visit https://threatenedtaxa.org/index.php/JoTT/about/submissions#onlineSubmissions For Policies against Scientific Misconduct, visit https://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-2 For reprints, contact <ravi@threatenedtaxa.org> Communication Prevalence and morphotype diversity of Trichuris species and other soil-transmitted helminths in captive non-human primates in northern Nigeria Joshua Kamani, James P. Yidawi, Aliyu Sada, Emmanuel G. Msheliza & Usman A. Turaki 26 July 2020 \| Vol. 12 \| No. 10 \| Pages: 16239–16244 DOI: 10.11609/jott.4552.12.10.16239-16244 Member Publisher & Host PLATINUM OPEN ACCESS The opinions expressed by the authors do not reflect the views of the Journal of Threatened Taxa, Wildlife Information Liaison Development Society, Zoo Outreach Organization, or any of the partners. The journal, the publisher, the host, and the part- ners are not responsible for the accuracy of the political boundaries shown in the maps by the authors. The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication. www.threatenedtaxa.org ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) Building evidence for conservation globally Journal of Threatened Taxa Communication Journal of Threatened Taxa For Focus, Scope, Aims, Policies, and Guidelines visit https://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-0 For Article Submission Guidelines, visit https://threatenedtaxa.org/index.php/JoTT/about/submissions#onlineSubmissions For Policies against Scientific Misconduct, visit https://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-2 For reprints, contact <ravi@threatenedtaxa.org> For Focus, Scope, Aims, Policies, and Guidelines visit https://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-0 For Article Submission Guidelines, visit https://threatenedtaxa.org/index.php/JoTT/about/submissions#onlineSubmissions For Policies against Scientific Misconduct, visit https://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-2 For reprints, contact <ravi@threatenedtaxa.org> For Focus, Scope, Aims, Policies, and Guidelines visit https://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-0 For Article Submission Guidelines, visit https://threatenedtaxa.org/index.php/JoTT/about/submissions#onlineSubmissions For Policies against Scientific Misconduct, visit https://threatenedtaxa.org/index.php/JoTT/about/editorialPolicies#custom-2 For reprints, contact <ravi@threatenedtaxa.org> The opinions expressed by the authors do not reflect the views of the Journal of Threatened Taxa, Wildlife Information Liaison Development Society, Zoo Outreach Organization, or any of the partners. The journal, the publisher, the host, and the part- ners are not responsible for the accuracy of the political boundaries shown in the maps by the authors. Threatened Taxa Publisher & Host Publisher & Host Member Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 July 2020 \| 12(10): 16239–16244 ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) #4552 \| Received 04 September 2018 \| Final received 03 June 2020 \| Finally accepted 30 June 2020 DOI: https://doi.org/10.11609/jott.4552.12.10.16239-16244 PLATINUM OPEN ACCESS Prevalence and morphotype diversity of Trichuris species and other soil-transmitted helminths in captive non-human primates in northern Nigeria Parasitology Division, National Veterinary Research Institute, Vom Nigeria. J.P. Yidawi—diagnosis and research of parasitic diseases of Aves. Mohamet Lawan College of Agriculture, Maiduguri, Borno state, Nigeria. A. Sada—epidemiology of parasitic livestock and wildlife diseases. National Veterinary Research Institute, Vom Nigeria. Emmanuel G. Msheliza—investigation of ecto and endo parasite of animals. National Veterinary Research Institute, Vom Nigeria. Usman A. Turaki—research and teaching of parasitic diseases of animals. Federal University of Kashere Gombe state. NiNigeria h Author contribution: JK—conceive and design study, Investigation and data analyses, writing of the manuscript. JPY—sample collection, data analyses, writing of manuscript. AS—sample collection, data analysis. EGM—Sample collection, investigation, writing of manuscript. UAT—sample collection, data analyses, writing of manuscript. Acknowledgements: The authors are grateful to the staff and management of the Zoos and Parks for the permission and assistance in fecal sample collections. Technical assistance of staff of Helminthology Lab NVRI Vom is highly appreciated Editor: Rupika S. Rajakaruna, University of Peradeniya, Peradeniya, Sri Lanka. Date of publication: 26 July 2020 (online & print) Date of publication: 26 July 2020 (online & print) Citation: Kamani, J., J.P. Yidawi, A. Sada, E.G. Msheliza & U.A. Turaki (2020). Prevalence and morphotype diversity of Trichuris species and other soil-transmitted hel- minths in captive non-human primates in northern Nigeria. Journal of Threatened Taxa 12(10): 16239–16244. https://doi.org/10.11609/jott.4552.12.10.16239-16244 Kamani et al. 2020. Creative Commons Attribution 4.0 International License. JoTT allows unrestricted use, reproduction, and distribution of this medium by providing adequate credit to the author(s) and the source of publication. Funding: This study did not receive any funding from government or private sources. Competing interests: The authors declare no competing interests. Author details: J. Kamani—investigation of parasitic and vector-borne zoonoses. Parasitology Division, National Veterinary Research Institute, Vom Nigeria. J.P. Yidawi—diagnosis and research of parasitic diseases of Aves. Mohamet Lawan College of Agriculture, Maiduguri, Borno state, Nigeria. A. Sada—epidemiology of parasitic livestock and wildlife diseases. National Veterinary Research Institute, Vom Nigeria. Emmanuel G. Msheliza—investigation of ecto and endo parasite of animals. National Veterinary Research Institute, Vom Nigeria. Usman A. Turaki—research and teaching of parasitic diseases of animals. Federal University of Kashere Gombe state. NiNigeria h Author contribution: JK—conceive and design study, Investigation and data analyses, writing of the manuscript. JPY—sample collection, data analyses, writing of manuscript. AS—sample collection, data analysis. EGM—Sample collection, investigation, writing of manuscript. UAT—sample collection, data analyses, writing of manuscript. Prevalence and morphotype diversity of Trichuris species and other soil-transmitted helminths in captive non-human primates in northern Nigeria ani 1 , James P. Yidawi 2 , Aliyu Sada 3 , Emmanuel G. Msheliza 4 & Usman A. Turaki 5 Joshua Kamani 1 , James P. Yidawi 2 , Aliyu Sada 3 , Emmanuel G. Msheliza 4 & Usman 1,4 Parasitology Division, National Veterinary Research Institute, PMB 01 Vom, Plateau State, Nigeria. 2,3 Mohamet Lawan College of Agriculture, PMB 1427 Maiduguri, Borno State, Nigeria. 5 Department of Animal Science Federal University Kashere, Gombe State, Nigeria. 1 mshelizakj@gmail.com (corresponding author), 2 jamesyidawi198@gmail.com, 3 aliyuvet@yahoo.co.uk, 4 egmshel@yahoo.com, 5 usmanturakialiyu@gmail.com Abstract: A study to determine the prevalence and morphotype diversity of soil-transmitted helminths in captive non-human primates (NHPs) in northern Nigeria was conducted. Simple flotation and sedimentation methods were used to examine fecal samples. A Morphometric analysis was done on Trichuris spp. eggs to determine the diversity of whipworm circulating in NHPs in the study area. High prevalence (60%) of infection was recorded in captive NHPs; Patas Monkey (n=17), Tantalus Monkey (n=9), Mona Monkey (n=7), Vervet Monkey (n=2), Mangabey Monkey (n=1), Baboon (n=14), and Chimpanzee (n=8) from parks and zoological gardens located in four Nigerian states (Borno, Gombe, Kano, and Plateau) and the Federal Capital Territory (FCT), Abuja. Captive NHPs examined were infected with helminths either as single, double or triple infections. Four zoonotic soil transmitted helminth (STH) genera, Trichuris, Strongyloides, Ancylostoma, and Enterobius were detected in the examined animals. Eggs of Trichuris spp. were the most prevalent with four morphotypes suggesting several morphotypes of whipworm were circulating among the NHPs in this region. Further studies are required to elucidate the epidemiologic and public health implications of these findings. Keywords: Helminths, morphotype, non-human primates, northern Nigeria, zoonosis. Editor: Rupika S. Rajakaruna, University of Peradeniya, Peradeniya, Sri Lanka. Date of publication: 26 July 2020 (online & print) Citation: Kamani, J., J.P. Yidawi, A. Sada, E.G. Msheliza & U.A. Turaki (2020). Prevalence and morphotype diversity of Trichuris species and other soil-transmitted hel- minths in captive non-human primates in northern Nigeria. Journal of Threatened Taxa 12(10): 16239–16244. https://doi.org/10.11609/jott.4552.12.10.16239-16244 Copyright: © Kamani et al. 2020. Creative Commons Attribution 4.0 International License. JoTT allows unrestricted use, reproduction, and distribution of this article in any medium by providing adequate credit to the author(s) and the source of publication. Funding: This study did not receive any funding from government or private sources. Competing interests: The authors declare no competing interests. Author details: J. Kamani—investigation of parasitic and vector-borne zoonoses. Sampling sites Non-human primates (NHPs) enclosures in zoological gardens or parks in Nigeria are among the most popular attractions to visitors, especially children; however, the maintenance of wild animals in captivity is fraught with numerous challenges particularly that of parasitic disease conditions due to high contamination of the environment (Rao & Acharjyo 1984; Vanitha et al. 2011). This is exacerbated by lowered immunity of the animals due to the stress of captivity, consequently diminishing their resistance to parasitic diseases (Gracenea et al. 2002; Perez Cordon et al. 2008). Therefore, gastrointestinal parasite infections are among the most common diseases found in non-human primates (Bezjian et al. 2008; Strait et al. 2012). Although the captive animals do not show alarming signs of parasitism, it has been reported that some of the helminth parasites they harbor have zoonotic potential and are, therefore, considered to be a threat to public health (Gillespie et al. 2008; Klaus et al. 2017). Soil-transmitted helminths (STHs) such as Ascaris spp., Trichuris spp., Ancylostoma spp., and Strongyloides spp. that can easily be transferred from NHPs to humans and vice versa through contaminated environments are a major concern (Ranglack & Yeager 1986; Bethony et al. 2006; Lynn 2010). Single or mixed infections of zoonotic STHs have often been recorded in NHPs from different countries: Bangladesh (Raja et al. 2014), Central African Republic (Hasegawa et al. 2014), Tanzania (Petrželkova et al. 2010), China (Li et al. 2017), India (Hussain et al. 2013), Sri Lanka (Aviruppola et al. 2016), Malaysia (Klaus et al. 2017), and Spain (Perez Corden et al. 2008). Several studies have been carried out on the prevalence of helminth infection in NHPs in various zoological gardens and/or parks in the southern part of Nigeria: Oyo State (Adedokun et al. 2002; Emikpe et al. 2002; Adetunji 2014), Ondo State (Egbetade et al. 2014), Cross River State (Mbaya & Udendeye 2011), and Imo state (Opara et al. 2010). Comparatively, only a few studies have been conducted on the helminth infections of NHPs in northern Nigeria (Nwosu 1995; Mbaya & Nwosu 2006; Mbaya et al. 2006a,b; Dawet et al. 2013). The aim of this study, therefore, was to determine the prevalence and diversity of helminths in captive NHPs in northern Nigeria and to discuss the public health implications. The study was conducted in Zoological Gardens and Parks in four northern states and Abuja (9.0760N, 7.3980E) the Federal Capital Territory (FCT) of Nigeria. Sampling sites The four states; Borno (11.8310N, 13.1510E), Gombe (10.2790N, 11.1730E), Kano (12.0020N, 8.5920E) and Plateau (9.8960N, 8.8580E) were selected based on convenience and accessibility of NHPs for sample collection. MATERIALS AND METHODS MATERIALS AND METHODS Sample collection Fecal samples were collected opportunistically from individual captive NHP with the help of the caretakers over a period of six months (November 2017 to April 2018). Fresh feces were collected from the ground under the nest of individual NHPs. Approximately 5g of feces was scooped from the surface of each fecal mass using a disposable hand glove and transferred into a screw capped bottle. Each sample was labeled appropriately and transported in a cold box to the laboratory for analysis. Fecal analysis Samples were processed and analyzed in the Helminthology Research Laboratory, National Veterinary Research Institute (NVRI), Vom, Plateau State, Nigeria. First, each sample was examined macroscopically for the presence of helminths or taenid segments. Microscopic analysisli Simple tube flotation and sedimentation: Fecal samples were individually processed by simple tube flotation in saturated sodium chloride solution (SG 1.20) and simple sedimentation techniques (Greiner & McIntosh 2009). The preparation was then examined using direct light microscope (100X and 400X magnifications) for the presence of parasite eggs. The identification of the parasites was based on egg morphology, shape, size and color according to standard keys (Samuel et al. 2001; Hasegawa et al. 2009). Prevalence and morphotype diversity of Trichuris species and other soil-transmitted helminths in captive non-human primates in northern Nigeria Acknowledgements: The authors are grateful to the staff and management of the Zoos and Parks for the permission and assistance in fecal sample collections. Technical assistance of staff of Helminthology Lab NVRI Vom is highly appreciated 16239 Soil transmitted helminths in non-human primates Kamani et al. RESULTS RESULTS Fifty-eight captive NHPs in five zoological gardens located in Jos (n=15), Kano (n=9), Maiduguri (n=3), Gombe (n=9) & FCT Abuja (n=4) and two Parks located in Jos (n=8) & FCT Abuja (n=10) were examined. Seven NHP species including 17 Patas Monkey Erythrocebus patas, 14 Baboons Papio sp., nine Tantalus Monkey Chlorocebus tantalus, eight Chimpanzees Pan troglodytes, seven Mona Monkeys Cercopithecus mona, two Vervet Monkeys Chlorocebus pygerythrus, and one Mangabey Monkey Cercocebus sp. were sampled during this study (Table 1). Diversity of helminths Four helminth genera: Trichuris, Ancylostoma, Strongyloides, and Enterobius and one unidentified parasite egg were detected from the feces of NHPs from northern Nigeria. Trichuris spp. eggs were detected with a high prevalence (31/44) across all the NHP species screened in the study. This was followed by Strongyloides spp. (4/44) and Ancylostoma spp. (3/44) both affecting three NHP species each. An unidentified egg that resembled an egg of Opisthorchis sp. was detected in Prevalence of helminths in NHPs from northern Nigeria Helminth eggs were detected in fecal samples of NHPs from zoos or parks in all the states studied. Overall, 60% of the animals studied had helminth eggs in their feces. The highest prevalence (100%, n=4/4) was recorded in samples from the Abuja Children Park Table 1. Prevalence of helminths in captive non-human primates in zoological gardens and parks in northern Nigeria. Study location Habitat Number of animal positive/ no. screened according to NHP species Total (%) Patas Monkey Erythrocebus patas Tantalus Monkey Chlorocebus tantalus Mona Monkey Cercopithecus mona Vervet Monkey Chlorocebus pygerythrus Mangabey Monkey Cercocebus sp. Baboon Papio sp. Chimpanzee Pan troglodytes Jos Zoo 0/4 0 2/3 1/2 0 3/3 0/3 6/15 (40) Jos Wildlife Park 2/3 1/1 1/1 0 0 2/2 0/1 6/8 (75) Gombe Zoo 5/5 0 0 0 0 1/2 2/2 8/9 (89) Maiduguri Zoo 0/1 0/1 0 0 0 1/1 0 1/3 (33) Abuja Park/ Zoo 1/1 1/1 1/1 0 0 1/1 0 4/4 (100) Abuja National Park 0/1 4/5 0/1 0 0 1/3 0 5/10 (50) Kano Zoo 0/2 1/1 1/1 0 0/1 1/2 2/2 5/9 (56) Total 8/17 7/9 5/7 1/2 0/1 10/14 4/8 35/58 (60) Table 1. Prevalence of helminths in captive non-human primates in zoological gardens and parks in northern Nigeria. Table 2. Prevalence of soil transmitted helminths in different species of non-human primates in northern Nigeria. Host species No of NHP tested No positive (%) Single infection Dual infection Triple infection Patas Monkey Erythrocebus patas 17 8 (47) 7 1 0 Tantalus Monkey Chlorocebus tantalus 9 7 (78) 5 2 0 Mona Monkeys Cercopithecus mona 7 5 (71) 2 2 1 Vervet Monkey Chlorocebus pygerythrus 2 1 (50) 1 0 0 Baboon Papio sp. 14 10 (71) 1 3 0 Chimpanzee Pan troglodytes 8 4 (50) 10 0 0 Mangabey Monkey Cercocebus sp. 1 0 (0) 0 0 0 Total 58 35 26 8 1 able 2. Prevalence of soil transmitted helminths in different species of non-human primates in northern Nigeria. Morphometry analysis Helminth eggs were measured (length and width) by using a calibrated light microscope. Mean values of measurements were given in micrometers (µm) ± standard deviations (SD). Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 July 2020 \| 12(10): 16239–16244 16240 Soil transmitted helminths in non-human primates Kamani et al. and Zoo, followed by (89%, n=8/9) in samples from the Gombe Zoo and the lowest prevalence (33%, n=1/3) was recorded in samples from the Maiduguri Zoological Garden (Table 1). Prevalence according to NHP species was highest (78%) for Tantalus Monkeys Chlorocebus tantalus. The lowest prevalence (47%, n=8/17) was observed in Patas Monkeys while the fecal sample of the only Mangabey Monkey Cercocebus sp. screened in this study was negative for helminth eggs (Table 1). Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 July 2020 \| 12(10): 16239–16244 Egg morphotypes detected in NHP examined Egg morphotypes detected in NHP examined Four morphotypes of Trichuris spp. eggs (T1–T4) were detected in this study. All the trichurid egg morphotypes were thick-shelled, with prominent bipolar plugs but varied in shape, size, and colour. Egg morphotypes T1 and T2 appear to be more common in the various species of monkeys examined and occur as mixed infections, while T3 and T4 were commonly associated with baboons and chimpanzees. The eggs of other helminth genera, Ancylostoma, Strongyloides, and Enterobius detected in this study occurred as single morphotypes (Table 3). Trichuris eggs were the most prevalent (70.5%) in the infected NHPs in this study. This finding is in accord with an earlier survey of NHPs in northern (Dawet et al. 2013) and southern (Mbaya & Udendeye, 2011; Adetunji 2014) parts of Nigeria. High prevalence of Trichuris sp. was also observed in NHPs in Côte d’Ivoire (Kouassi et al. 2015), Sri Lanka (Aviruppola et al. 2016), Peru (Kimberley et al. 2004), Malaysia (Lim et al. 2008; Klaus et al. 2017), China (Li et al. 2017), India (Singh et al. 2009), and Spain (Perez Cordon et al. 2008) signifying its global distribution among NHP population. This study provides additional information on the metric details of the trichurid eggs present in the NHPs in northern Nigeria suggesting the diversity of this parasite in the region. It appears that NHPs in northern Nigeria are infected with various Trichuris spp. based on the morphology and dimensions of the eggs detected during this study. A similar observation was earlier made in a study in south west Nigeria but the authors did not provide any metric details of the Trichuris eggs detected (Egbetade et al. 2014). Therefore, our study is the first to provide morphometric analyses of Trichuris eggs infecting NHPs in Nigeria. Four morphotypes (T1– T4) of Trichuris eggs, with mean size ranging from 43– 53 x 22–30 µm (length x width) were detected in this study suggesting a diversity of this parasite in the NHPs examined. Indeed, even among the NHP species there is variability in the morphology of Trichuris eggs detected. Similar observations have been reported (Petrzelkova et al. 2010; Klaus et al. 2017). Thus, our findings agree with several reports on morphometric and molecular Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 July 2020 \| 12(10): 16239–16244 16241 Soil transmitted helminths in non-human primates Soil transmitted helminths in non-human primates Table 3. Morphologic and morphometric analyses of helminth eggs detected in non-human primates in northern Nigeria Helminth genera Morphology and morphometric characteristics of helminth eggs Morphotype Egg size (um + SD) Egg shape Egg shell appearance and color Length Width Trichuris sp. T1 T2 T3 T4 43.1 ± 5 50.8 ± 2.5 54.7 ± 0.1 58.2 ± 1.0 25.1 ± 1 30.5 ±1.5 22.9 ± 5.7 22.4 ± 3.2 Ellipsoidal Rounded lemon Barrel-like Lemon Thick with prominent bipolar plugs, dark brown Thick less prominent bipolar plugs, light brown Thick prominent bipolar plugs, dark brown Thick, flat bipolar transparent plugs, dark brown Ancylostoma sp. A 73.5 ± 5.3 31.5 ± 2.3 Ellipsoidal Thin transparent light color Strongyloides sp. S 48.3 ± 8.8 34.3 ± 4.7 Ellipsoidal Thin transparent folded motile larva light color Enterobius sp. E 54.5 ± 5.3 30.5 ± 2.3 Irregular Thin with folded larva light color 3. Morphologic and morphometric analyses of helminth eggs detected in non-human primates in northern Nigeria the fecal sediments of a female baboon from Abuja. Parasite order richness (POR) ranged from 0 to 3 per fecal sample. Most of the positive samples harboured eggs of a single parasite genus (45%), followed by dual infections (12%) and triple infections in only 2% of the samples (Table 2). soil-transmitted helminth infections globally (Bethony et al. 2006). The differences in prevalence of these helminths observed in the study locations could be attributed to differences in host species susceptibility to helminth infections and the variations in climatic conditions between the study sites. Such observation on the effects of climate on parasite prevalence have been reported (Cordon et al. 2008; Wren et al. 2015). DISCUSSION Captive NHPs in Nigerian zoological gardens and parks attract attention due to their agility and playfulness. Thus, their wellbeing and survival is paramount to conservationists, veterinarians and zoo administrators. Parasitic diseases, particularly helminth infections have been reported to constitute a challenge to the health of NHPs (Samuel et al. 2001; Vanitha et al. 2011; Wren et al. 2015). The results from this survey showed that more than half (60%) of the examined NHPs in northern Nigeria were infected with helminth parasites. Four helminth genera of zoonotic importance, viz., Trichuris, Ancylostoma, Strongyloides, and Enterobius were detected in the NHPs examined in this study. These helminths have a high potential for transmission to humans because of their simple life cycles (Li et al. 2017). Thus, they are listed among the major cause of Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 July 2020 \| 12(10): 16239–16244 16242 Soil transmitted helminths in non-human primates Kamani et al. studies of trichurid eggs in NHPs conducted in areas with different climatic conditions from Nigeria (Hasegawa et al. 1983; Dupain et al. 2009; Ghai et al. 2014; Raja et al. 2014; Cavallero et al. 2015; Klaus et al. 2017; Li et al. 2017). studies of trichurid eggs in NHPs conducted in areas with different climatic conditions from Nigeria (Hasegawa et al. 1983; Dupain et al. 2009; Ghai et al. 2014; Raja et al. 2014; Cavallero et al. 2015; Klaus et al. 2017; Li et al. 2017). Nigeria. Nigeria Veterinary Journal 23: 56–59. Adetunji, V.E. (2014). Prevalence of gastro-intestinal parasites in primates and their keepers from two zoological gardens in Ibadan, Nigeria. Sokoto Journal of Veterinary Science 12(2): 25–30. Aviruppola, A.J.M.K., R.P.V.J. Rajapakse & R.S. Rajakaruna (2016). Coprological survey of gastrointestinal parasites of mammals in Dehiwala National Zoological Gardens, Sri Lanka. Ceylon Journal of Science 45(1): 83–96. https://doi.org/10.4038/cjs.v45i1.7367 It is noteworthy that the dimensions of some of the eggs of Trichuris spp. detected in this study are similar to those of human T. trichiura, suggesting zoonotic or reverse zoonotic transmissions. This finding has implications for veterinarians, public health workers, and wildlife managers, in terms of the epidemiology of the disease and the choice of treatment and control measures to be adopted (Melfi & Poyser 2007). Therefore, the assumption hitherto, among wildlife parasitologists that all the Trichuris infecting NHPs are the same as T. DISCUSSION trichiura of humans should be reconsidered, however, the other three zoonotic helminths genera; Ancylostoma, Strongyloides and Enterobius detected in this study occurred as monotypes each with dimensions similar to those of the species infecting human, suggesting possible circulation of these worms between humans and NHPs in the study area. i g y f Science 45(1): 83–96. https://doi.org/10.4038/cjs.v45i1.7367 Bethony, J., S. Brooker, M. Albonico, S.M. Geiger, A. Loukas, D. Diemert & P.J. Hotez (2006). Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 367: 1521–32 Bezjian, M., T.R. Gillespie, C.A. Chapman & E.C. Greiner (2008). Coprologic evidence of gastrointestinal helminths of forest baboons, Papio anubis, in Kibale National Park, Uganda. Journal of Wildlife Diseases 44: 878–887. Cavallero, S., C. De Liberato, K.G. Friedrich, D. Di Cave, V. Masella, S. D’amelio & F. Berrilli (2015). Genetic heterogeneity and phylogeny of Trichuris spp. from captive non-human primates based on ribosomal DNA sequence data. Infection, Genetics and Evolution. 34: 450–456. Cordon, G.P., A.H. Prados, D. Romero, M.S. Moreno, A. Pontes, A. Osuna & M.J. Rosales (2008). Intestinal parasitism in the animals of the zoological garden ‘‘Pena Escrita’’ (Almunecar, Spain). Veterinary Parasitology 156: 302–309.i Dawet, A., D.P. Yakubu & H.M. Butu (2013). Survey of gastrointestinal parasites of non-human primates in Jos Zoological Garden. Journal of Primatology 2: 108. https://doi.org/10.4172/2167–6801.1000108 Dupain J., C. Nell, K. J. Petrzelkova, P. Garcia, D. Modry & F.P. Gordo (2009). Gastrointestinal parasites of bonobos in the Lomako Forest, Democratic Republic of Congo, pp. 297–310. In: Huffman M.A. & C. Chapman (eds.). Primate parasite ecology, the dynamics and study of host parasite relationships. Cambridge University Press, UK. Taken together, our results demonstrate the presence of zoonotic helminths and a diversity of Trichuris sp. infection amongst NHPs. Therefore, a comprehensive study to elucidate the genetic diversity of Trichurids infection NHPs in Nigeria is desirable. This will assist to distinguish the species and genotypes of this parasite in NHPs in northern Nigeria and to determine their pathogenicity. Therefore, molecular studies on pinworm diversity in Nigerian NHPs are needed in order to elucidate the species and morphotypes circulating in the country. Egbetade, A., O. Akinkuotu, O. Jayeola, A.N. Niniola, N. Emmanuel, E.S. Olugbogi & S. Onadeko (2014). Gastrointestinal helminths of resident wildlife at the Federal University of Agriculture Zoological Park, Abeokuta. Sokoto Journal of Veterinary Science 12(3): 26–31. Emikpe, B.O., J.O. Ayoade, O.G. Ohore, O.O. Olaniyan, & M.O. Akusu (2002). DISCUSSION Fatal trichuriosis in a captive baboon (Papio anubis) in Ibadan. Tropical Veterinarian 20: 36–39. Ghai, R.R., N.D. Simons, C.A. Chapman, P.A. Omeja, T.J. Davies, N. Ting & T.L. Goldberg (2014). Hidden population structure and cross- species transmission of whipworms (Trichuris sp.) in humans and non-human primates in Uganda. PLoS Neglected Tropical Disease 8: e3256. https://doi.org/10.1371/journal.pntd.0003256i Gillespie, T.R., C.L. Nunn & F.H. Leendertz (2008). Integrative approaches to the study of primate infectious disease: implications for biodiversity conservation and global health. American Journal of Physical Anthropology Suppl 47: 53–69. https://doi.org/10.1002/ ajpa.20949 CONCLUSION Gracenea, M., M.S. Gomez, J. Torres, E. Carne & J. Fernandez-Moran (2002). Transmission dynamics of Cryptosporidium in primates and herbivores at the Barcelona zoo: a long-term study. Veterinary Parasitology 104: 19–26.ii Captive NHPs in zoological gardens and parks in northern Nigeria are infected with helminths of public health significance. High prevalence of Trichuris spp. coupled with a diversity of their egg morphotypes were observed in the NHPs examined. Further investigation using modern tools like molecular phylogenetics in order to fully understand their epidemiology and zoonotic potentials is warranted. Greiner, E.C & A. McIntosh (2009). Collection methods and diagnostic procedures for primate parasitology, pp. 3–27. In: Huffman, M.A. & C.A. Chapman (eds.). Primates Parasite Ecology. The Dynamics and Study of Host-parasite Relationships. Cambridge University Press, 531pp. Hasegawa, H., C.A. Chapman & M.A. Huffman (2009). Useful diagnostic references and images found in wild primates, pp. 507– 513. In: Huffman M.A. & C. Chapman (editors). Primates Parasite Ecology. The Dynamics and Study of Host-parasite Relationships. Cambridge University Press, 531pp. t Hasegawa, H., D. Modrý, M. Kitagawa, K.A. Shutt, A. Todd, B. Kalousova, I. Profousova & K.J. Petrzelkova (2014). Humans and great apes cohabiting the forest ecosystem in Central African Republic harbour the same hookworms. PLoS Neglected Tropical Disease 8: e2715. https://doi.org/10.1371/journal.pntd.0002715 Adedokun, O.A., R.A.M. Adedokun, B.O. Emikpe, O.G. Ohore, D.O. Oluwayele & O.I. Ajayi (2002). Concurrent fatal helminthosis and balantidiosis in a red monkey (Erythrocebus pattas) in Ibadan, REFERENCES Adedokun, O.A., R.A.M. Adedokun, B.O. Emikpe, O.G. Ohore, D.O. Oluwayele & O.I. Ajayi (2002). Concurrent fatal helminthosis and balantidiosis in a red monkey (Erythrocebus pattas) in Ibadan, t Hasegawa, H., T. Kano & M. Mulavwa (1983). A parasitological survey Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 July 2020 \| 12(10): 16239–16244 16243 Soil transmitted helminths in non-human primates Kamani et al. Mbaya, A.W., M.M. Aliyu & C.O. Nwosu (2006b). Outbreak of balantidiosis among captive chimpanzees (Pan troglodytes) at the Sanda Kyarimi Park, Maiduguri, Nigeria. Sahel Journal of Veterinary Science 5: 55–58. on the feces of pygmy chimpanzees, Pan paniscus, at Wamba, Zaire. Primates 24: 419–423. on the feces of pygmy chimpanzees, Pan paniscus, at Wamba, Zaire. Primates 24: 419–423. Hussain, S., M.S. Ram, A. Kumar, S. Shivaji & G. Umapathy (2013). Human presence increases parasitic load in endangered Lion-tailed Macaques (Macaca silenus) in its fragmented rainforest habitats in southern India. PLoS One 8: e63685. https://doi.org/10.1371/ journal.pone.0063685 Melfi, V. & F. Poyser (2007). Trichuris burdens in zoo-housed Colobus guereza. International Journal of Primatology 28: 1449–1456. Nwosu, C.O. (1995). Helminth parasites of captive wild animals in Borno State Nigeria. Tropical Veterinarian 13: 1–13.i Kimberley, A.P., E.H. Meghan, W.G. Brian & Y. Mirtha (2004). Survey of the gastrointestinal parasites of the primate community at Tambopata National Reserve, Peru. Journal of Zoology 264: 149– 151. Opara, M.N., C.T. Osuji & J.A. Opara (2010). Gastrointestinal parasitism in captive animals at the zoological garden, Nekede Owerri, southeast Nigeria. Report and Opinion 2: 21–28. Petrželkova, K.J., H. Hasegawa, C.C. Appleton, M.A. Huffman, C.E. Archer, L.R. Moscovice, M.I. Mapua, J. Singh & T. Kaur (2010). Gastrointestinal parasites of the chimpanzee population introduced onto Rubondo Island National Park, Tanzania. American Journal of Primatology 72: 307–316. Klaus, A., E. Zimmermann, M.K. Roper, U. Radespiel, S. Nathan, B. Goossens & C. Strube (2017). Co-infection patterns of intestinal parasites in arboreal primates (proboscis monkeys, Nasalis larvatus) in Borneo. International Journal of Parasitology, Parasite and Wildlife 6: 320–329. Raja, M.M.R.U., A.R. Dey, M. Begum, U.K. Kundu & A.F. Ashad (2014). Coprological prevalence of gastrointestinal parasites in carnivores and small mammals at Dhaka Zoo, Bangladesh. Journal of Threatened Taxa 6(3): 5574–5579. https://doi.org/10.11609/ JoTT.o3569.5574-9 Kouassi, R.Y., S.W. Mcgraw, P.K. Yao, A. Abou-Bacar, J. Brunet, B. Pesson, B. Bonfoh, E.K. N’goran & E. Candolfi (2015). Diversity and prevalence of gastrointestinal parasites in seven non-human primates of the Taï National Park, Côte d’Ivoire. Parasite 22: 1. REFERENCES Li, J., H. Dong, R. Wang, F. Yu, Y. Wu, Y. Chang, C. Wang, M. Qi & L. Zhang (2017). An investigation of parasitic infections and review of molecular characterization of the intestinal protozoa in nonhuman primates in China from 2009 to 2015. International Journal of Parasitology, Parasite and Wildlife 6: 8–15. Ranglack, G.S & C.P. Yeager (1986). Survey of intestinal parasites found in Proboscis Monkeys (Nasalis larvatus) and Long-tailed Macaques (Macaca fascicularis). Primate Reports 14: 249. ii Rao, A.T & L.N. Acharjyo (1984). Diagnosis and classification of common diseases of captive animals at Nandankanan Zoo in Orissa (India). Indian Journal of Animal Health 33: 147–152.i Lim, Y.A.L., R. Ngui, J. Shukri, M. Rohela & N.H.R. Mat (2008). Intestinal parasites in various animals at a zoo in Malaysia. Veterinary Parasitology 157: 154–159. Samuel, W.M., M. J. Pybus & A. Kocan (eds.). (2001). Parasitic Diseases of Wild Mammals 2nd ed. Iowa State University Press, Ames, 568pp. Lynn, M.S. (2010). Primate viability in a fragmented landscape: Genetic diversity and parasite burden of long-tailed macaques and proboscis monkeys in the lower Kinabatangan Flood plain, Sabah, Malaysia. PhD Thesis. Cardiff University, Cardiff, 228pp. Singh, P., L.D. Singla, M.P. Gupta, S. Sharma & D.R. Sharma (2009). Epidemiology and chemotherapy of parasitic infections in wild omnivores in the Mechendra Choudhury Zoological park, Chhat Bir, Punjab. Journal of Threatened Taxa 1(1): 62– 64. https://doi.org/10.11609/JoTT.o1767a.62-4 i Mbaya, A.W. & E.U.J. Udendey (2011). Gastrointestinal parasites of captive and free-roaming primates at the Afi mountain Primate Conservation area in Calabar, Nigeria and their zoonotic implications. Pakistan Journal of Biological Science 14(13): 709–714. t Strait K., J. Else & M.L. Eberhard (2012). Parasitic diseases of nonhuman primates, pp. 197–298. In: Abee C.R., K. Mansfield, S.D. Tardif & T. Morris (eds.) Nonhuman Primates in Biomedical Research: Diseases. Academic Press, London, 536pp. Mbaya, A.W. & C.O. Nwosu (2006). Outbreak of clinical amoebiasis among captive chimpanzees (Pan troglodytes) at the Sanda Kyarimi Park, Maiduguri, Nigeria. Nigerian Veterinary Journal 1: 68–73. Vanitha, V., K. Thiyagesan & N. Baskaran (2011). Prevalence of intestinal parasites among captive Asian Elephants Elephas maximus: effect of season, host demography, and management systems in Tamil Nadu, India. Journal of Threatened Taxa 3(2): 1527– 1534. https://doi.org/10.11609/JoTT.o2488.1527-34 Mbaya, A.W., C.O. Nwosu, M.M. Aliyu & T.A. Ahmed (2006a). Comparative study of gastrointestinal parasites of captive and free-living wild animals in the semi-arid zone of north-eastern Nigeria. Short Communications Short Communications A threat assessment of Three-striped Palm Squirrel Funambulus palmarum (Mammalia: Rodentia: Sciuridae) from roadkills in Sigur Plateau, Mudumalai Tiger Reserve, Tamil Nadu, India – Arockianathan Samson, Balasundaram Ramakrishnan & Jabamalainathan Leonaprincy, Pp. 16347–16351 Impact of vehicular traffic on birds in Tiruchirappalli District, Tamil Nadu, India – T. Siva & P. Neelanarayanan, Pp. 16352–16356 Ichthyofaunal diversity of Manjeera Reservoir, Manjeera Wildlife Sanctuary, Telangana, India – Kante Krishna Prasad, Mohammad Younus & Chelmala Srinivasulu, Pp. 16357–16367 New distribution record of the endemic and critically endangered Giant Staghorn Fern Platycerium grande (Fee) Kunze (Polypodiaceae) in central Mindanao – Cherie Cano-Mangaoang & Charissa Joy Arroyo Gumban, Pp. 16368–16372 Notes First photographic record of the Dhole Cuon alpinus (Mammalia: Carnivora: Canidae) from the Sirumalai Hills in Tamil Nadu, India – B.M. Krishnakumar & M. Eric Ramanujam, Pp. 16373–16376 Tracing heavy metals in urban ecosystems through the study of bat guano - a preliminary study from Kerala, India – Jithin Johnson & Moncey Vincent, Pp. 16377–16379 Population dynamics and management strategies for the invasive African Catfish Clarias gariepinus (Burchell, 1822) in the Western Ghats hotspot – Kuttanelloor Roshni, Chelapurath Radhakrishnan Renjithkumar, Rajeev Raghavan, Neelesh Dahanukar & Kutty Ranjeet, Pp. 16380–16384 First records of the black widow spider Latrodectus elegans Thorell, 1898 (Araneae: Theridiidae) from Nepal – Binu Shrestha & Tobias Dörr, Pp. 16385–16388 First report of the assassin bug Epidaus wangi (Heteroptera: Reduviidae: Harpactorinae) from India – Swapnil S. Boyane & Hemant V. Ghate, Pp. 16389–16391 Observations of the damselfly Platylestes cf. platystylus Rambur, 1842 (Insecta: Odonata: Zygoptera: Lestidae) from peninsular India – K.J. Rison & A. Vivek Chandran, Pp. 16392–16395 Herminium longilobatum (Orchidaceae), a new record for Bhutan – Ugyen Dechen, Tandin Wangchuk & Lam Norbu, Pp. 16396–16398 Recent record of a threatened holoparasitic plant Sapria himalayana Griff. in Mehao Wildlife Sanctuary, Arunachal Pradesh, India – Arif Ahmad, Amit Kumar, Gopal Singh Rawat & G.V. Gopi , Pp. 16399–16401 Eleven new records of lichens to the state of Kerala, India – Sonia Anna Zachariah, Sanjeeva Nayaka, Siljo Joseph, Pooja Gupta & Scaria Kadookunnel Varghese, Pp. 16402–16406 Publisher & Host Pakshirajan Lakshminarasimhan: a plant taxonomist who loved plants and people alike – Mandar N. Datar, Pp. 16195–16203 Ecology of the Critically Endangered Singidia Tilapia (Teleostei: Cichlidae: Oreochromis esculentus) of lake Kayanja, Uganda and its conservation implications – Richard Olwa, Herbert Nakiyende, Elias Muhumuza, Samuel Bassa, Anthony Taabu- Munyaho & Winnie Nkalubo, Pp. REFERENCES Bower & J. Robert Britton, Pp. 16257– 16261 The identity and distribution of Bhavania annandalei Hora, 1920 (Cypriniformes: Balitoridae), a hillstream loach endemic to the Western Ghats of India – Remya L. Sundar, V.K. Anoop, Arya Sidharthan, Neelesh Dahanukar & Rajeev Raghavan, Pp. 16262–16271 Records of two toads Duttaphrynus scaber and D. stomaticus (Amphibia: Anura: Bufonidae) from southeastern India – S.R. Ganesh, M. Rameshwaran, Naveen A. Joseph, Ahamed M. Jerith & Sushil K. Dutta, Pp. 16272–16278 Some rare damselflies and dragonflies (Odonata: Zygoptera and Anisoptera) in Ukraine: new records, notes on distribution, and habitat preferences – Alexander V. Martynov, Pp. 16279–16294 Floristic diversity of Anjaneri Hills, Maharashtra, India – Sanjay Gajanan Auti, Sharad Suresh Kambale, Kumar Vinod Chhotupuri Gosavi & Arun Nivrutti Chandore, Pp. 16295–16313 A checklist of macrofungi (mushroom) diversity and distribution in the forests of Tripura, India — Sanjit Debnath, Ramesh Chandra Upadhyay, Rahul Saha, Koushik Majumdar, Panna Das & Ajay Krishna Saha, Pp. 16314–16346 PLATINUM OPEN ACCESS ISSN 0974-7907 (Online) \| ISSN 0974-7893 (Print) July 2020 \| Vol. 12 \| No. 10 \| Pages: 16195–16406 Date of Publication: 26 July 2020 (Online & Print) DOI: 10.11609/jott.2020.12.10.16195-16406 REFERENCES Nigerian Journal of Experimental and Applied Biology 7: 185–193. Wren, B.T., T.R. Gillespie, J.W. Camp & M.J. Remis (2015). Helminths of Vervet Monkeys, Chlorocebus aethiops, from Loskop Dam Nature Reserve, South Africa. Comparative Parasitology 82: 101–108. Threatened Taxa Journal of Threatened Taxa \| www.threatenedtaxa.org \| 26 July 2020 \| 12(10): 16239–16244 16244 The Journal of Threatened Taxa (JoTT) is dedicated to building evidence for conservation globally by publishing peer-reviewed articles online every month at a reasonably rapid rate at www.threatenedtaxa.org. All articles published in JoTT are registered under Creative Commons Attribution 4.0 International License unless otherwise mentioned. JoTT allows allows unrestricted use, reproduction, and distribution of articles in any medium by providing adequate credit to the author(s) and the source of publication. ISSN 0974- July 2020 \| Date of Pub DOI: 10. www.threatenedtaxa.org The Journal of Threa publishing peer-review All articles published i unless otherwise men in any medium by prov Editorial Pakshirajan Lakshminarasimhan: a plant taxonomist who loved plants and people alike – Mandar N. Datar, Pp. 16195–16203 Communications The worrisome conservation status of ecosystems within the distribution range of the Spectacled Bear Tremarctos ornatus (Mammalia: Carnivora: Ursidae) in Ecuador – José Guerrero-Casado & Ramón H. Zambrano, Pp. 16204–16209 Living with Leopard Panthera pardus fusca (Mammalia: Carnivora: Felidae): livestock depredation and community perception in Kalakkad-Mundanthurai Tiger Reserve, southern Western Ghats – Bawa Mothilal Krishnakumar, Rajarathinavelu Nagarajan & Kanagaraj Muthamizh Selvan, Pp. 16210–16218 An updated checklist of mammals of Odisha, India – Subrat Debata & Himanshu Shekhar Palei, Pp. 16219–16229 Negative human-wildlife interactions in traditional agroforestry systems in Assam, India – Yashmita-Ulman, Manoj Singh, Awadhesh Kumar & Madhubala Sharma, Pp. 16230–16238 Prevalence and morphotype diversity of Trichuris species and other soil-transmitted helminths in captive non-human primates in northern Nigeria – Joshua Kamani, James P. Yidawi, Aliyu Sada, Emmanuel G. Msheliza & Usman A. Turaki, Pp. 16239–16244 Detection of hemoparasites in bats, Bangladesh – Shariful Islam, Rakib Uddin Ahmed, Md. Kaisar Rahman, Jinnat Ferdous, Md. Helal Uddin, Sazeda Akter, Abdullah Al Faruq, Mohammad Mahmudul Hassan, Ausraful Islam & Ariful Islam, Pp. 16245–16250 Ecology of the Critically Endangered Singidia Tilapia (Teleostei: Cichlidae: Oreochromis esculentus) of lake Kayanja, Uganda and its conservation implications – Richard Olwa, Herbert Nakiyende, Elias Muhumuza, Samuel Bassa, Anthony Taabu- Munyaho & Winnie Nkalubo, Pp. 16251–16256 Length-weight relationships of two conservation-concern mahseers (Teleostei: Cyprinidae: Tor) of the river Cauvery, Karnataka, India – Adrian C. Pinder, Rajeev Raghavan, Shannon D. Short Communications 16251–16256 Length-weight relationships of two conservation-concern mahseers (Teleostei: Cyprinidae: Tor) of the river Cauvery, Karnataka, India – Adrian C. Pinder, Rajeev Raghavan, Shannon D. Bower & J. Robert Britton, Pp. 16257– 16261 The identity and distribution of Bhavania annandalei Hora, 1920 (Cypriniformes: Balitoridae), a hillstream loach endemic to the Western Ghats of India – Remya L. Sundar, V.K. Anoop, Arya Sidharthan, Neelesh Dahanukar & Rajeev Raghavan, Pp. 16262–16271 Records of two toads Duttaphrynus scaber and D. stomaticus (Amphibia: Anura: Bufonidae) from southeastern India – S.R. Ganesh, M. Rameshwaran, Naveen A. Joseph, Ahamed M. Jerith & Sushil K. Dutta, Pp. 16272–16278 Records of two toads Duttaphrynus scaber and D. stomaticus (Amphibia: Anura: Bufonidae) from southeastern India – S.R. Ganesh, M. Rameshwaran, Naveen A. Joseph, Ahamed M. Jerith & Sushil K. Dutta, Pp. 16272–16278 Some rare damselflies and dragonflies (Odonata: Zygoptera and Anisoptera) in Ukraine: new records, notes on distribution, and habitat preferences – Alexander V. Martynov, Pp. 16279–16294 Eleven new records of lichens to the state of Kerala, India – Sonia Anna Zachariah, Sanjeeva Nayaka, Siljo Joseph, Pooja Gupta & Scaria Kadookunnel Varghese, Pp. 16402–16406 Some rare damselflies and dragonflies (Odonata: Zygoptera and Anisoptera) in Ukraine: new records, notes on distribution, and habitat preferences – Alexander V. Martynov, Pp. 16279–16294 Floristic diversity of Anjaneri Hills, Maharashtra, India – Sanjay Gajanan Auti, Sharad Suresh Kambale, Kumar Vinod Chhotupuri Gosavi & Arun Nivrutti Chandore, Pp. 16295–16313 A checklist of macrofungi (mushroom) diversity and distribution in the forests of Tripura, India — Sanjit Debnath, Ramesh Chandra Upadhyay, Rahul Saha, Koushik Majumdar, Panna Das & Ajay Krishna Saha, Pp. 16314–16346 A checklist of macrofungi (mushroom) diversity and distribution in the forests of Tripura, India Threatened Taxa Publisher & Host Publisher & Host — Sanjit Debnath, Ramesh Chandra Upadhyay, Rahul Saha, Koushik Majumdar, Panna Das & Ajay Krishna Saha, Pp. 16314–16346 Member Threatened Taxa
https://openalex.org/W2280654418	https://europepmc.org/articles/pmc2971844?pdf=render	Latin	null	Tetrakis(μ<sub>2</sub>-5-methylpyrazine-2-carboxylato)-1:2κ<sup>3</sup><i>N</i><sup>1</sup>,<i>O</i>:<i>O</i>;2:3κ<sup>3</sup><i>O</i>:<i>N</i><sup>1</sup>,<i>O</i>;1:2κ<sup>2</sup><i>O</i>:<i>O</i>′;3:4κ<sup>2</sup><i>O</i>:<i>O</i>′-octaoctyl-1κ<sup>2</sup><i>C</i>,2κ<sup>2</sup><i>C</i>,3κ<sup>2</sup><i>C</i>,4κ<sup>2</sup><i>C</i>-di-μ<sub>3</sub>-oxido-1:2:3κ<sup>3</sup><i>O</i>;1:3:4κ<sup>3</sup><i>O</i>-tetratin(IV)	Acta crystallographica. Section E	2,009	cc-by	5,518	Zhongjun Gaoa* and Fahui Lib aDepartment of Chemistry, Jining University, Shandong 273155, People’s Republic of China, and bMarine Drug and Food Institute, Ocean University of China, Qingdao 266003, People’s Republic of China Correspondence e-mail: zhongjungao@yahoo.cn Received 3 November 2009; accepted 9 November 2009 Key indicators: single-crystal X-ray study; T = 295 K; mean (C–C) = 0.025 A˚; R factor = 0.064; wR factor = 0.223; data-to-parameter ratio = 17.4. Table 1 Selected bond lengths (A˚ ). Sn1—O5 1.993 (6) Sn1—C21 2.133 (16) Sn1—C13 2.135 (13) Sn1—O4i 2.158 (7) Sn1—O1 2.202 (6) Sn2—C37 2.090 (12) Sn2—C29 2.093 (13) Sn2—O5i 2.111 (6) Sn2—O5 2.151 (6) Sn2—O1 2.455 (7) Sn2—O3 2.472 (7) Symmetry code: (i) x þ 1; y þ 2; z þ 2. The title compound, [Sn4(C8H17)8O2(C6H5N2O2)4], is a tetra- nuclear SnIV complex, built up by inversion symmetry around the central Sn2O2 ring. The SnIV coordination geometries are distorted SnO3C2 trigonal-bipyramidal and distorted SnO4C2 octahedral. The three-coordinate 3-oxido bridging O atom in the Sn2O2 ring is attached to three Sn atoms. All non-H atoms, with the exception of the Sn-bonded octyl groups, lie approximately on a non-crystallographic mirror plane. Data collection: SMART (Bruker 1998); cell reﬁnement: SAINT (Bruker 1998); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXL97 and PLATON (Spek, 2009). Related literature For biological activity of organotin derivatives of carboxylic acid ligands, see: Gielen et al. (1988). For related 3-oxo bridged SnIV structures, see: Vollano et al. (1984); Yin et al. (2003). We acknowledge the ﬁnancial support of the Science Foundation of Shandong. Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: SI2220). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: SI2220). metal-organic compounds metal-organic compounds Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Experimental Crystal data [Sn4(C8H17)8O2(C6H5N2O2)4] Mr = 1960.97 Triclinic, P1 a = 12.406 (4) A˚ b = 13.282 (4) A˚ c = 16.223 (5) A˚ = 76.623 (4) = 73.361 (4) = 86.522 (3) V = 2491.7 (13) A˚ 3 Z = 1 Mo K radiation = 1.05 mm1 T = 295 K 0.63 0.54 0.49 mm Data collection Bruker SMART CCD diffractometer Absorption correction: multi-scan (SADABS; Sheldrick, 1996) Tmin = 0.602, Tmax = 0.623 12764 measured reﬂections 8613 independent reﬂections 4935 reﬂections with I > 2(I) Rint = 0.041 Reﬁnement R[F 2 > 2(F 2)] = 0.064 wR(F 2) = 0.223 S = 1.11 8613 reﬂections 496 parameters H-atom parameters constrained max = 3.10 e A˚ 3 min = 1.14 e A˚ 3 = 86.522 (3) V = 2491.7 (13) A˚ 3 Z = 1 Mo K radiation = 1.05 mm1 T = 295 K 0.63 0.54 0.49 mm ISSN 1600-5368 Tetrakis(l2-5-methylpyrazine-2-carboxyl- ato)-1:2j3N1,O:O;2:3j3O:N1,O;- 1:2j2O:O000;3:4j2O:O000-octaoctyl- 1j2C,2j2C,3j2C,4j2C-di-l3-oxido- 1:2:3j3O;1:3:4j3O-tetratin(IV) Bruker, (1998). SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Gielen, M., Vanbellinghen, C., Gelan, J. & Willem, R. (1988). Bull. Soc. Chim. Belg. 97, 873–876. Sheldrick, G. M. (1996). SADABS. University of Go¨ttingen, Germany. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Vollano, J. F., Day, R. O. & Holmes, R. R. (1984). Organometallics, 3, 745–750. Yin, H. D., Wang, C. H. & Ma, C. L. (2003). Chin. J. Org. Chem. 23, 475–478. References Bruker, (1998). SMART and SAINT. Bruker AXS Inc., Madison, Wisconsin, USA. Gielen, M., Vanbellinghen, C., Gelan, J. & Willem, R. (1988). Bull. Soc. Chim. Belg. 97, 873–876. Gao and Li m1579 Gao and Li m1579 Acta Cryst. (2009). E65, m1579 doi:10.1107/S1600536809047345 Experimental A mixture of dicapryltin oxide (2.0 mmol,0.722 g) and 2-methylpyrazine-5-carboxylic acid (2.0 mmol, 0.276 g) in methanol (80 ml) was heated under reflux for 8 h. The obtained clear solution was evaporated under vacuum. The product was crystallized from a mixture of dichloromethane/ethanol (1:1) to yield blocks of (I). Yield 0.804 g, 82%, m.p. 368 K, analysis, calculated for C88H156N8O10Sn4: C 53.90, H, 8.02; N 5.71%; found: C 53.92, H 8.09, N, 5.76%. Comment Self-assembled organotin derivatives of carboxylic acid ligands have been extensively studied due to their biological activ- ities (Gielen, et al., 1988). 2-Methylpyrazine-5-carboxylic acid is a good bridging ligand that can sometimes be used to generate unexpected and interesting coordination polymers, and small changes in experimental conditions can lead to very different architectures. The title compound, (Fig. 1), is a tetranuclear tin(IV) complex containing a total of 110 non-H atoms. The molecule is centrosymmetric with a central core Sn2O2; the structure is similar to those seen previously in resemble compounds (Yin et al., 2003). The µ3-bridging O5 atom in the Sn2O2 ring is also attached to a capryl2SnO2 unit (capryl is the trivial name of the octyl group). In addition, the carboxylate group coordinates to two Sn atoms in a bridging mode. Sn—O and Sn—C bond lenghts are shown in Table 1. The geometries of both the exocyclic Sn atoms are distorted trigonal- bipyramidal. For the Sn1, atoms O1 and O7 are in axial positions [O1—Sn1—O4 = 168.0 (3)°] and the C atoms of the two capryl groups and O5 are in equatorial positions. The sum of the equatorial C—Sn—C and O—Sn—C angles is 339°, indicating a significant distortion from coplanarity for these atoms. The geometry around the endocyclic atom Sn2 is different from that of Sn1 and is a distorted octahedron. Here, O3 and O5 are in axial positions [O3—Sn2—O5 =158.9 (3)°] and the C atoms of the two capryl groups, O5i [symmetry code: -x + 1, -y + 2, -z + 2] and O1 are in equatorial positions. The sum of the equatorial C—Sn—C and O—Sn—C angles is 353.7°, indicating a significant distortion from coplanarity for these atoms. This distortion may arise because of a short Sn2···N1 contact of 2.829 (6)Å (sum of the van der Waals radii = 3.81 Å). Related µ3 oxo-bridhed tin(IV) ladder structures were reported by Vollano et al., 1984) (Fig. 2). H atoms were positioned geometrically [0.93 (CH), 0.97 (CH2) and 0.96 (CH3) Å] and constrained to ride on their parent atoms with Uiso(H) = 1.2(1.5 for methyl)Ueq(C/N). supplementary materials supplementary materials Acta Cryst. (2009). E65, m1579 [ doi:10.1107/S1600536809047345 ] supplementary materials φ and ω scans θmax = 25.0°, θmin = 1.6° Absorption correction: multi-scan (SADABS; Sheldrick, 1996) h = −11→14 Tmin = 0.602, Tmax = 0.623 k = −15→15 12764 measured reflections l = −19→18 θmax = 25.0°, θmin = 1.6° h = −11→14 k = −15→15 l = −19→18 Primary atom site location: structure-invariant direct methods Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.064 wR(F2) = 0.223 S = 1.11 8613 reflections 496 parameters 0 restraints Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo 2) + (0.0784P)2 + 14.9991P] where P = (Fo 2 + 2Fc 2)/3 (Δ/σ)max = 0.001 Δρmax = 3.10 e Å−3 Δρmin = −1.14 e Å−3 Refinement H atoms were positioned geometrically [0.93 (CH), 0.97 (CH2) and 0.96 (CH3) Å] and constrained to ride on their parent atoms with Uiso(H) = 1.2(1.5 for methyl)Ueq(C/N). sup-1 supplementary materials Figures Fig. 1. The molecular structure of (I) with 30% displacement ellipsoids (H atoms omitted for clarity) and the short Sn···N contacts indicated by dashed lines. Symmetry codes as in Table 1. Fig. 2. The same view as in Figure 1, but the n-capryl groups have been omitted for clarity. Tetrakis(µ2-5-methylpyrazine-2-carboxylato)- 1:4κ3N1,O:O;2:3κ3O:N1,O; 1:2κ2O:O';3:4κ2O:4κO'-octaoctyl- 1κ2C,2κ2C,3κ2C,4κ2C-di-µ3-oxido- 1:2:3κ3O;1:3:4κ3O-tetratin(IV) Fig. 1. The molecular structure of (I) with 30% displacement ellipsoids (H atoms omitted for clarity) and the short Sn···N contacts indicated by dashed lines. Symmetry codes as in Table 1. Fig. 1. The molecular structure of (I) with 30% displacement ellipsoids (H atoms omitted for clarity) and the short Sn···N contacts indicated by dashed lines. Symmetry codes as in Table 1. Tetrakis(µ2-5-methylpyrazine-2-carboxylato)- 1:4κ3N1,O:O;2:3κ3O:N1,O; 1:2κ2O:O';3:4κ2O:4κO'-octaoctyl- 1κ2C,2κ2C,3κ2C,4κ2C-di-µ3-oxido- 1:2:3κ3O;1:3:4κ3O-tetratin(IV) Tetrakis(µ2-5-methylpyrazine-2-carboxylato)- 1:4κ3N1,O:O;2:3κ3O:N1,O; 1:2κ2O:O';3:4κ2O:4κO'-octaoctyl- 1κ2C,2κ2C,3κ2C,4κ2C-di-µ3-oxido- 1:2:3κ3O;1:3:4κ3O-tetratin(IV) Tetrakis(µ2-5-methylpyrazine-2-carboxylato)- 1:4κ3N1,O:O;2:3κ3O:N1,O; 1:2κ2O:O';3:4κ2O:4κO'-octaoctyl- 1κ2C,2κ2C,3κ2C,4κ2C-di-µ3-oxido- 1:2:3κ3O;1:3:4κ3O-tetratin(IV) Crystal data [Sn4(C8H17)8O2(C6H5N2O2)4] Z = 1 Mr = 1960.97 F(000) = 1020 Triclinic, P1 Dx = 1.307 Mg m−3 Hall symbol: -P 1 Mo Kα radiation, λ = 0.71073 Å a = 12.406 (4) Å Cell parameters from 3961 reflections b = 13.282 (4) Å θ = 2.2–26.6° c = 16.223 (5) Å µ = 1.05 mm−1 α = 76.623 (4)° T = 295 K β = 73.361 (4)° Block, colourless γ = 86.522 (3)° 0.63 × 0.54 × 0.49 mm V = 2491.7 (13) Å3 Data collection Bruker SMART CCD diffractometer 8613 independent reflections Radiation source: fine-focus sealed tube 4935 reflections with I > 2σ(I) graphite Rint = 0.041 sup-2 supplementary materials Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance mat- rix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, convention- al R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R- factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq Sn1 0.75921 (6) 1.02020 (5) 1.00407 (5) 0.0425 (3) Sn2 0.54331 (5) 0.88620 (5) 0.97148 (5) 0.0352 (2) N1 0.6754 (7) 0.7190 (6) 0.9181 (6) 0.048 (2) N2 0.8219 (9) 0.5630 (7) 0.8661 (7) 0.064 (3) N3 0.3417 (7) 0.5913 (7) 0.9200 (7) 0.053 (3) N4 0.1270 (10) 0.5904 (8) 0.9034 (9) 0.082 (4) O1 0.7472 (6) 0.8839 (5) 0.9515 (5) 0.0458 (18) O2 0.9195 (7) 0.8183 (6) 0.9440 (6) 0.065 (2) O3 0.4103 (6) 0.7570 (5) 0.9680 (5) 0.051 (2) O4 0.2668 (6) 0.8471 (5) 0.9371 (5) 0.052 (2) O5 0.6013 (5) 1.0223 (5) 0.9969 (5) 0.0377 (16) C1 0.8261 (10) 0.8085 (8) 0.9410 (8) 0.050 (3) C2 0.7819 (10) 0.7240 (9) 0.9119 (8) 0.055 (3) sup-3 supplementary materials supplementary materials pp y 6 C38 0.6816 (11) 0.9497 (11) 0.7698 (9) 0.074 (4) H38A 0.7279 0.8882 0.7735 0.088 H38B 0.7174 1.0023 0.7858 0.088* C39 0.6774 (13) 0.9875 (12) 0.6759 (10) 0.086 (4) H39A 0.6394 0.9350 0.6617 0.104* H39B 0.6305 1.0487 0.6735 0.104* C40 0.7846 (15) 1.0136 (14) 0.6054 (11) 0.104 (5) H40A 0.8313 0.9523 0.6058 0.124* H40B 0.8238 1.0657 0.6192 0.124* C41 0.7724 (15) 1.0529 (15) 0.5138 (12) 0.109 (6) H41A 0.7236 1.0052 0.5040 0.131* H41B 0.7341 1.1189 0.5121 0.131* C42 0.8757 (16) 1.0673 (16) 0.4391 (13) 0.120 (7) H42A 0.9127 1.0009 0.4380 0.144* H42B 0.9264 1.1131 0.4490 0.144* C43 0.8560 (17) 1.1118 (17) 0.3492 (13) 0.127 (7) H43A 0.8058 1.0654 0.3394 0.153* H43B 0.8178 1.1775 0.3510 0.153* C44 0.9569 (19) 1.1282 (19) 0.2737 (15) 0.165 (10) H44A 0.9357 1.1556 0.2204 0.247* H44B 0.9949 1.0636 0.2701 0.247* H44C 1.0062 1.1763 0.2811 0.247* Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 Sn1 0.0267 (4) 0.0374 (4) 0.0702 (6) 0.0005 (3) −0.0202 (4) −0.0176 (4) Sn2 0.0282 (4) 0.0306 (4) 0.0502 (5) −0.0008 (3) −0.0144 (3) −0.0113 (3) N1 0.038 (5) 0.040 (5) 0.078 (7) 0.002 (4) −0.030 (5) −0.020 (5) N2 0.065 (7) 0.048 (6) 0.094 (9) 0.010 (5) −0.029 (6) −0.038 (6) N3 0.038 (5) 0.044 (5) 0.085 (8) 0.000 (4) −0.028 (5) −0.017 (5) N4 0.070 (8) 0.049 (6) 0.152 (12) 0.008 (6) −0.056 (8) −0.043 (7) O1 0.037 (4) 0.036 (4) 0.073 (5) 0.004 (3) −0.021 (4) −0.020 (4) O2 0.046 (5) 0.054 (5) 0.113 (8) 0.004 (4) −0.037 (5) −0.035 (5) O3 0.041 (4) 0.044 (4) 0.083 (6) −0.003 (3) −0.031 (4) −0.023 (4) O4 0.042 (4) 0.044 (4) 0.083 (6) −0.002 (4) −0.031 (4) −0.023 (4) O5 0.030 (4) 0.027 (3) 0.064 (5) −0.003 (3) −0.025 (3) −0.010 (3) C1 0.053 (7) 0.050 (7) 0.062 (8) −0.013 (6) −0.022 (6) −0.033 (6) C2 0.049 (7) 0.057 (7) 0.072 (9) 0.000 (6) −0.031 (6) −0.020 (6) C3 0.051 (8) 0.057 (8) 0.078 (9) 0.001 (6) −0.024 (7) −0.019 (7) C4 0.053 (8) 0.052 (7) 0.072 (9) −0.001 (6) −0.025 (7) −0.021 (6) C5 0.053 (8) 0.053 (7) 0.077 (9) −0.007 (6) −0.027 (7) −0.024 (7) C6 0.075 (10) 0.061 (8) 0.093 (11) −0.008 (7) −0.032 (8) −0.027 (8) C7 0.040 (6) 0.044 (6) 0.082 (9) −0.002 (5) −0.031 (6) −0.023 (6) C8 0.044 (7) 0.044 (6) 0.079 (9) −0.007 (5) −0.029 (6) −0.026 (6) C9 0.055 (8) 0.047 (7) 0.099 (11) 0.010 (6) −0.022 (7) −0.025 (7) C10 0.056 (8) 0.046 (7) 0.087 (10) −0.004 (6) −0.028 (7) −0.025 (7) C11 0.056 (8) 0.049 (7) 0.087 (10) −0.001 (6) −0.028 (7) −0.024 (7) supplementary materials sup-4 C3 0.8574 (11) 0.6445 (9) 0.8875 (9) 0.060 (3) H3 0.9324 0.6488 0.8863 0.072* C4 0.7144 (10) 0.5611 (9) 0.8713 (8) 0.056 (3) C5 0.6447 (10) 0.6354 (9) 0.8977 (8) 0.057 (3) H5 0.5690 0.6284 0.9021 0.068* C6 0.6730 (12) 0.4696 (10) 0.8488 (10) 0.073 (4) H6A 0.7351 0.4253 0.8308 0.109* H6B 0.6389 0.4933 0.8017 0.109* H6C 0.6185 0.4318 0.8998 0.109* C7 0.3250 (9) 0.7600 (8) 0.9431 (8) 0.051 (3) C8 0.2775 (9) 0.6749 (8) 0.9274 (8) 0.051 (3) C9 0.1718 (11) 0.6705 (9) 0.9178 (9) 0.066 (4) H9 0.1274 0.7291 0.9218 0.079* C10 0.1861 (11) 0.5135 (9) 0.8960 (9) 0.059 (3) C11 0.2959 (10) 0.5123 (9) 0.9025 (9) 0.061 (3) H11 0.3396 0.4540 0.8943 0.073* C12 0.1371 (12) 0.4235 (10) 0.8802 (10) 0.075 (4) H12A 0.1070 0.3754 0.9354 0.112* H12B 0.1944 0.3902 0.8420 0.112* H12C 0.0780 0.4464 0.8530 0.112* C13 0.7885 (12) 0.9313 (11) 1.1232 (9) 0.073 (4) H13A 0.7954 0.8591 1.1200 0.087* H13B 0.8596 0.9529 1.1275 0.087* C14 0.6980 (13) 0.9405 (12) 1.2060 (10) 0.081 (4) H14A 0.6257 0.9258 1.1993 0.097* H14B 0.6965 1.0114 1.2126 0.097* C15 0.7133 (13) 0.8685 (12) 1.2899 (10) 0.086 (5) H15A 0.7168 0.7976 1.2831 0.104* H15B 0.7843 0.8846 1.2981 0.104* C16 0.6188 (15) 0.8776 (13) 1.3717 (11) 0.096 (5) H16A 0.6163 0.9483 1.3790 0.116* H16B 0.5477 0.8629 1.3630 0.116* C17 0.6319 (16) 0.8052 (14) 1.4551 (12) 0.103 (5) H17A 0.7018 0.8213 1.4650 0.124* H17B 0.6366 0.7347 1.4474 0.124* C18 0.5347 (16) 0.8129 (14) 1.5359 (12) 0.110 (6) H18A 0.4646 0.7974 1.5262 0.132* H18B 0.5305 0.8830 1.5446 0.132* C19 0.5503 (17) 0.7374 (16) 1.6189 (13) 0.121 (7) H19A 0.5559 0.6673 1.6100 0.145* H19B 0.6194 0.7538 1.6297 0.145* C20 0.4504 (19) 0.7450 (17) 1.6984 (14) 0.145 (8) H20A 0.4610 0.6981 1.7501 0.218* H20B 0.3824 0.7273 1.6879 0.218* H20C 0.4452 0.8145 1.7070 0.218* C21 0.8774 (14) 1.0941 (13) 0.8843 (11) 0.098 (5) H21A 0.9513 1.0669 0.8859 0.117* H21B 0.8596 1.0736 0.8360 0.117* C22 0.8853 (17) 1.2086 (15) 0.8628 (13) 0.125 (7) sup-4 supplementary materials H22A 0.9007 1.2301 0.9116 0.150* H22B 0.8127 1.2366 0.8582 0.150* C23 0.9729 (17) 1.2552 (16) 0.7797 (14) 0.127 (7) H23A 0.9758 1.3290 0.7758 0.152* H23B 1.0453 1.2270 0.7848 0.152* C24 0.9587 (17) 1.2402 (16) 0.6965 (14) 0.125 (7) H24A 0.8811 1.2543 0.6962 0.150* H24B 0.9735 1.1683 0.6935 0.150* C25 1.0353 (18) 1.3088 (16) 0.6151 (15) 0.131 (7) H25A 1.0141 1.3804 0.6147 0.158* H25B 1.1120 1.3010 0.6194 0.158* C26 1.0324 (18) 1.2861 (17) 0.5299 (15) 0.131 (7) H26A 0.9557 1.2926 0.5260 0.157* H26B 1.0554 1.2150 0.5296 0.157* C27 1.108 (2) 1.3568 (18) 0.4499 (16) 0.144 (8) H27A 1.0823 1.4274 0.4494 0.173* H27B 1.1834 1.3527 0.4559 0.173* C28 1.111 (2) 1.3339 (19) 0.3636 (17) 0.166 (10) H28A 1.1636 1.3801 0.3170 0.249* H28B 1.0379 1.3431 0.3547 0.249* H28C 1.1350 1.2637 0.3635 0.249* C29 0.5414 (11) 0.7814 (10) 1.0901 (9) 0.068 (4) H29A 0.5512 0.7128 1.0778 0.082* H29B 0.6074 0.7957 1.1062 0.082* C30 0.4425 (13) 0.7758 (12) 1.1704 (10) 0.083 (4) H30A 0.3759 0.7564 1.1576 0.099* H30B 0.4297 0.8438 1.1837 0.099* C31 0.4592 (14) 0.6980 (12) 1.2516 (11) 0.089 (5) H31A 0.4744 0.6306 1.2374 0.107* H31B 0.5249 0.7187 1.2650 0.107* C32 0.3609 (15) 0.6884 (13) 1.3323 (11) 0.101 (5) H32A 0.2954 0.6660 1.3198 0.122* H32B 0.3446 0.7558 1.3463 0.122* C33 0.3816 (16) 0.6122 (14) 1.4119 (12) 0.109 (6) H33A 0.3944 0.5443 1.3984 0.131* H33B 0.4497 0.6327 1.4219 0.131* C34 0.2882 (17) 0.6041 (16) 1.4950 (13) 0.121 (6) H34A 0.2200 0.5815 1.4865 0.145* H34B 0.2742 0.6717 1.5091 0.145* C35 0.3176 (18) 0.5265 (16) 1.5727 (14) 0.131 (7) H35A 0.3170 0.4565 1.5643 0.157* H35B 0.3924 0.5412 1.5745 0.157* C36 0.232 (2) 0.5354 (19) 1.6591 (16) 0.169 (10) H36A 0.2496 0.4865 1.7071 0.253* H36B 0.1580 0.5211 1.6569 0.253* H36C 0.2342 0.6042 1.6679 0.253* C37 0.5683 (10) 0.9247 (10) 0.8349 (8) 0.061 (3) H37A 0.5380 0.8677 0.8202 0.073* H37B 0.5208 0.9840 0.8230 0.073* sup-5 supplementary materials Atomic displacement parameters (Å2) sup-6 supplementary materials sup C12 0.081 (10) 0.058 (8) 0.102 (12) −0.011 (7) −0.041 (9) −0.029 (8) C13 0.072 (9) 0.075 (9) 0.080 (10) 0.000 (8) −0.034 (8) −0.020 (8) C14 0.079 (11) 0.084 (10) 0.085 (11) 0.004 (8) −0.036 (9) −0.016 (9) C15 0.086 (11) 0.088 (11) 0.088 (12) 0.000 (9) −0.032 (10) −0.015 (9) C16 0.095 (13) 0.098 (12) 0.094 (13) −0.002 (10) −0.030 (11) −0.014 (10) C17 0.102 (14) 0.103 (13) 0.099 (14) −0.003 (11) −0.028 (11) −0.012 (11) C18 0.109 (15) 0.112 (14) 0.103 (15) −0.006 (12) −0.026 (12) −0.011 (12) C19 0.118 (17) 0.122 (16) 0.109 (16) −0.007 (13) −0.022 (13) −0.008 (13) C20 0.14 (2) 0.15 (2) 0.121 (18) −0.015 (16) −0.015 (15) −0.012 (15) C21 0.085 (12) 0.093 (12) 0.103 (13) −0.013 (10) −0.011 (10) −0.015 (10) C22 0.109 (15) 0.117 (16) 0.120 (17) −0.015 (13) −0.002 (13) −0.005 (13) C23 0.112 (16) 0.122 (16) 0.120 (18) −0.017 (13) −0.005 (14) −0.005 (14) C24 0.109 (16) 0.124 (16) 0.119 (18) −0.017 (13) −0.008 (14) −0.007 (14) C25 0.116 (17) 0.130 (17) 0.122 (18) −0.016 (14) −0.008 (14) −0.003 (15) C26 0.116 (17) 0.130 (17) 0.120 (18) −0.015 (14) −0.007 (14) −0.006 (15) C27 0.131 (19) 0.14 (2) 0.13 (2) −0.016 (16) −0.008 (16) −0.004 (17) C28 0.15 (2) 0.17 (2) 0.14 (2) −0.020 (18) −0.001 (18) −0.008 (19) C29 0.066 (9) 0.067 (8) 0.080 (10) −0.002 (7) −0.029 (8) −0.019 (7) C30 0.080 (11) 0.081 (10) 0.089 (12) 0.000 (8) −0.029 (9) −0.015 (9) C31 0.091 (12) 0.089 (11) 0.090 (12) −0.006 (9) −0.031 (10) −0.014 (10) C32 0.101 (14) 0.098 (13) 0.098 (14) −0.003 (11) −0.026 (11) −0.010 (11) C33 0.108 (15) 0.108 (14) 0.103 (15) −0.007 (12) −0.026 (12) −0.008 (12) C34 0.120 (16) 0.118 (16) 0.111 (16) −0.006 (13) −0.023 (13) −0.007 (13) C35 0.126 (18) 0.128 (17) 0.117 (18) −0.006 (14) −0.017 (14) −0.004 (14) C36 0.16 (2) 0.17 (2) 0.14 (2) −0.017 (19) −0.009 (18) −0.005 (18) C37 0.056 (8) 0.062 (8) 0.075 (9) −0.005 (6) −0.026 (7) −0.022 (7) C38 0.066 (9) 0.075 (9) 0.081 (11) −0.005 (8) −0.021 (8) −0.018 (8) C39 0.079 (11) 0.094 (11) 0.083 (12) −0.011 (9) −0.019 (9) −0.017 (9) C40 0.093 (13) 0.108 (14) 0.097 (14) −0.011 (11) −0.014 (11) −0.009 (11) C41 0.100 (14) 0.115 (14) 0.098 (15) −0.013 (11) −0.012 (12) −0.011 (12) C42 0.108 (15) 0.125 (16) 0.106 (16) −0.013 (13) −0.010 (13) −0.006 (13) C43 0.115 (17) 0.135 (18) 0.108 (17) −0.015 (14) −0.009 (14) −0.007 (14) C44 0.14 (2) 0.18 (2) 0.13 (2) −0.017 (18) 0.002 (17) 0.002 (17) Geometric parameters (Å, °) Sn1—O5 1.993 (6) C21—H21B 0.9700 Sn1—C21 2.133 (16) C22—C23 1.50 (2) Sn1—C13 2.135 (13) C22—H22A 0.9700 Sn1—O4i 2.158 (7) C22—H22B 0.9700 Sn1—O1 2.202 (6) C23—C24 1.47 (2) Sn2—C37 2.090 (12) C23—H23A 0.9700 Sn2—C29 2.093 (13) C23—H23B 0.9700 Sn2—O5i 2.111 (6) C24—C25 1.52 (2) Sn2—O5 2.151 (6) C24—H24A 0.9700 Sn2—O1 2.455 (7) C24—H24B 0.9700 Sn2—O3 2.472 (7) C25—C26 1.49 (3) Sn2—N1 2.829 (9) C25—H25A 0.9700 N1—C2 1.300 (13) C25—H25B 0.9700 sup-7 supplementary materials supplementary materials 1.334 (13) C26—C27 1.313 (14) C26—H26A 1.343 (14) C26—H26B 1.334 (13) C27—C28 1.345 (14) C27—H27A 1.229 (15) C27—H27B 1.326 (15) C28—H28A 1.363 (13) C28—H28B 1.189 (12) C28—H28C 1.231 (12) C29—C30 1.326 (12) C29—H29A 2.158 (7) C29—H29B 2.111 (6) C30—C31 1.500 (14) C30—H30A 1.418 (16) C30—H30B 0.9300 C31—C32 1.336 (16) C31—H31A 1.504 (15) C31—H31B 0.9300 C32—C33 0.9600 C32—H32A 0.9600 C32—H32B 0.9600 C33—C34 1.411 (14) C33—H33A 1.370 (15) C33—H33B 0.9300 C34—C35 1.395 (16) C34—H34A 1.478 (15) C34—H34B 0.9300 C35—C36 0.9600 C35—H35A 0.9600 C35—H35B 0.9600 C36—H36A 1.508 (19) C36—H36B 0.9700 C36—H36C 0.9700 C37—C38 1.526 (19) C37—H37A 0.9700 C37—H37B 0.9700 C38—C39 1.52 (2) C38—H38A 0.9700 C38—H38B 0.9700 C39—C40 1.51 (2) C39—H39A 0.9700 C39—H39B 0.9700 C40—C41 1.52 (2) C40—H40A 0.9700 C40—H40B 0.9700 C41—C42 1.53 (2) C41—H41A 0.9700 C41—H41B N1—C5 1.334 (13) C26—C27 N2—C4 1.313 (14) C26—H26A N2—C3 1.343 (14) C26—H26B N3—C8 1.334 (13) C27—C28 N3—C11 1.345 (14) C27—H27A N4—C10 1.229 (15) C27—H27B N4—C9 1.326 (15) C28—H28A O1—C1 1.363 (13) C28—H28B O2—C1 1.189 (12) C28—H28C O3—C7 1.231 (12) C29—C30 O4—C7 1.326 (12) C29—H29A O4—Sn1i 2.158 (7) C29—H29B O5—Sn2i 2.111 (6) C30—C31 C1—C2 1.500 (14) C30—H30A C2—C3 1.418 (16) C30—H30B C3—H3 0.9300 C31—C32 C4—C5 1.336 (16) C31—H31A C4—C6 1.504 (15) C31—H31B C5—H5 0.9300 C32—C33 C6—H6A 0.9600 C32—H32A C6—H6B 0.9600 C32—H32B C6—H6C 0.9600 C33—C34 C7—C8 1.411 (14) C33—H33A C8—C9 1.370 (15) C33—H33B C9—H9 0.9300 C34—C35 C10—C11 1.395 (16) C34—H34A C10—C12 1.478 (15) C34—H34B C11—H11 0.9300 C35—C36 C12—H12A 0.9600 C35—H35A C12—H12B 0.9600 C35—H35B C12—H12C 0.9600 C36—H36A C13—C14 1.508 (19) C36—H36B C13—H13A 0.9700 C36—H36C C13—H13B 0.9700 C37—C38 C14—C15 1.526 (19) C37—H37A C14—H14A 0.9700 C37—H37B C14—H14B 0.9700 C38—C39 C15—C16 1.52 (2) C38—H38A C15—H15A 0.9700 C38—H38B C15—H15B 0.9700 C39—C40 C16—C17 1.51 (2) C39—H39A C16—H16A 0.9700 C39—H39B C16—H16B 0.9700 C40—C41 C17—C18 1.52 (2) C40—H40A C17—H17A 0.9700 C40—H40B C17—H17B 0.9700 C41—C42 C18—C19 1.53 (2) C41—H41A C18—H18A 0.9700 C41—H41B 1.334 (13) C26—C27 1.51 (3) 1.313 (14) C26—H26A 0.9700 1.343 (14) C26—H26B 0.9700 1.334 (13) C27—C28 1.49 (3) 1.345 (14) C27—H27A 0.9700 1.229 (15) C27—H27B 0.9700 1.326 (15) C28—H28A 0.9600 1.363 (13) C28—H28B 0.9600 1.189 (12) C28—H28C 0.9600 1.231 (12) C29—C30 1.503 (19) 1.326 (12) C29—H29A 0.9700 2.158 (7) C29—H29B 0.9700 2.111 (6) C30—C31 1.532 (19) 1.500 (14) C30—H30A 0.9700 1.418 (16) C30—H30B 0.9700 0.9300 C31—C32 1.50 (2) 1.336 (16) C31—H31A 0.9700 1.504 (15) C31—H31B 0.9700 0.9300 C32—C33 1.52 (2) 0.9600 C32—H32A 0.9700 0.9600 C32—H32B 0.9700 0.9600 C33—C34 1.49 (2) 1.411 (14) C33—H33A 0.9700 1.370 (15) C33—H33B 0.9700 0.9300 C34—C35 1.55 (2) 1.395 (16) C34—H34A 0.9700 1.478 (15) C34—H34B 0.9700 0.9300 C35—C36 1.52 (3) 0.9600 C35—H35A 0.9700 0.9600 C35—H35B 0.9700 0.9600 C36—H36A 0.9600 1.508 (19) C36—H36B 0.9600 0.9700 C36—H36C 0.9600 0.9700 C37—C38 1.500 (17) 1.526 (19) C37—H37A 0.9700 0.9700 C37—H37B 0.9700 0.9700 C38—C39 1.503 (19) 1.52 (2) C38—H38A 0.9700 0.9700 C38—H38B 0.9700 0.9700 C39—C40 1.48 (2) 1.51 (2) C39—H39A 0.9700 0.9700 C39—H39B 0.9700 0.9700 C40—C41 1.50 (2) 1.52 (2) C40—H40A 0.9700 0.9700 C40—H40B 0.9700 0.9700 C41—C42 1.48 (2) 1.53 (2) C41—H41A 0.9700 0.9700 C41—H41B 0.9700 1.334 (13) C26—C27 1.313 (14) C26—H26A 1.343 (14) C26—H26B 1.334 (13) C27—C28 1.345 (14) C27—H27A 1.229 (15) C27—H27B 1.326 (15) C28—H28A 1.363 (13) C28—H28B 1.189 (12) C28—H28C 1.231 (12) C29—C30 1.326 (12) C29—H29A 2.158 (7) C29—H29B 2.111 (6) C30—C31 1.500 (14) C30—H30A 1.418 (16) C30—H30B 0.9300 C31—C32 1.336 (16) C31—H31A 1.504 (15) C31—H31B 0.9300 C32—C33 0.9600 C32—H32A 0.9600 C32—H32B 0.9600 C33—C34 1.411 (14) C33—H33A 1.370 (15) C33—H33B 0.9300 C34—C35 1.395 (16) C34—H34A 1.478 (15) C34—H34B 0.9300 C35—C36 0.9600 C35—H35A 0.9600 C35—H35B 0.9600 C36—H36A 1.508 (19) C36—H36B 0.9700 C36—H36C 0.9700 C37—C38 1.526 (19) C37—H37A 0.9700 C37—H37B 0.9700 C38—C39 1.52 (2) C38—H38A 0.9700 C38—H38B 0.9700 C39—C40 1.51 (2) C39—H39A 0.9700 C39—H39B 0.9700 C40—C41 1.52 (2) C40—H40A 0.9700 C40—H40B 0.9700 C41—C42 1.53 (2) C41—H41A 0.9700 C41—H41B sup-8 supplementary materials supplementary materials 0.9700 C42—C43 1.52 (2) 1.53 (2) C42—H42A 0.9700 0.9700 C42—H42B 0.9700 0.9700 C43—C44 1.46 (2) 0.9600 C43—H43A 0.9700 0.9600 C43—H43B 0.9700 0.9600 C44—H44A 0.9600 1.48 (2) C44—H44B 0.9600 0.9700 C44—H44C 0.9600 114.0 (5) H21A—C21—H21B 107.1 116.2 (4) C21—C22—C23 115.4 (18) 129.3 (6) C21—C22—H22A 108.4 92.8 (3) C23—C22—H22A 108.4 95.7 (5) C21—C22—H22B 108.4 88.1 (4) C23—C22—H22B 108.4 75.7 (2) H22A—C22—H22B 107.5 92.0 (5) C24—C23—C22 117.1 (19) 94.2 (4) C24—C23—H23A 108.0 168.0 (3) C22—C23—H23A 108.0 152.6 (5) C24—C23—H23B 108.0 96.3 (4) C22—C23—H23B 108.0 104.8 (4) H23A—C23—H23B 107.3 102.9 (4) C23—C24—C25 113.4 (19) 99.4 (4) C23—C24—H24A 108.9 74.7 (3) C25—C24—H24A 108.9 91.0 (4) C23—C24—H24B 108.9 82.9 (4) C25—C24—H24B 108.9 142.4 (2) H24A—C24—H24B 107.7 67.8 (2) C26—C25—C24 114.3 (19) 83.7 (4) C26—C25—H25A 108.7 81.0 (4) C24—C25—H25A 108.7 84.7 (2) C26—C25—H25B 108.7 158.9 (3) C24—C25—H25B 108.7 132.8 (2) H25A—C25—H25B 107.6 78.1 (4) C25—C26—C27 113 (2) 75.8 (4) C25—C26—H26A 108.9 158.1 (2) C27—C26—H26A 108.9 127.1 (2) C25—C26—H26B 108.9 59.3 (2) C27—C26—H26B 108.9 73.7 (2) H26A—C26—H26B 107.7 114.8 (10) C28—C27—C26 115 (2) 115.7 (7) C28—C27—H27A 108.5 129.5 (7) C26—C27—H27A 108.5 116.1 (10) C28—C27—H27B 108.5 116.2 (9) C26—C27—H27B 108.5 117.3 (11) H27A—C27—H27B 107.5 125.7 (6) C27—C28—H28A 109.5 C18—H18B 0.9700 C42—C43 1.52 (2) C19—C20 1.53 (2) C42—H42A 0.9700 C19—H19A 0.9700 C42—H42B 0.9700 C19—H19B 0.9700 C43—C44 1.46 (2) C20—H20A 0.9600 C43—H43A 0.9700 C20—H20B 0.9600 C43—H43B 0.9700 C20—H20C 0.9600 C44—H44A 0.9600 C21—C22 1.48 (2) C44—H44B 0.9600 C21—H21A 0.9700 C44—H44C 0.9600 O5—Sn1—C21 114.0 (5) H21A—C21—H21B 107.1 O5—Sn1—C13 116.2 (4) C21—C22—C23 115.4 (18) C21—Sn1—C13 129.3 (6) C21—C22—H22A 108.4 O5—Sn1—O4i 92.8 (3) C23—C22—H22A 108.4 C21—Sn1—O4i 95.7 (5) C21—C22—H22B 108.4 C13—Sn1—O4i 88.1 (4) C23—C22—H22B 108.4 O5—Sn1—O1 75.7 (2) H22A—C22—H22B 107.5 C21—Sn1—O1 92.0 (5) C24—C23—C22 117.1 (19) C13—Sn1—O1 94.2 (4) C24—C23—H23A 108.0 O4i—Sn1—O1 168.0 (3) C22—C23—H23A 108.0 C37—Sn2—C29 152.6 (5) C24—C23—H23B 108.0 C37—Sn2—O5i 96.3 (4) C22—C23—H23B 108.0 C29—Sn2—O5i 104.8 (4) H23A—C23—H23B 107.3 C37—Sn2—O5 102.9 (4) C23—C24—C25 113.4 (19) C29—Sn2—O5 99.4 (4) C23—C24—H24A 108.9 O5i—Sn2—O5 74.7 (3) C25—C24—H24A 108.9 C37—Sn2—O1 91.0 (4) C23—C24—H24B 108.9 C29—Sn2—O1 82.9 (4) C25—C24—H24B 108.9 O5i—Sn2—O1 142.4 (2) H24A—C24—H24B 107.7 O5—Sn2—O1 67.8 (2) C26—C25—C24 114.3 (19) C37—Sn2—O3 83.7 (4) C26—C25—H25A 108.7 C29—Sn2—O3 81.0 (4) C24—C25—H25A 108.7 O5i—Sn2—O3 84.7 (2) C26—C25—H25B 108.7 O5—Sn2—O3 158.9 (3) C24—C25—H25B 108.7 O1—Sn2—O3 132.8 (2) H25A—C25—H25B 107.6 C37—Sn2—N1 78.1 (4) C25—C26—C27 113 (2) C29—Sn2—N1 75.8 (4) C25—C26—H26A 108.9 O5i—Sn2—N1 158.1 (2) C27—C26—H26A 108.9 O5—Sn2—N1 127.1 (2) C25—C26—H26B 108.9 O1—Sn2—N1 59.3 (2) C27—C26—H26B 108.9 O3—Sn2—N1 73.7 (2) H26A—C26—H26B 107.7 C2—N1—C5 114.8 (10) C28—C27—C26 115 (2) C2—N1—Sn2 115.7 (7) C28—C27—H27A 108.5 C5—N1—Sn2 129.5 (7) C26—C27—H27A 108.5 C4—N2—C3 116.1 (10) C28—C27—H27B 108.5 C8—N3—C11 116.2 (9) C26—C27—H27B 108.5 C10—N4—C9 117.3 (11) H27A—C27—H27B 107.5 C1—O1—Sn1 125.7 (6) C27—C28—H28A 109.5 sup-9 supplementary materials supplementary materials 132.8 (6) C27—C28—H28B 98.3 (3) H28A—C28—H28B 135.6 (7) C27—C28—H28C 137.4 (7) H28A—C28—H28C 137.1 (3) H28B—C28—H28C 116.6 (3) C30—C29—Sn2 105.3 (3) C30—C29—H29A 122.6 (9) Sn2—C29—H29A 127.3 (11) C30—C29—H29B 109.4 (9) Sn2—C29—H29B 121.7 (10) H29A—C29—H29B 120.5 (11) C29—C30—C31 117.5 (10) C29—C30—H30A 120.7 (11) C31—C30—H30A 119.6 C29—C30—H30B 119.6 C31—C30—H30B 121.5 (10) H30A—C30—H30B 116.8 (11) C32—C31—C30 121.6 (11) C32—C31—H31A 125.0 (11) C30—C31—H31A 117.5 C32—C31—H31B 117.5 C30—C31—H31B 109.5 H31A—C31—H31B 109.5 C31—C32—C33 109.5 C31—C32—H32A 109.5 C33—C32—H32A 109.5 C31—C32—H32B 109.5 C33—C32—H32B 118.4 (9) H32A—C32—H32B 125.3 (10) C34—C33—C32 116.1 (9) C34—C33—H33A 116.8 (9) C32—C33—H33A 117.2 (10) C34—C33—H33B 126.0 (11) C32—C33—H33B 126.0 (11) H33A—C33—H33B 117.0 C33—C34—C35 117.0 C33—C34—H34A 120.5 (11) C35—C34—H34A 117.9 (12) C33—C34—H34B 121.6 (12) C35—C34—H34B 123.1 (11) H34A—C34—H34B 118.4 C36—C35—C34 118.4 C36—C35—H35A 109.5 C34—C35—H35A 109.5 C36—C35—H35B 109.5 C34—C35—H35B 109.5 H35A—C35—H35B 109.5 C35—C36—H36A 132.8 (6) C27—C28—H28B 109.5 98.3 (3) H28A—C28—H28B 109.5 135.6 (7) C27—C28—H28C 109.5 137.4 (7) H28A—C28—H28C 109.5 137.1 (3) H28B—C28—H28C 109.5 116.6 (3) C30—C29—Sn2 120.7 (9) 105.3 (3) C30—C29—H29A 107.2 122.6 (9) Sn2—C29—H29A 107.2 127.3 (11) C30—C29—H29B 107.2 109.4 (9) Sn2—C29—H29B 107.2 121.7 (10) H29A—C29—H29B 106.8 120.5 (11) C29—C30—C31 112.8 (13) 117.5 (10) C29—C30—H30A 109.0 120.7 (11) C31—C30—H30A 109.0 119.6 C29—C30—H30B 109.0 119.6 C31—C30—H30B 109.0 121.5 (10) H30A—C30—H30B 107.8 116.8 (11) C32—C31—C30 114.3 (14) 121.6 (11) C32—C31—H31A 108.7 125.0 (11) C30—C31—H31A 108.7 117.5 C32—C31—H31B 108.7 117.5 C30—C31—H31B 108.7 109.5 H31A—C31—H31B 107.6 109.5 C31—C32—C33 112.7 (15) 109.5 C31—C32—H32A 109.0 109.5 C33—C32—H32A 109.0 109.5 C31—C32—H32B 109.0 109.5 C33—C32—H32B 109.0 118.4 (9) H32A—C32—H32B 107.8 125.3 (10) C34—C33—C32 114.6 (17) 116.1 (9) C34—C33—H33A 108.6 116.8 (9) C32—C33—H33A 108.6 117.2 (10) C34—C33—H33B 108.6 126.0 (11) C32—C33—H33B 108.6 126.0 (11) H33A—C33—H33B 107.6 117.0 C33—C34—C35 110.9 (17) 117.0 C33—C34—H34A 109.5 120.5 (11) C35—C34—H34A 109.5 117.9 (12) C33—C34—H34B 109.5 121.6 (12) C35—C34—H34B 109.5 123.1 (11) H34A—C34—H34B 108.0 118.4 C36—C35—C34 109.5 (19) 118.4 C36—C35—H35A 109.8 109.5 C34—C35—H35A 109.8 109.5 C36—C35—H35B 109.8 109.5 C34—C35—H35B 109.8 109.5 H35A—C35—H35B 108.2 109.5 C35—C36—H36A 109.5 sup-10 C1—O1—Sn2 132.8 (6) C27—C28—H28B Sn1—O1—Sn2 98.3 (3) H28A—C28—H28B C7—O3—Sn2 135.6 (7) C27—C28—H28C C7—O4—Sn1i 137.4 (7) H28A—C28—H28C Sn1—O5—Sn2i 137.1 (3) H28B—C28—H28C Sn1—O5—Sn2 116.6 (3) C30—C29—Sn2 Sn2i—O5—Sn2 105.3 (3) C30—C29—H29A O2—C1—O1 122.6 (9) Sn2—C29—H29A O2—C1—C2 127.3 (11) C30—C29—H29B O1—C1—C2 109.4 (9) Sn2—C29—H29B N1—C2—C3 121.7 (10) H29A—C29—H29B N1—C2—C1 120.5 (11) C29—C30—C31 C3—C2—C1 117.5 (10) C29—C30—H30A N2—C3—C2 120.7 (11) C31—C30—H30A N2—C3—H3 119.6 C29—C30—H30B C2—C3—H3 119.6 C31—C30—H30B N2—C4—C5 121.5 (10) H30A—C30—H30B N2—C4—C6 116.8 (11) C32—C31—C30 C5—C4—C6 121.6 (11) C32—C31—H31A N1—C5—C4 125.0 (11) C30—C31—H31A N1—C5—H5 117.5 C32—C31—H31B C4—C5—H5 117.5 C30—C31—H31B C4—C6—H6A 109.5 H31A—C31—H31B C4—C6—H6B 109.5 C31—C32—C33 H6A—C6—H6B 109.5 C31—C32—H32A C4—C6—H6C 109.5 C33—C32—H32A H6A—C6—H6C 109.5 C31—C32—H32B H6B—C6—H6C 109.5 C33—C32—H32B O3—C7—O4 118.4 (9) H32A—C32—H32B O3—C7—C8 125.3 (10) C34—C33—C32 O4—C7—C8 116.1 (9) C34—C33—H33A N3—C8—C9 116.8 (9) C32—C33—H33A N3—C8—C7 117.2 (10) C34—C33—H33B C9—C8—C7 126.0 (11) C32—C33—H33B N4—C9—C8 126.0 (11) H33A—C33—H33B N4—C9—H9 117.0 C33—C34—C35 C8—C9—H9 117.0 C33—C34—H34A N4—C10—C11 120.5 (11) C35—C34—H34A N4—C10—C12 117.9 (12) C33—C34—H34B C11—C10—C12 121.6 (12) C35—C34—H34B N3—C11—C10 123.1 (11) H34A—C34—H34B N3—C11—H11 118.4 C36—C35—C34 C10—C11—H11 118.4 C36—C35—H35A C10—C12—H12A 109.5 C34—C35—H35A C10—C12—H12B 109.5 C36—C35—H35B H12A—C12—H12B 109.5 C34—C35—H35B C10—C12—H12C 109.5 H35A—C35—H35B H12A—C12—H12C 109.5 C35—C36—H36A C1—O1—Sn2 132.8 (6) C27—C28—H28B 109.5 Sn1—O1—Sn2 98.3 (3) H28A—C28—H28B 109.5 C7—O3—Sn2 135.6 (7) C27—C28—H28C 109.5 C7—O4—Sn1i 137.4 (7) H28A—C28—H28C 109.5 Sn1—O5—Sn2i 137.1 (3) H28B—C28—H28C 109.5 Sn1—O5—Sn2 116.6 (3) C30—C29—Sn2 120.7 (9) Sn2i—O5—Sn2 105.3 (3) C30—C29—H29A 107.2 O2—C1—O1 122.6 (9) Sn2—C29—H29A 107.2 O2—C1—C2 127.3 (11) C30—C29—H29B 107.2 O1—C1—C2 109.4 (9) Sn2—C29—H29B 107.2 N1—C2—C3 121.7 (10) H29A—C29—H29B 106.8 N1—C2—C1 120.5 (11) C29—C30—C31 112.8 (13) C3—C2—C1 117.5 (10) C29—C30—H30A 109.0 N2—C3—C2 120.7 (11) C31—C30—H30A 109.0 N2—C3—H3 119.6 C29—C30—H30B 109.0 C2—C3—H3 119.6 C31—C30—H30B 109.0 N2—C4—C5 121.5 (10) H30A—C30—H30B 107.8 N2—C4—C6 116.8 (11) C32—C31—C30 114.3 (14) C5—C4—C6 121.6 (11) C32—C31—H31A 108.7 N1—C5—C4 125.0 (11) C30—C31—H31A 108.7 N1—C5—H5 117.5 C32—C31—H31B 108.7 C4—C5—H5 117.5 C30—C31—H31B 108.7 C4—C6—H6A 109.5 H31A—C31—H31B 107.6 C4—C6—H6B 109.5 C31—C32—C33 112.7 (15) H6A—C6—H6B 109.5 C31—C32—H32A 109.0 C4—C6—H6C 109.5 C33—C32—H32A 109.0 H6A—C6—H6C 109.5 C31—C32—H32B 109.0 H6B—C6—H6C 109.5 C33—C32—H32B 109.0 O3—C7—O4 118.4 (9) H32A—C32—H32B 107.8 O3—C7—C8 125.3 (10) C34—C33—C32 114.6 (17) O4—C7—C8 116.1 (9) C34—C33—H33A 108.6 N3—C8—C9 116.8 (9) C32—C33—H33A 108.6 N3—C8—C7 117.2 (10) C34—C33—H33B 108.6 C9—C8—C7 126.0 (11) C32—C33—H33B 108.6 N4—C9—C8 126.0 (11) H33A—C33—H33B 107.6 N4—C9—H9 117.0 C33—C34—C35 110.9 (17) C8—C9—H9 117.0 C33—C34—H34A 109.5 N4—C10—C11 120.5 (11) C35—C34—H34A 109.5 N4—C10—C12 117.9 (12) C33—C34—H34B 109.5 C11—C10—C12 121.6 (12) C35—C34—H34B 109.5 N3—C11—C10 123.1 (11) H34A—C34—H34B 108.0 N3—C11—H11 118.4 C36—C35—C34 109.5 (19) C10—C11—H11 118.4 C36—C35—H35A 109.8 C10—C12—H12A 109.5 C34—C35—H35A 109.8 C10—C12—H12B 109.5 C36—C35—H35B 109.8 H12A—C12—H12B 109.5 C34—C35—H35B 109.8 C10—C12—H12C 109.5 H35A—C35—H35B 108.2 H12A—C12—H12C 109.5 C35—C36—H36A 109.5 sup-10 supplementary materials supplementary materials s H12B—C12—H12C 109.5 C35—C36—H36B 109.5 C14—C13—Sn1 114.5 (9) H36A—C36—H36B 109.5 C14—C13—H13A 108.6 C35—C36—H36C 109.5 Sn1—C13—H13A 108.6 H36A—C36—H36C 109.5 C14—C13—H13B 108.6 H36B—C36—H36C 109.5 Sn1—C13—H13B 108.6 C38—C37—Sn2 123.2 (9) H13A—C13—H13B 107.6 C38—C37—H37A 106.5 C13—C14—C15 114.1 (13) Sn2—C37—H37A 106.5 C13—C14—H14A 108.7 C38—C37—H37B 106.5 C15—C14—H14A 108.7 Sn2—C37—H37B 106.5 C13—C14—H14B 108.7 H37A—C37—H37B 106.5 C15—C14—H14B 108.7 C37—C38—C39 113.7 (12) H14A—C14—H14B 107.6 C37—C38—H38A 108.8 C16—C15—C14 112.7 (13) C39—C38—H38A 108.8 C16—C15—H15A 109.0 C37—C38—H38B 108.8 C14—C15—H15A 109.0 C39—C38—H38B 108.8 C16—C15—H15B 109.0 H38A—C38—H38B 107.7 C14—C15—H15B 109.0 C40—C39—C38 118.6 (14) H15A—C15—H15B 107.8 C40—C39—H39A 107.7 C17—C16—C15 113.2 (14) C38—C39—H39A 107.7 C17—C16—H16A 108.9 C40—C39—H39B 107.7 C15—C16—H16A 108.9 C38—C39—H39B 107.7 C17—C16—H16B 108.9 H39A—C39—H39B 107.1 C15—C16—H16B 108.9 C39—C40—C41 115.1 (15) H16A—C16—H16B 107.7 C39—C40—H40A 108.5 C16—C17—C18 112.6 (16) C41—C40—H40A 108.5 C16—C17—H17A 109.1 C39—C40—H40B 108.5 C18—C17—H17A 109.1 C41—C40—H40B 108.5 C16—C17—H17B 109.1 H40A—C40—H40B 107.5 C18—C17—H17B 109.1 C42—C41—C40 118.0 (17) H17A—C17—H17B 107.8 C42—C41—H41A 107.8 C17—C18—C19 111.0 (16) C40—C41—H41A 107.8 C17—C18—H18A 109.4 C42—C41—H41B 107.8 C19—C18—H18A 109.4 C40—C41—H41B 107.8 C17—C18—H18B 109.4 H41A—C41—H41B 107.2 C19—C18—H18B 109.4 C41—C42—C43 114.4 (17) H18A—C18—H18B 108.0 C41—C42—H42A 108.7 C20—C19—C18 109.9 (17) C43—C42—H42A 108.7 C20—C19—H19A 109.7 C41—C42—H42B 108.7 C18—C19—H19A 109.7 C43—C42—H42B 108.7 C20—C19—H19B 109.7 H42A—C42—H42B 107.6 C18—C19—H19B 109.7 C44—C43—C42 116 (2) H19A—C19—H19B 108.2 C44—C43—H43A 108.3 C19—C20—H20A 109.5 C42—C43—H43A 108.3 C19—C20—H20B 109.5 C44—C43—H43B 108.3 H20A—C20—H20B 109.5 C42—C43—H43B 108.3 C19—C20—H20C 109.5 H43A—C43—H43B 107.4 H20A—C20—H20C 109.5 C43—C44—H44A 109.5 H20B—C20—H20C 109.5 C43—C44—H44B 109.5 sup- 109.5 C35—C36—H36B 109.5 114.5 (9) H36A—C36—H36B 109.5 108.6 C35—C36—H36C 109.5 108.6 H36A—C36—H36C 109.5 108.6 H36B—C36—H36C 109.5 108.6 C38—C37—Sn2 123.2 (9) 107.6 C38—C37—H37A 106.5 114.1 (13) Sn2—C37—H37A 106.5 108.7 C38—C37—H37B 106.5 108.7 Sn2—C37—H37B 106.5 108.7 H37A—C37—H37B 106.5 108.7 C37—C38—C39 113.7 (12) 107.6 C37—C38—H38A 108.8 112.7 (13) C39—C38—H38A 108.8 109.0 C37—C38—H38B 108.8 109.0 C39—C38—H38B 108.8 109.0 H38A—C38—H38B 107.7 109.0 C40—C39—C38 118.6 (14) 107.8 C40—C39—H39A 107.7 113.2 (14) C38—C39—H39A 107.7 108.9 C40—C39—H39B 107.7 108.9 C38—C39—H39B 107.7 108.9 H39A—C39—H39B 107.1 108.9 C39—C40—C41 115.1 (15) 107.7 C39—C40—H40A 108.5 112.6 (16) C41—C40—H40A 108.5 109.1 C39—C40—H40B 108.5 109.1 C41—C40—H40B 108.5 109.1 H40A—C40—H40B 107.5 109.1 C42—C41—C40 118.0 (17) 107.8 C42—C41—H41A 107.8 111.0 (16) C40—C41—H41A 107.8 109.4 C42—C41—H41B 107.8 109.4 C40—C41—H41B 107.8 109.4 H41A—C41—H41B 107.2 109.4 C41—C42—C43 114.4 (17) 108.0 C41—C42—H42A 108.7 109.9 (17) C43—C42—H42A 108.7 109.7 C41—C42—H42B 108.7 109.7 C43—C42—H42B 108.7 109.7 H42A—C42—H42B 107.6 109.7 C44—C43—C42 116 (2) 108.2 C44—C43—H43A 108.3 109.5 C42—C43—H43A 108.3 109.5 C44—C43—H43B 108.3 109.5 C42—C43—H43B 108.3 109.5 H43A—C43—H43B 107.4 109.5 C43—C44—H44A 109.5 109.5 C43—C44—H44B 109.5 sup-11 supplementary materials C22—C21—Sn1 118.2 (13) H44A—C44—H44B 109.5 C22—C21—H21A 107.8 C43—C44—H44C 109.5 Sn1—C21—H21A 107.8 H44A—C44—H44C 109.5 C22—C21—H21B 107.8 H44B—C44—H44C 109.5 Sn1—C21—H21B 107.8 Symmetry codes: (i) −x+1, −y+2, −z+2. supplementary materials supplementary materials supplementary materials sup-12 supplementary materials Fig. 1 sup-13 supplementary materials Fig. 2 Fig. 2 Fig. 2 sup-14
https://openalex.org/W2221030204	https://univoak.eu/islandora/object/islandora%3A64171/datastream/PDF/view	English	null	Insights into the Role of the Habenular Circadian Clock in Addiction	Frontiers in psychiatry	2,016	cc-by	7,877	Abbreviations: CPP, conditioned preference place; DA, dopamine; DBS, deep brain stimulation; FR, fasciculus retroflexus; Hb, habenula; LHb, lateral habenula; MHb, median habenula; mPFC, medial prefrontal cortex; NAc, nucleus accumbens; PK2, prokineticine2; RMTg, rostromedian tegmentum nucleus; SCN, suprachiasmatic nucleus; VTA, ventral tegmental area. Edited by: Ryan Wellington Logan, University of Pittsburgh, USA Reviewed by: Shashank Tandon, University of Utah, USA Luigi Janiri, Università Cattolica del Sacro Cuore, Italy Anton Ilango, Leibniz Institute for Neurobiology, Germany Correspondence: Jorge Mendoza jmendoza@inci-cnrs.unistra.fr †Nora L. Salaberry and Jorge Mendoza have contributed equally to this work. Reviewed by: Shashank Tandon, University of Utah, USA Luigi Janiri, Università Cattolica del Sacro Cuore, Italy Anton Ilango, Leibniz Institute for Neurobiology, Germany Correspondence: Jorge Mendoza jmendoza@inci-cnrs.unistra.fr †Nora L. Salaberry and Jorge Mendoza have contributed equally to this work. Reviewed by: Shashank Tandon, University of Utah, USA Luigi Janiri, Università Cattolica del Sacro Cuore, Italy Anton Ilango, Leibniz Institute for Neurobiology, Germany Reviewed by: Shashank Tandon, University of Utah, USA Luigi Janiri, Università Cattolica del Sacro Cuore, Italy Anton Ilango, Leibniz Institute for Neurobiology, Germany I CNRS UPR-3212, Institute of Cellular and Integrative Neurosciences, University of Strasbourg, Strasbourg, France Drug addiction is a brain disease involving alterations in anatomy and functional neu- ral communication. Drug intake and toxicity show daily rhythms in both humans and rodents. Evidence concerning the role of clock genes in drug intake has been previously reported. However, the implication of a timekeeping brain locus is much less known. The epithalamic lateral habenula (LHb) is now emerging as a key nucleus in drug intake and addiction. This brain structure modulates the activity of dopaminergic neurons from the ventral tegmental area, a central part of the reward system. Moreover, the LHb has circadian properties: LHb cellular activity (i.e., firing rate and clock genes expression) oscillates in a 24-h range, and the nucleus is affected by photic stimulation and has ana- tomical connections with the main circadian pacemaker, the suprachiasmatic nucleus. Here, we describe the current insights on the role of the LHb as a circadian oscillator and its possible implications on the rhythmic regulation of the dopaminergic activity and drug intake. These data could inspire new strategies to treat drug addiction, considering circadian timing as a principal factor. Keywords: circadian system, habenula, drug of abuse, addiction, dopamine Edited by: Edited by: Ryan Wellington Logan, University of Pittsburgh, USA THE CIRCADIAN SYSTEM Environmental signals are rhythmic and organisms have to adapt to daily changes imposed by the day–night alternation. Species have, therefore, developed timekeeping mechanisms that are regulated by circadian (circa = close to, dien = day) clocks in almost any cell. These clocks are able to oscillate in a self-sustained manner with a periodicity close to a day (24 h), and to transmit time information to the rest of the body through specific output pathways (1).h Specialty section: This article was submitted to Addictive Disorders and Behavioral Dyscontrol, a section of the journal Frontiers in Psychiatry Received: 04 September 2015 Accepted: 07 December 2015 Published: 05 January 2016 Citation: Salaberry NL and Mendoza J (2016) Insights into the Role of the Habenular Circadian Clock in Addiction. Front. Psychiatry 6:179. doi: 10.3389/fpsyt.2015.00179 Specialty section: This article was submitted to Addictive Disorders and Behavioral Dyscontrol, a section of the journal Frontiers in Psychiatry Received: 04 September 2015 Accepted: 07 December 2015 Published: 05 January 2016 Citation: Salaberry NL and Mendoza J (2016) Insights into the Role of the Habenular Circadian Clock in Addiction. Front. Psychiatry 6:179. doi: 10.3389/fpsyt.2015.00179 Specialty section: This article was submitted to Addictive Disorders and Behavioral Dyscontrol, a section of the journal Frontiers in Psychiatry Received: 04 September 2015 Accepted: 07 December 2015 Published: 05 January 2016 The mammalian hypothalamus contains the principal circadian clock in the suprachiasmatic nucleus [SCN; (2)], which controls most of the behavioral and physiological rhythms (e.g., locomo- tor activity, hormone secretion). In every SCN cell, the clockwork is dependent on an oscillatory mechanism formed from transcription-translational feedback loops in which the expression of clock genes like, Clock, Bmal1, Per (1–3), Cry (1–2), and Rev-erbα, and their respective proteins, runs around 24 h (3). Mini Review published: 05 January 2016 doi: 10.3389/fpsyt.2015.00179 The LHb in the Dopamine Circuitry: Implications in Addictionh The reward system is an intricate network involving diverse struc- tures. The keystone of this circuit is the communication between the ventral tegmental area (VTA) and the nucleus accumbens (NAc). Dopaminergic neurons of the lateral VTA, which project to the lateral part of the NAc, encode for reward or for aversion depending on the dopamine (DA) receptors involved (39). Few studies reported NAc projections to LHb cells (40), although this has to be confirmed. Furthermore, the medial prefrontal cortex (mPFC) receives DAergic fibers from the medial VTA and sends glutamatergic projections to the NAc and LHb [(36, 39, 41); Figure 1].h In rats, cocaine self-administration or alcohol intake shows a daily rhythm with higher consumption during the night (20–22). Nonetheless, for cocaine, daily rhythms are observed only when access is restricted to two or three intakes per hour. Over three intakes/h drug intake increases and the rhythm is disrupted (20). Interestingly, under constant darkness conditions, a circadian rhythm of cocaine intake emerges with a higher consumption at night, indicating that an endogenous clock regulates the circadian activity of cocaine intake (23). The VTA is known to be a DAergic source, although some VTA neurons also contain GABA and glutamate. In this regard, some studies reported that the LHb is innervated by DAergic fibers and also by glutamate and GABAergic projections from the medial and lateral part of the VTA, respectively (42–45). In humans, there is a functional correlation between the LHb and VTA during aversion process, indicating a strong coupling of these structures (46). Thus, VTA-GABAergic transmission to the LHb may encode reward, while glutamatergic transmission results in aversion [(47); Figures 1A,B]. Circadian genes from the molecular clockwork regulate drug intake and drug-related behaviors. Mutations of the genes Per2 or Clock (e.g., ClockΔ19 mice) lead to higher behavioral responses (e.g., CPP and behavioral sensitization) to chronic injections of cocaine, and an increase in alcohol consumption (15, 24–26). Interestingly, animals lacking the gene Per1 or Npas2 (a homolog of the Clock gene) are behaviorally less sensitive to cocaine (15, 27), showing opposite effects to drug intake to those observed in Per2 and Clock mutants. Together, these data suggest an important link between drug intake, the circadian system, and the master clock in the SCN. THE HABENULA In humans and animals, drug intake often results in disruptions of daily rhythms [e.g., locomotor activity, sleep–wake cycle, eating habits; (5, 8)]. In rodents, several drugs of abuse like metham- phetamine, cocaine, ethanol, and morphine affect the period of circadian rhythms of behavior and physiology [e.g., locomotion, body temperature (5, 8, 9)]. The habenula (Hb) is a brain region, which with the pineal gland forms the epithalamus. It is an interesting structure involved in the control of behavior (32). The Hb is located dorsally along the third ventricle close to the dorso-medial thalamus. It is mainly divided into two regions: the median Hb (MHb) and the lateral Hb (LHb) nuclei. Both regions receive forebrain information via the fibers of the stria medullaris and project to the midbrain via the efferent fibers of the fasciculus retroflexus (FR). The MHb and LHb contain sub-nuclei that differ in their receptors, cell morphology, anatomical and functional inputs and outputs, and neurotransmitters (33–36).h Furthermore, people with perturbations of the circadian system [e.g., shift-workers, people experiencing frequent jet-lags; (10)], or animal models with disruptions of the body clock [e.g., chronic jet-lag exposure; (11)] show an increase of drug intake (e.g., psychostimulants). For example, shift-workers use metham- phetamine to avoid sleepiness and perform tasks accurately dur- ing their working hours. Methamphetamine intake occurs mainly at the beginning of the active phase regardless of the day–night period (10), suggesting that drug intake may have a synchronizer role for the circadian clock (8). The LHb is a relay structure and, according to Hikosaka (37), its role is mainly related to the suppression of motor activity. However, several studies showed that the LHb is involved in other brain functions, such as reward, aversion, cognition, maternal behavior, sleep, and circadian rhythms, and in brain dysfunc- tions, such as addiction, depression, and schizophrenia (35–38). Reciprocally, the circadian system modulates the behavioral responses to drug intake. In some clinical reports drug overdoses and toxicity have been showed to be clock-dependent (12–14). In rodents, behavioral sensitization and reward responses (measured by a conditioned place preference, CPP) to chronic injections of cocaine are significantly higher when animals are treated in the early day than at dusk or night (15, 16). Furthermore, some studies showed that the hormone melatonin, which is secreted at night, modulates the day–night variations in cocaine sensitiza- tion and CPP in mice (16–19). Citation: Front. Psychiatry 6:179. doi: 10.3389/fpsyt.2015.00179 January 2016 \| Volume 6 \| Article 179 Frontiers in Psychiatry \| www.frontiersin.org 1 Clocks, Drugs, and the Habenula Salaberry and Mendoza (28). More importantly, in brain structures regulating reward and behavior, circadian oscillations with a similar molecular machinery to the SCN have been reported (28–30). This suggests that, beyond the SCN, a multi-oscillatory circadian system in the mammalian brain composed of the reward centers, inter alia, may regulate behavior and drug intake (31). Light is the most important synchronizer for the SCN, this relays on a direct pathway from the photosensitive retinal gan- glion cells that use the photopigment melanopsin (4). Besides light stimulation, non-photic time cues, such as exercise, food restriction, or drug intake, are able to affect or synchronize the SCN clock activity as well (5–7). Frontiers in Psychiatry \| www.frontiersin.org The LHb in the Dopamine Circuitry: Implications in Addictionh LHb neurons, which are mainly glutamatergic, are activated by an aversive cue, an unrewarding task or an error of prediction (i.e., absence of expected reward), and they are inhibited by an expected or unexpected reward (32, 48). Interestingly, DAergic VTA neurons behave the other way round in the presence of a reward or an aversive stimulus (32, 49, 50). Glutamatergic LHb Although the SCN is the principal pacemaker, other circa- dian oscillators in the brain and peripheral organs are present January 2016 \| Volume 6 \| Article 179 2 Salaberry and Mendoza Clocks, Drugs, and the Habenula i it f d d i f th FIGURE 1 \| Simplified circuitry of reward and aversive responses of the LHb to drug intake. (A) Acute injections of cocaine activate the lateral VTA inducing DA release to the lateral NAc (shell) leading in a reward response. The LHb receives GABAergic and DAergic fibers from the VTA that inhibit the LHb. (B) Cocaine also has a delayed aversive effect that follows the reward response. Here, the LHb is activated by several glutamatergic excitatory inputs from BG, mPFC, and medial VTA that will be facilitated by cocaine. BG, basal ganglia; Med, median; mPFC, medial prefrontal cortex; Lat, lateral. LHb to drug intake. (A) Acute injections of cocaine activate the lateral VTA inducing FIGURE 1 \| Simplified circuitry of reward and aversive responses of the LHb to drug intake. (A) Acute injections of cocaine activate the lateral VTA inducing DA release to the lateral NAc (shell) leading in a reward response. The LHb receives GABAergic and DAergic fibers from the VTA that inhibit the LHb. (B) Cocaine also has a delayed aversive effect that follows the reward response. Here, the LHb is activated by several glutamatergic excitatory inputs from BG, mPFC, and medial VTA that will be facilitated by cocaine. BG, basal ganglia; Med, median; mPFC, medial prefrontal cortex; Lat, lateral. are no more rewarding effects (60). Voluntary ethanol intake is higher in rats with LHb lesions than that in sham control animals (61). In sum, these results show the important role of the LHb in the regulation of drug-directed behaviors and in the mediation of drug intake effects.h projections terminate mostly on the rostromedial tegmentum nucleus (RMTg), which is a GABAergic nucleus inhibiting the lateral VTA, which then projects to the NAc (41, 51). Thus, the LHb–RMTg–VTA circuit predicts appetitive or aversive outcomes. The LHb in the Dopamine Circuitry: Implications in Addictionh There are also few LHb glutamatergic fibers directly innervating DAergic neurons of the medial VTA which modu- late the activity of the mPFC to drive aversion [(41, 43, 51); Figures 1A,B].f f The MHb seems to play a role in the regulation of behavior and drug addiction as well. Mainly the MHb has been implicated in nicotine addiction (62). However, in mice with lesions of the dorsal part of the MHb, voluntary motor activity and sucrose preference are affected, suggesting an important role of the MHb in diverse motivated behaviors (63). Moreover, MHb inhibition by the antagonists of nicotinic receptors or optogenetic manipu- lations blocks drug self-administration (e.g., cocaine, morphine, alcohol) or leads to an aversive response, respectively (62, 63).f Cocaine intake has an initial rewarding effect, which depends on the DAergic circuit to the NAc, which is followed by a negative effect in which glutamate might play an important role. Due to the close functional relationship of the LHb with the DAergic reward system, its implication in positive and negative effects of drug intake has been extensively studied. The LHb is a structure very sensitive to cocaine. A low dose of cocaine increases the glucose uptake in this region in rats (52). In vivo and in vitro cocaine exposure leads to an inhibitory (reward effect) or a biphasic firing rate response of the LHb. This LHb biphasic response (inhibitory then an excitatory effect) represents the reward and aversive states after cocaine intake, respectively [(53); Figures 1A,B]. Moreover, cocaine induces an increase of AMPA receptors in the LHb that facilitates glutamatergic inputs (from the VTA, basal ganglia, or mPFC), leading to LHb hyperactivity and hyperexcit- ability (Figure 1B). This LHb hyperactivity might have a feedback role to prevent a higher activation of the VTA [(36, 43, 54–59); Figure 1B].h Hence, habenular nuclei (LHb and MHb) may have differ- ent impact on behavior and drug intake. This difference might depend on their particular function, the circadian properties of each structure (i.e., period, phase, and amplitude), and/or the time of drug exposure. Frontiers in Psychiatry \| www.frontiersin.org The Circadian Clock in the Lateral Habenula 3V, ventral third ventricule; BNST, bed nucleus of the stria terminalis; D3V, dorsal third ventricule; OC, optic chiasma. An indirect pathway can drive the message from the SCN to the LHb by a hormonal input (e.g., melatonin), or by a neuronal pathway with an intermediate structure, such as the raphe nuclei that project to both the SCN and LHb [(77–79); Figure 2]. Interestingly, serotonin release from the raphe nuclei is also rhythmic; it modulates SCN activity and dampens excitatory inputs from the basal ganglia to the LHb (54, 80, 81). Thus, serotonin may be a good candidate to modulate the rhythmic activity of the LHb. Reciprocally, the LHb projects to the raphe nuclei (82), and drugs of abuse act on serotonin transporters (83). Serotonin levels are altered in depression and mood signs are often associated symptoms of addicted behaviors. Therefore, the mood changes during addiction may be dependent on dis- turbance in the circadian link between the LHb and the raphe nuclei (84). Other studies report the expression of Per1–2 and Clock mRNA and protein in the whole Hb complex (68, 69). Using transgenic mice in which a luciferase reporter is coupled to the protein PER2, authors showed that the expression of PER2 in a small selected part in the mid-LHb has a sustained rhythmic activity (64). Moreover, c-Fos expression in the LHb is also rhythmic in mice, hamsters, and rats (70). Interestingly, denervations of the FR, the main output of the LHb, alter the circadian rest-activity cycle in hamsters (71). Thus, the LHb clock modulates the intensity or amount of activity in the day–night rhythms of locomotion.h The LHb receives environmental light information from melanopsin ganglion cells of the retina that project to the border of the LHb; thus, some interneurons may link the retino-LHb pathway [(72); Figure 2]. In addition, firing rate of LHb cells is affected by light in vivo, and this photic response is significantly larger in amplitude in LHb cells during the night than during the day (67).hh What May Be the Clock-Outputs of the LHb to Control Rhythmic Drug Intake? The VTA expresses clock genes and tyrosine hydroxylase (the limiting enzyme in DA synthesis) co-localizes with clock proteins [REV-ERB, BMAL1, PER1; (85–87)]. DA release and cell firing in the VTA are rhythmic (88–90). Frontiers in Psychiatry \| www.frontiersin.org The Circadian Clock in the Lateral Habenula The main SCN clock contacts the LHb by a direct neural pathway in which PK2 is positioned as the main clock-output factor. However, possible indirect pathways via the pineal gland, raphe nuclei, or BNST may link the SCN to the LHb as well. The LHB modulates midbrain DA activity from the VTA, and reciprocally DA may affect LHb activity. Under normal conditions (black oscillation), this pathway may result in a circadian release of DA in the striatum and several behavioral outputs, such as motivated behavior, voluntary motor activity, or sleep–wake cycles. On the other hand, under acute cocaine conditions, circadian rhythms of the LHb may change in amplitude, phase, or period (pink oscillations), which can lead in modifications of the VTA activity. However, under chronic cocaine situations beyond changes in LHb oscillations, denervations of the FR (pink cross) may lead to a dysregulation in the DAergic system and related behavioral outputs. Thus, symptoms of addictive behaviors may appear due to loss of rhythmic control of LHb. 3V, ventral third ventricule; BNST, bed nucleus of the stria terminalis; D3V, dorsal third ventricule; OC, optic chiasma. FIGURE 2 \| Circadian circuitry of LHb for the control of behavior. The circadian oscillator in the LHb may be affected by light information from retinal projections (in bold), which arrive at the border of the LHb. The main SCN clock contacts the LHb by a direct neural pathway in which PK2 is positioned as the main clock-output factor. However, possible indirect pathways via the pineal gland, raphe nuclei, or BNST may link the SCN to the LHb as well. The LHB modulates midbrain DA activity from the VTA, and reciprocally DA may affect LHb activity. Under normal conditions (black oscillation), this pathway may result in a circadian release of DA in the striatum and several behavioral outputs, such as motivated behavior, voluntary motor activity, or sleep–wake cycles. On the other hand, under acute cocaine conditions, circadian rhythms of the LHb may change in amplitude, phase, or period (pink oscillations), which can lead in modifications of the VTA activity. However, under chronic cocaine situations beyond changes in LHb oscillations, denervations of the FR (pink cross) may lead to a dysregulation in the DAergic system and related behavioral outputs. Thus, symptoms of addictive behaviors may appear due to loss of rhythmic control of LHb. The Circadian Clock in the Lateral Habenula Beyond the role of the Hb on behavioral control, recent data have shown an implication of the Hb in the regula- tion of sleep and circadian rhythms. Electrophysiological recordings showed that both LHb and MHb cells present a circadian variation of their spontaneous firing rate [(64–67); Figure 2]. This rhythmic activity disappears in both the MHb and LHb in animals with a molecular circadian clock mutation (65, 66). The role of the LHb in drug intake has also been highlighted in studies using lesions. LHb-ablated animals do not extinct drug- seeking behavior while cocaine intake is not affected, suggesting that animals are not able to decrease drug-seeking even if there January 2016 \| Volume 6 \| Article 179 3 Salaberry and Mendoza Clocks, Drugs, and the Habenula FIGURE 2 \| Circadian circuitry of LHb for the control of behavior. The circadian oscillator in the LHb may be affected by light information from retinal projections (in bold), which arrive at the border of the LHb. The main SCN clock contacts the LHb by a direct neural pathway in which PK2 is positioned as the main clock-output factor. However, possible indirect pathways via the pineal gland, raphe nuclei, or BNST may link the SCN to the LHb as well. The LHB modulates midbrain DA activity from the VTA, and reciprocally DA may affect LHb activity. Under normal conditions (black oscillation), this pathway may result in a circadian release of DA in the striatum and several behavioral outputs, such as motivated behavior, voluntary motor activity, or sleep–wake cycles. On the other hand, under acute cocaine conditions, circadian rhythms of the LHb may change in amplitude, phase, or period (pink oscillations), which can lead in modifications of the VTA activity. However, under chronic cocaine situations beyond changes in LHb oscillations, denervations of the FR (pink cross) may lead to a dysregulation in the DAergic system and related behavioral outputs. Thus, symptoms of addictive behaviors may appear due to loss of rhythmic control of LHb. 3V, ventral third ventricule; BNST, bed nucleus of the stria terminalis; D3V, dorsal third ventricule; OC, optic chiasma. FIGURE 2 \| Circadian circuitry of LHb for the control of behavior. The circadian oscillator in the LHb may be affected by light information from retinal projections (in bold), which arrive at the border of the LHb. The Circadian Clock in the Lateral Habenula Moreover, the PER2 protein expression in the striatum follows a circadian rhythm (peak of expression rises at night) that is entrained by DA via D2 type receptors (91). However, in isolated cultured VTA from Per1: Luciferase rats, no oscillations were found (92). Thus, VTA circadian oscillations must be under the control of a brain self- sustained circadian oscillator. The SCN clock innervates the LHb (Figure 2). The SCN uses different neuropeptide, such as vasopressin, vasoactive intestinal peptide, and prokineticin 2 (PK2), to communicate time infor- mation to other brain structures (73). The LHb receives PK2 fibers from the SCN and contains PK2 receptors [(74); Figure 2]. A recent study reported that PK2 is able to inhibit the LHb firing rate in vitro (66). Some vasopressin fibers run around the ventral part of the LHb even in SCN-lesioned rats, suggesting that vaso- pressin source might come from other brain structure (75); the bed nucleus of the stria terminalis, or the paraventricular nucleus of the hypothalamus (76) for example. January 2016 \| Volume 6 \| Article 179 4 Salaberry and Mendoza Clocks, Drugs, and the Habenula Despite the prominent actions of DBS as a treatment, the technique still remains an invasive approach that may produce side effects. Therefore, other alternative treatments for addiction have to be considered. Day–night differences of tyrosine hydroxylase and DA transporter in striatum are dampened but not abolished in SCN- lesioned rats (93). Therefore, another circadian clock beyond the SCN regulates rhythmic activity of the DAergic system. The LHb clock could play an important role for the regulation of DAergic rhythms (Figure 2).h In chronic drug intake, degeneration of FR may be due, in part, to a reduction of the GABA receptors expression, leading to a LHb disinhibition, and an increase of glutamate receptors allowing an excitotoxic effect on LHb cells (58, 96, 99). LHb disinhibition may be restored by a GABAB receptor agonist, a molecule that is already proposed as a possible treatment for drug addiction (101). Hence, LHb inhibition by GABA agonists at the circadian optimal time could rescue a daily rhythm of LHb activity and reduce addiction states.h The circadian role of the LHb might be to inhibit DAergic neurons at a specific time during the 24-h cycle. In rats, the LHb peak of electrical activity occurs during the day, when the release of DA to the striatum is low (67, 91). CONCLUSION In this review, we addressed the circadian relevance of the LHb for the regulation of rhythmic brain activity and behavior. This work may help to understand the role of LHb, as a circadian clock, in the development of psychiatric pathologies and addic- tion. The vast literature devoted to LHb indicates that these nuclei have a relevant role in different brain functions, and dys- functions (e.g., depression and addiction) (106). Some studies also highlight the clock properties of the LHb in the regulation of behavior (70, 71, 107). Therefore, to understand the specific neurobiological role of the Hb (both the MHb and LHb nuclei) in brain physiology and pathophysiological conditions, the circadian properties of this brain locus should be considered as an important factor. Possible Therapies for Addiction: The Habenula as a Target Low frequency stimulations, which mimic inputs from the VTA or raphe nucleus to the LHb, inhibit the LHb activity and induce cocaine intake in rats (35–38, 60). However, high frequencies, which mimic aversive and excitatory input from the basal ganglia, do not reduce cocaine intake (35–38, 60). Interestingly, deep brain stimulation (DBS) is associated with reward and aversive inputs (low and high frequencies), and reduces cocaine consumption in rats (60). DBS is a technical approach that has been used for the treatment of some psychiatric disorders, such as depression and obsessive–compulsive disorder (97). The Circadian Clock in the Lateral Habenula In mice, however, there is a circadian rhythm of LHb firing rate with a peak at night time, which correlates with VTA lower electrical activity (64, 94). Thus, it is necessary to determine the circadian activity (i.e., firing rate, clock gene expression) of the LHb in vivo in both rats and mice. The Hb expresses melatonin receptors (102) and projects to the pineal gland, the site of melatonin production (103). Melatonin receptors mediate the behavioral sensitization to methamphetamine (104). Moreover, activation of melatonin receptors (with melatonin or the agonist agomelatine) reduces relapse-like alcohol intake in rats (105). More importantly, since there is a strong rhythmic activity of melatonin, it is possible that the effects of the hormone on the circadian activity of the Hb are phase (time)-dependent. Thus, melatonin might have effects to modulate drug-seeking and intake via an activation of its recep- tors in the Hb at specific times of the day. Since LHb activity controls DA neuronal activity, it is also possible that LHb modulates rhythmic behaviors related to drug intake through the regulation of the DAergic system. In fact, behavioral sensitization [which has a rhythmic profile; (15, 16)] is altered by LHb lesions in rats (95). This suggests that the LHb clock could modulate the rhythmic patterns of behavioral sensitization or reward effects of cocaine. Moreover, cocaine self- administration shows a daily rhythm in phase (with a maximum at the night) with the peak of DA release in the striatum. Both self-administration and DA release are correlated with the high- est LHb firing rate (20, 23, 67, 91). Importantly, chronic intake of cocaine, which leads to FR degeneration, is able to disrupt circadian rhythms probably due to the lack of LHb control on monoamine nuclei [(20, 96); Figure 2]. ACKNOWLEDGMENTS The LHb has been proposed as a target for DBS to treat addiction because of its close functional relationship with the reward system (98). However, chronic exposure to cocaine, amphetamine, methamphetamine, MDMA, or nicotine results in the degeneration of the FR, which blocks the effect of DBS on drug intake (96, 99, 100). As long as FR is able to transmit information, the LHb can be considered as a main target for DBS in the treatment of addiction. Moreover, if circadian properties of the LHb are an important factor for DBS, it then becomes possible to potentiate the beneficial effect, or avoid side effects of DBS, by applying stimulation at a specific time of the day and consider a genuine type of DBS chronotherapy (i.e., time- dependent treatment). We would like to thank Dr. Matthew Beymer, Dr. Domitille Boudard, and Milagros M Arietti for comments and sugges- tions on the manuscript. We thank the three reviewers for all the comments and suggestions that certainly improved our manuscript. REFERENCES Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain? Eur J Neurosci (2007) 25:3195–216. doi:10.1111/j.1460-9568.2007.05581.x 7. Hughes ATL, Piggins HD. Feedback actions of locomotor activity to the circadian clock. Prog Brain Res (2012) 199:305–36. doi:10.1016/ B978-0-444-59427-3.00018-6 30. Webb IC, Lehman MN, Coolen LM. Diurnal and circadian regulation of reward-related neurophysiology and behavior. Physiol Behav (2015) 143:58–69. doi:10.1016/j.physbeh.2015.02.034 8. Kosobud AEK, Gillman AG, Leffel JK, Pecoraro NC, Rebec GV, Timberlake W. Drugs of abuse can entrain circadian rhythms. ScientificWorldJournal (2007) 7:203–12. doi:10.1100/tsw.2007.234 31. Mendoza J, Challet E. Circadian insights into dopamine mechanisms. Neuroscience (2014) 282C:230–42. doi:10.1016/j.neuroscience.2014.07.081 9. Stowie AC, Amicarelli MJ, Prosser RA, Glass JD. Chronic cocaine causes long- term alterations in circadian period and photic entrainment in the mouse. Neuroscience (2015) 284:171–9. doi:10.1016/j.neuroscience.2014.08.057 32. Matsumoto M, Hikosaka O. Lateral habenula as a source of negative reward signals in dopamine neurons. Nature (2007) 447:1111–5. doi:10.1038/ nature05860 10. Kirkpatrick MG, Haney M, Vosburg SK, Comer SD, Foltin RW, Hart CL. Methamphetamine self-administration by humans subjected to abrupt shift and sleep schedule changes. Psychopharmacology (Berl) (2009) 203:771–80. doi:10.1007/s00213-008-1423-1 33. Aizawa H, Kobayashi M, Tanaka S, Fukai T, Okamoto H. Molecular characterization of the subnuclei in rat habenula. J Comp Neurol (2012) 520:4051–66. doi:10.1002/cne.23167 11. Doyle SE, Feng H, Garber G, Menaker M, Lynch WJ. Effects of circadian disruption on methamphetamine consumption in methamphetamine-ex- posed rats. Psychopharmacology (Berl) (2015) 232:2169–79. doi:10.1007/ s00213-014-3845-2 34. Andres KH, von Düring M, Veh RW. Subnuclear organization of the rat habenular complexes. J Comp Neurol (1999) 407:130–50. doi:10.1002/ (SICI)1096-9861(19990428)407:1<130::AID-CNE10>3.0.CO;2-8 35. Sutherland RJ. The dorsal diencephalic conduction system: a review of the anatomy and functions of the habenular complex. Neurosci Biobehav Rev (1982) 6:1–13. doi:10.1016/0149-7634(82)90003-3 12. Morris RW. Circadian and circannual rhythms of emergency room drug-overdose admissions. Prog Clin Biol Res (1987) 227B:451–7. 13. Raymond RC, Warren M, Morris RW, Leikin JB. Periodicity of presentations of drugs of abuse and overdose in an emergency department. J Toxicol Clin Toxicol (1992) 30:467–78. doi:10.3109/15563659209021561 36. Lecourtier L, Kelly PH. A conductor hidden in the orchestra? Role of the habenular complex in monoamine transmission and cognition. Neurosci Biobehav Rev (2007) 31:658–72. doi:10.1016/j.neubiorev.2007.01.004 14. Erickson TB, Lee J, Zautcke JL, Morris R. Analysis of cocaine chronotox- icology in an urban ED. Am J Emerg Med (1998) 16:568–71. doi:10.1016/ S0735-6757(98)90220-0 37. Hikosaka O. The habenula: from stress evasion to value-based decision-mak- ing. Nat Rev Neurosci (2010) 11:503–13. REFERENCES 22. Perreau-Lenz S, Vengeliene V, Noori HR, Merlo-Pich EV, Corsi MA, Corti C, et al. Inhibition of the casein-kinase-1-ε/δ/prevents relapse-like alcohol drinking. Neuropsychopharmacology (2012) 37:2121–31. doi:10.1038/ npp.2012.62 1. Pittendrigh CS. Temporal organization: reflections of a Darwinian clock-watcher. Annu Rev Physiol (1993) 55:16–54. doi:10.1146/annurev. ph.55.030193.000313 1. Pittendrigh CS. Temporal organization: reflections of a Darwinian clock-watcher. Annu Rev Physiol (1993) 55:16–54. doi:10.1146/annurev. ph.55.030193.000313 23. Bass CE, Jansen HT, Roberts DCS. Free-running rhythms of cocaine self-ad- ministration in rats held under constant lighting conditions. Chronobiol Int (2010) 27:535–48. doi:10.3109/07420521003664221 2. Welsh DK, Takahashi JS, Kay SA. Suprachiasmatic nucleus: cell autonomy and network properties. Annu Rev Physiol (2010) 72:551–77. doi:10.1146/ annurev-physiol-021909-135919 h 2. Welsh DK, Takahashi JS, Kay SA. Suprachiasmatic nucleus: cell autonomy and network properties. Annu Rev Physiol (2010) 72:551–77. doi:10.1146/ annurev-physiol-021909-135919 h 24. Spanagel R, Pendyala G, Abarca C, Zghoul T, Sanchis-Segura C, Magnone MC, et al. The clock gene Per2 influences the glutamatergic system and mod- ulates alcohol consumption. Nat Med (2005) 11:35–42. doi:10.1038/nm1163 3. Takahashi JS, Hong H-K, Ko CH, McDearmon EL. The genetics of mamma- lian circadian order and disorder: implications for physiology and disease. Nat Rev Genet (2008) 9:764–75. doi:10.1038/nrg2430 25. Falcón E, McClung CA. A role for the circadian genes in drug addic- tion. Neuropharmacology (2009) 56(Suppl 1):91–6. doi:10.1016/j. neuropharm.2008.06.054 4. Lucas RJ, Lall GS, Allen AE, Brown TM. How rod, cone, and melanopsin photoreceptors come together to enlighten the mammalian circadian clock. Prog Brain Res (2012) 199:1–18. doi:10.1016/B978-0-444-59427-3.00001-0 h 26. Ozburn AR, Falcon E, Mukherjee S, Gillman A, Arey R, Spencer S, et al. The role of clock in ethanol-related behaviors. Neuropsychopharmacology (2013) 38:2393–400. doi:10.1038/npp.2013.138 5. Honma K, Honma S. The SCN-independent clocks, methamphet- amine and food restriction. Eur J Neurosci (2009) 30:1707–17. doi:10.1111/j.1460-9568.2009.06976.x 5. Honma K, Honma S. The SCN-independent clocks, methamphet- amine and food restriction. Eur J Neurosci (2009) 30:1707–17. doi:10.1111/j.1460-9568.2009.06976.x 27. Ozburn AR, Falcon E, Twaddle A, Nugent AL, Gillman AG, Spencer SM, et al. Direct regulation of diurnal Drd3 expression and cocaine reward by NPAS2. Biol Psychiatry (2015) 77:425–33. doi:10.1016/j.biopsych.2014.07.030 6. Glass JD, Brager AJ, Stowie AC, Prosser RA. Cocaine modulates pathways for photic and nonphotic entrainment of the mammalian SCN circadian clock. Am J Physiol Regul Integr Comp Physiol (2012) 302:R740–50. doi:10.1152/ ajpregu.00602.2011 28. Albrecht U. Timing to perfection: the biology of central and peripheral cir- cadian clocks. Neuron (2012) 74:246–60. doi:10.1016/j.neuron.2012.04.006 29. Guilding C, Piggins HD. FUNDING This work was supported by Agence Nationale de la Recherche (grant ANR-14-CE13-0002-01 ADDiCLOCK JCJC to JM and NLS PhD fellow) and the Centre Nationale de la Recherche Scientifique (CNRS). January 2016 \| Volume 6 \| Article 179 Frontiers in Psychiatry \| www.frontiersin.org Clocks, Drugs, and the Habenula Salaberry and Mendoza REFERENCES doi:10.1038/nrn2866 h 38. Bianco IH, Wilson SW. The habenular nuclei: a conserved asymmetric relay station in the vertebrate brain. Philos Trans R Soc Lond B Biol Sci (2009) 364:1005–20. doi:10.1098/rstb.2008.0213 15. Abarca C, Albrecht U, Spanagel R. Cocaine sensitization and reward are under the influence of circadian genes and rhythm. Proc Natl Acad Sci U S A (2002) 99:9026–30. doi:10.1073/pnas.142039099 39. Russo SJ, Nestler EJ. The brain reward circuitry in mood disorders. Nat Rev Neurosci (2013) 14:609–25. doi:10.1038/nrn3381 16. Uz T, Javaid JI, Manev H. Circadian differences in behavioral sensitization to cocaine: putative role of arylalkylamine N-acetyltransferase. Life Sci (2002) 70:3069–75. doi:10.1016/S0024-3205(02)01559-X 40. Li YQ, Takada M, Mizuno N. Demonstration of habenular neurons which receive afferent fibers from the nucleus accumbens and send their axons to the midbrain periaqueductal gray. Neurosci Lett (1993) 158:55–8. doi:10.1016/0304-3940(93)90611-N 17. Uz T, Akhisaroglu M, Ahmed R, Manev H. The pineal gland is critical for circadian Period1 expression in the striatum and for circadian cocaine sensi- tization in mice. Neuropsychopharmacology (2003) 28:2117–23. doi:10.1038/ sj.npp.1300254 41. Lammel S, Lim BK, Ran C, Huang KW, Betley MJ, Tye KM, et al. Input- specific control of reward and aversion in the ventral tegmental area. Nature (2012) 491:212–7. doi:10.1038/nature11527 j 18. Akhisaroglu M, Ahmed R, Kurtuncu M, Manev H, Uz T. Diurnal rhythms in cocaine sensitization and in Period1 levels are common across rodent species. Pharmacol Biochem Behav (2004) 79:37–42. doi:10.1016/j.pbb.2004.06.014 42. Gruber C, Kahl A, Lebenheim L, Kowski A, Dittgen A, Veh RW. Dopaminergic projections from the VTA substantially contribute to the mesohabenular pathway in the rat. Neurosci Lett (2007) 427:165–70. doi:10.1016/j. neulet.2007.09.016 19. Kurtuncu M, Arslan AD, Akhisaroglu M, Manev H, Uz T. Involvement of the pineal gland in diurnal cocaine reward in mice. Eur J Pharmacol (2004) 489:203–5. doi:10.1016/j.ejphar.2004.03.010 43. Hnasko TS, Hjelmstad GO, Fields HL, Edwards RH. Ventral tegmental area glutamate neurons: electrophysiological properties and projections. J Neurosci (2012) 32:15076–85. doi:10.1523/JNEUROSCI.3128-12.2012 f 20. Morgan D, Roberts DCS. Sensitization to the reinforcing effects of cocaine following binge-abstinent self-administration. Neurosci Biobehav Rev (2004) 27:803–12. doi:10.1016/j.neubiorev.2003.11.004 j 21. Rosenwasser AM, Fecteau ME, Logan RW. Effects of ethanol intake and eth- anol withdrawal on free-running circadian activity rhythms in rats. Physiol Behav (2005) 84:537–42. doi:10.1016/j.physbeh.2005.01.016 44. Root DH, Mejias-Aponte CA, Zhang S, Wang H-L, Hoffman AF, Lupica CR, et al. Single rodent mesohabenular axons release glutamate and GABA. Nat Neurosci (2014) 17:1543–51. REFERENCES Eur J Neurosci (2011) 34:478–88. doi:10.1111/j.1460-9568.2011.07762.x 72. Hattar S, Kumar M, Park A, Tong P, Tung J, Yau K-W, et al. Central projections of melanopsin-expressing retinal ganglion cells in the mouse. J Comp Neurol (2006) 497:326–49. doi:10.1002/cne.20970 52. London ED, Wilkerson G, Goldberg SR, Risner ME. Effects of L-cocaine on local cerebral glucose utilization in the rat. Neurosci Lett (1986) 68:73–8. doi:10.1016/0304-3940(86)90232-6 73. Kalsbeek A, Palm IF, La Fleur SE, Scheer FA, Perreau-Lenz S, Ruiter M, et al. SCN outputs and the hypothalamic balance of life. J Biol Rhythms (2006) 21:458–69. doi:10.1177/0748730406293854 f 53. Jhou TC, Good CH, Rowley CS, Xu S-P, Wang H, Burnham NW, et al. Cocaine drives aversive conditioning via delayed activation of dopamine-re- sponsive habenular and midbrain pathways. J Neurosci (2013) 33:7501–12. doi:10.1523/JNEUROSCI.3634-12.2013 74. Zhang C, Truong KK, Zhou Q-Y. Efferent projections of prokineticin 2 expressing neurons in the mouse suprachiasmatic nucleus. PLoS One (2009) 4:e7151. doi:10.1371/journal.pone.0007151 i 54. Shabel SJ, Proulx CD, Trias A, Murphy RT, Malinow R. Input to the lateral habenula from the basal ganglia is excitatory, aversive, and suppressed by serotonin. Neuron (2012) 74:475–81. doi:10.1016/j.neuron.2012.02.037 i 75. Hoorneman EM, Buijs RM. Vasopressin fiber pathways in the rat brain following suprachiasmatic nucleus lesioning. Brain Res (1982) 243:235–41. doi:10.1016/0006-8993(82)90246-3 55. Maroteaux M, Mameli M. Cocaine evokes projection-specific synaptic plas- ticity of lateral habenula neurons. J Neurosci (2012) 32:12641–6. doi:10.1523/ JNEUROSCI.2405-12.2012 76. Hernández VS, Vázquez-Juárez E, Márquez MM, Jáuregui-Huerta F, Barrio RA, Zhang L. Extra-neurohypophyseal axonal projections from individual vasopressin-containing magnocellular neurons in rat hypothalamus. Front Neuroanat (2015) 9:130. doi:10.3389/fnana.2015.00130 56. Zuo W, Chen L, Wang L, Ye J-H. Cocaine facilitates glutamatergic transmis- sion and activates lateral habenular neurons. Neuropharmacology (2013) 70:180–9. doi:10.1016/j.neuropharm.2013.01.008 77. Leak RK, Moore RY. Topographic organization of suprachiasmatic nucleus projection neurons. J Comp Neurol (2001) 433:312–34. doi:10.1002/cne.1142 57. Neumann PA, Ishikawa M, Otaka M, Huang YH, Schlüter OM, Dong Y. Increased excitability of lateral habenula neurons in adolescent rats following cocaine self-administration. Int J Neuropsychopharmacol (2015) 18:pyu109. doi:10.1093/ijnp/pyu109 78. Kalsbeek A, Palm IF, Buijs RM. Central vasopressin systems and steroid hormones. Prog Brain Res (2002) 139:57–73. doi:10.1016/ S0079-6123(02)39007-1 79. Vertes RP, Linley SB. Efferent and afferent connections of the dorsal and median raphe nuclei in the rat. In: Monti JM, Pandi-Perumal SR, Jacobs BL, Nutt DJ, editors. Serotonin and Sleep: Molecular, Functional and Clinical Aspects. Basel: Birkhäuser (2008). p. 69–102. 58. REFERENCES doi:10.1038/nn.3823 January 2016 \| Volume 6 \| Article 179 Frontiers in Psychiatry \| www.frontiersin.org 6 Salaberry and Mendoza Clocks, Drugs, and the Habenula 45. Taylor SR, Badurek S, Dileone RJ, Nashmi R, Minichiello L, Picciotto MR. GABAergic and glutamatergic efferents of the mouse ventral tegmental area. J Comp Neurol (2014) 522:3308–34. doi:10.1002/cne.23603 65. Sakhi K, Belle MDC, Gossan N, Delagrange P, Piggins HD. Daily variation in the electrophysiological activity of mouse medial habenula neurones. J Physiol (2014) 592:587–603. doi:10.1113/jphysiol.2013.263319 66. Sakhi K, Wegner S, Belle MDC, Howarth M, Delagrange P, Brown TM, et al. Intrinsic and extrinsic cues regulate the daily profile of mouse lateral habenula neuronal activity. J Physiol (2014) 592:5025–45. doi:10.1113/ jphysiol.2014.280065 46. Hennigan K, D’Ardenne K, McClure SM. Distinct midbrain and habenula pathways are involved in processing aversive events in humans. J Neurosci (2015) 35:198–208. doi:10.1523/JNEUROSCI.0927-14.2015 47. Root DH, Mejias-Aponte CA, Qi J, Morales M. Role of glutamatergic projec- tions from ventral tegmental area to lateral habenula in aversive conditioning. J Neurosci (2014) 34:13906–10. doi:10.1523/JNEUROSCI.2029-14.2014 67. Zhao H, Rusak B. Circadian firing-rate rhythms and light responses of rat habenular nucleus neurons in vivo and in vitro. Neuroscience (2005) 132:519–28. doi:10.1016/j.neuroscience.2005.01.012 48. Matsumoto M, Hikosaka O. Representation of negative motivational value in the primate lateral habenula. Nat Neurosci (2009) 12:77–84. doi:10.1038/ nn.2233 68. Shieh K-R. Distribution of the rhythm-related genes rPERIOD1, rPERIOD2, and rCLOCK, in the rat brain. Neuroscience (2003) 118:831–43. doi:10.1016/ S0306-4522(03)00004-6 49. Matsumoto M, Hikosaka O. Two types of dopamine neuron distinctly con- vey positive and negative motivational signals. Nature (2009) 459:837–41. doi:10.1038/nature08028 69. Zhao Z, Xu H, Liu Y, Mu L, Xiao J, Zhao H. Diurnal expression of the Per2 gene and protein in the lateral habenular nucleus. Int J Mol Sci (2015) 16:16740–9. doi:10.3390/ijms160816740 50. Shumake J, Ilango A, Scheich H, Wetzel W, Ohl FW. Differential neu- romodulation of acquisition and retrieval of avoidance learning by the lateral habenula and ventral tegmental area. J Neurosci (2010) 30:5876–83. doi:10.1523/JNEUROSCI.3604-09.2010 70. Tavakoli-Nezhad M, Schwartz WJ. Hamsters running on time: is the lateral habenula a part of the clock? Chronobiol Int (2006) 23:217–24. doi:10.1080/07420520500521947 51. Brinschwitz K, Dittgen A, Madai VI, Lommel R, Geisler S, Veh RW. Glutamatergic axons from the lateral habenula mainly terminate on GABAergic neurons of the ventral midbrain. Neuroscience (2010) 168:463–76. doi:10.1016/j.neuroscience.2010.03.050 f 71. Paul MJ, Indic P, Schwartz WJ. A role for the habenula in the regu- lation of locomotor activity cycles. REFERENCES Meye FJ, Valentinova K, Lecca S, Marion-Poll L, Maroteaux MJ, Musardo S, et al. Cocaine-evoked negative symptoms require AMPA receptor trafficking in the lateral habenula. Nat Neurosci (2015) 18:376–8. doi:10.1038/nn.3923 59. Shen X, Ruan X, Zhao H. Stimulation of midbrain dopaminergic structures modifies firing rates of rat lateral habenula neurons. PLoS One (2012) 7:e34323. doi:10.1371/journal.pone.0034323 t 80. Dudley TE, DiNardo LA, Glass JD. Endogenous regulation of serotonin release in the hamster suprachiasmatic nucleus. J Neurosci (1998) 18:5045–52. 81. Glass JD, DiNardo LA, Ehlen JC. Dorsal raphe nuclear stimulation of SCN serotonin release and circadian phase-resetting. Brain Res (2000) 859:224–32. doi:10.1016/S0006-8993(00)01963-6 60. Friedman A, Lax E, Dikshtein Y, Abraham L, Flaumenhaft Y, Sudai E, et al. Electrical stimulation of the lateral habenula produces enduring inhibitory effect on cocaine seeking behavior. Neuropharmacology (2010) 59:452–9. doi:10.1016/j.neuropharm.2010.06.008 82. Aghajanian GK, Wang RY. Habenular and other midbrain raphe afferents demonstrated by a modified retrograde tracing technique. Brain Res (1977) 122:229–42. doi:10.1016/0006-8993(77)90291-8 61. Haack AK, Sheth C, Schwager AL, Sinclair MS, Tandon S, Taha SA. Lesions of the lateral habenula increase voluntary ethanol consumption and operant self-administration, block yohimbine-induced reinstatement of ethanol seeking, and attenuate ethanol-induced conditioned taste aversion. PLoS One (2014) 9:e92701. doi:10.1371/journal.pone.0092701 83. Hall FS, Sora I, Drgonova J, Li X-F, Goeb M, Uhl GR. Molecular mecha- nisms underlying the rewarding effects of cocaine. Ann N Y Acad Sci (2004) 1025:47–56. doi:10.1196/annals.1316.006 84. Zhao H, Zhang B-L, Yang S-J, Rusak B. The role of lateral habenula-dorsal raphe nucleus circuits in higher brain functions and psychiatric illness. Behav Brain Res (2015) 277:89–98. doi:10.1016/j.bbr.2014.09.016 62. Viswanath H, Carter AQ, Baldwin PR, Molfese DL, Salas R. The medial habenula: still neglected. Front Hum Neurosci (2014) 7:931. doi:10.3389/ fnhum.2013.00931 85. McClung CA, Sidiropoulou K, Vitaterna M, Takahashi JS, White FJ, Cooper DC, et al. Regulation of dopaminergic transmission and cocaine reward by the Clock gene. Proc Natl Acad Sci U S A (2005) 102:9377–81. doi:10.1073/ pnas.0503584102 63. Hsu Y-WA, Wang SD, Wang S, Morton G, Zariwala HA, de la Iglesia HO, et al. Role of the dorsal medial habenula in the regulation of voluntary activity, motor function, hedonic state, and primary reinforcement. J Neurosci (2014) 34:11366–84. doi:10.1523/JNEUROSCI.1861-14.2014 86. Imbesi M, Yildiz S, Dirim Arslan A, Sharma R, Manev H, Uz T. Dopamine receptor-mediated regulation of neuronal “clock” gene expression. Neuroscience (2009) 158:537–44. doi:10.1016/j.neuroscience.2008.10.044 64. Guilding C, Hughes ATL, Piggins HD. Circadian oscillators in the epithalamus. Neuroscience (2010) 169:1630–9. REFERENCES doi:10.1016/j.neuroscience.2010.06.015 January 2016 \| Volume 6 \| Article 179 Frontiers in Psychiatry \| www.frontiersin.org 7 Salaberry and Mendoza Clocks, Drugs, and the Habenula control circuitry. Eur Neuropsychopharmacol (2002) 12:287–97. doi:10.1016/ S0924-977X(02)00020-2 87. Chung S, Lee EJ, Yun S, Choe HK, Park S-B, Son HJ, et al. Impact of circadian nuclear receptor REV-ERBα on midbrain dopamine production and mood regulation. Cell (2014) 157:858–68. doi:10.1016/j.cell.2014.03.039 control circuitry. Eur Neuropsychopharmacol (2002) 12:287–97. doi:10.1016/ S0924-977X(02)00020-2 100. Lax E, Friedman A, Croitoru O, Sudai E, Ben-Moshe H, Redlus L, et al. Neurodegeneration of lateral habenula efferent fibers after intermittent cocaine administration: implications for deep brain stimulation. Neuropharmacology (2013) 75:246–54. doi:10.1016/j.neuropharm.2013.06.034 88. Schade R, Vick K, Ott T, Sohr R, Pfister C, Bellach J, et al. Circadian rhythms of dopamine and cholecystokinin in nucleus accumbens and striatum of rats – influence on dopaminergic stimulation. Chronobiol Int (1995) 12:87–99. doi:10.3109/07420529509064504 101. Cousins MS, Roberts DCS, de Wit HGABA. (B) receptor agonists for the treatment of drug addiction: a review of recent findings. Drug Alcohol Depend (2002) 65:209–20. doi:10.1016/S0376-8716(01)00163-6 89. Shieh KR, Chu YS, Pan JT. Circadian change of dopaminergic neuron activ- ity: effects of constant light and melatonin. Neuroreport (1997) 8:2283–7. doi:10.1097/00001756-199707070-00037 102. Lacoste B, Angeloni D, Dominguez-Lopez S, Calderoni S, Mauro A, Fraschini F, et al. Anatomical and cellular localization of melatonin MT1 and MT2 receptors in the adult rat brain. J Pineal Res (2015) 58:397–417. doi:10.1111/ jpi.12224 90. Luo AH, Georges FE, Aston-Jones GS. Novel neurons in ventral tegmental area fire selectively during the active phase of the diurnal cycle. Eur J Neurosci (2008) 27:408–22. doi:10.1111/j.1460-9568.2007.05985.x 91. Hood S, Cassidy P, Cossette M-P, Weigl Y, Verwey M, Robinson B, et al. Endogenous dopamine regulates the rhythm of expression of the clock protein PER2 in the rat dorsal striatum via daily activation of D2 dopamine receptors. J Neurosci (2010) 30:14046–58. doi:10.1523/JNEUROSCI.2128-10.2010 103. Rønnekleiv OK, Kelly MJ, Wuttke W. Single unit recordings in the rat pineal gland: evidence for habenulo-pineal neural connections. Exp Brain Res (1980) 39:187–92. 104. Hutchinson AJ, Hudson RL, Dubocovich ML. Genetic deletion of MT(1) and MT(2) melatonin receptors differentially abrogates the development and expression of methamphetamine-induced locomotor sensitization during the day and the night in C3H/HeN mice. J Pineal Res (2012) 53:399–409. doi:10.1111/j.1600-079X.2012.01010.x 92. Abe M, Herzog ED, Yamazaki S, Straume M, Tei H, Sakaki Y, et al. Circadian rhythms in isolated brain regions. J Neurosci (2002) 22:350–6. f 93. Frontiers in Psychiatry \| www.frontiersin.org January 2016 \| Volume 6 \| Article 179 REFERENCES Sleipness EP, Sorg BA, Jansen HT. Diurnal differences in dopamine transporter and tyrosine hydroxylase levels in rat brain: dependence on the suprachiasmatic nucleus. Brain Res (2007) 1129:34–42. doi:10.1016/j. brainres.2006.10.063 105. Vengeliene V, Noori HR, Spanagel R. Activation of melatonin receptors reduces relapse-like alcohol consumption. Neuropsychopharmacology (2015) 40:2897–906. doi:10.1038/npp.2015.143 h 94. Stowie AC, Prosser RA, Glass JD. Cocaine modulation of the mammalian circadian clock: potential therapeutic targets. Ther Targets Neurol Dis (2015) 2:e607. doi:10.14800/ttnd.607 106. Lecca S, Meye FJ, Mameli M. The lateral habenula in addiction and depres- sion: an anatomical, synaptic and behavioral overview. Eur J Neurosci (2014) 39:1170–8. doi:10.1111/ejn.12480 ff 95. Gifuni AJ, Jozaghi S, Gauthier-Lamer A-C, Boye SM. Lesions of the lateral habenula dissociate the reward-enhancing and locomotor-stimulant effects of amphetamine. Neuropharmacology (2012) 63:945–57. doi:10.1016/j. neuropharm.2012.07.032 hf 107. Chastrette N, Pfaff DW, Gibbs RB. Effects of daytime and nighttime stress on Fos-like immunoreactivity in the paraventricular nucleus of the hypothala- mus, the habenula, and the posterior paraventricular nucleus of the thalamus. Brain Res (1991) 563:339–44. doi:10.1016/0006-8993(91)91559-J 96. Ellison G, Irwin S, Keys A, Noguchi K, Sulur G. The neurotoxic effects of continuous cocaine and amphetamine in Habenula: implications for the substrates of psychosis. NIDA Res Monogr (1996) 163:117–45. h 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. 97. Udupa K, Chen R. The mechanisms of action of deep brain stimulation and ideas for the future development. Prog Neurobiol (2015) 133:27–49. doi:10.1016/j.pneurobio.2015.08.001 Copyright © 2016 Salaberry and Mendoza. 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. 98. Pierce RC, Vassoler FM. Deep brain stimulation for the treatment of addiction: basic and clinical studies and potential mechanisms of action. Psychopharmacology (Berl) (2013) 229:487–91. doi:10.1007/ s00213-013-3214-6 99. Ellison G. Neural degeneration following chronic stimulant abuse reveals a weak link in brain, fasciculus retroflexus, implying the loss of forebrain January 2016 \| Volume 6 \| Article 179 Frontiers in Psychiatry \| www.frontiersin.org 8
https://openalex.org/W2779933443	https://jbiomedsem.biomedcentral.com/track/pdf/10.1186/s13326-017-0167-4	English	null	Integrating phenotype ontologies with PhenomeNET	Journal of biomedical semantics	2,017	cc-by	8,908	© 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. Abstract Background: Integration and analysis of phenotype data from humans and model organisms is a key challenge in building our understanding of normal biology and pathophysiology. However, the range of phenotypes and anatomical details being captured in clinical and model organism databases presents complex problems when attempting to match classes across species and across phenotypes as diverse as behaviour and neoplasia. We have previously developed PhenomeNET, a system for disease gene prioritization that includes as one of its components an ontology designed to integrate phenotype ontologies. While not applicable to matching arbitrary ontologies, PhenomeNET can be used to identify related phenotypes in different species, including human, mouse, zebrafish, nematode worm, fruit fly, and yeast. Results: Here, we apply the PhenomeNET to identify related classes from two phenotype and two disease ontologies using automated reasoning. We demonstrate that we can identify a large number of mappings, some of which require automated reasoning and cannot easily be identified through lexical approaches alone. Combining automated reasoning with lexical matching further improves results in aligning ontologies. Conclusions: PhenomeNET can be used to align and integrate phenotype ontologies. The results can be utilized for biomedical analyses in which phenomena observed in model organisms are used to identify causative genes and mutations underlying human disease. Keywords: Phenotype, PhenomeNET, Disease gene prioritization, OWL, Automated reasoning Keywords: Phenotype, PhenomeNET, Disease gene prioritization, OWL, Automated reasoning formally using ontologies, data integration and compari- son across species presents a major informatics challenge [2]. This task requires that related phenotypes which span levels of granularity as well as domains of knowledge, for example behaviour or neoplasia, in organisms as anatom- ically distinct as zebrafish and man, can be matched and compared so as to allow findings in one species to be related to others. Integrating phenotype ontologies with PhenomeNET Miguel Ángel Rodríguez-García1,2, Georgios V. Gkoutos3,4,5, Paul N. Schofield6 and Robert H *Correspondence: robert.hoehndorf@kaust.edu.sa 1Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology, 4700 KAUST, 23955-6900 Thuwal, Saudi Arabia 2Computer, Electrical and Mathematical Sciences & Engineering Division (CEMSE), King Abdullah University of Science and Technology, 4700 KAUST, PO Box 2882, 23955-6900 Thuwal, Saudi Arabia Full list of author information is available at the end of the article Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 DOI 10.1186/s13326-017-0167-4 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 DOI 10.1186/s13326-017-0167-4 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 DOI 10.1186/s13326-017-0167-4 RESEARCH Data sources and ontologies In our experiments, we use the Human Phenotype Ontology (HPO) [12], Mammalian Phenotype Ontology (MP) [13], Human Disease Ontology (DO) [15], and Orphanet Rare Disease Ontology (ORDO) [17] provided as part of the Ontology Alignment Evaluation Initiative 2016 competition. The HPO is an ontology of human phenotypes and con- sists of 11,787 classes that provide a standarized vocabu- lary for describing phenotypic abnormalities which have been commonly encountered in human monogenic dis- eases [18]. The MP is mainly used to characterize mouse phenotypes, but can also be applied to other organisms. It consists of 11,720 classes that have been organized into a directed acyclic graph (DAG) and can be used to describe abnormal phenotypes of physiological and anatomical sys- tems, behavior, and survival [19]. The PhenomeNET ontology was originally built by formally integrating species-specific phenotype ontolo- gies, permitting the relationship between classes of dif- ferent phenotype ontologies to be determined through deductive inference. For this purpose, PhenomeNET relies on the UBERON [9] ontology that identifies equivalences between anatomy ontologies of different species, the Gene Ontology (GO) [10] as a means to identify equivalent or related processes and func- tions, and the PATO ontology [11] to identify the qual- ities associated with anatomical entities or biological processes. DO provides a classification of human diseases accord- ing to multiple axes related to genetic disorders, infectious diseases, metabolic disorders. It consists 9247 classes that aim at unifying the representation of human diseases defined across a variety of developed biomedical vocabu- laries [20]. Here, we use the PhenomeNET ontology to identify alignments between phenotypes in different species. We present our results based on three versions of the Phe- nomeNET ontology: the first version consists of the plain ontology using only the axioms provided in the Human Phenotype Ontology (HPO) [12] and the Mammalian Phenotype Ontology (MP) [13]; in the second version, we extend our original ontology by adding additional lexical and structural mappings generated with the Agreement- MakerLight [14] system and represent them as equiv- alent class axioms in our ontology; and in the third version, we further generate mappings between classes in the PhenomeNET ontology, the Disease Ontology (DO) [15] and the Orphanet Rare Disease Ontology (ORDO) [16]. ORDO is derived from the Orphanet database of rare and orphan diseases and used to represent and catego- rize the diseases within Orphanet. Data sources and ontologies It consists of 12,960 classes which provides a structured vocabulary to repre- sent relationships between phenomes, diseases, genes and relevant features such genetic inheritance for analyzing rare diseases [17]. Background Understanding the functions of genes and gene prod- ucts is vital for our understanding of normal biology and pathophysiology. In recent years the amount of geno- type and phenotype data available for species as distinct as man and model organisms such as nematode worms has increased dramatically and continues to accelerate. Insights from non-human species have an important role to play in our understanding of human biology [1] and the challenge is to mobilise this data in a way in which it can be used to give meaningful insights into human physiol- ogy and disease. While much data is now being captured In response to this challenge we developed Phe- nomeNET. PhenomeNET [3] was built in 2011 as a system for disease gene discovery and prioritization. PhenomeNET consists of an ontology integrating species- specific phenotype ontologies based on the PATO ontol- ogy [4] and relations between anatomical structures and physiological processes, a database of gene-to-phenotype associations, and a measure of similarity between sets of phenotypes. Within PhenomeNET, species-specific phenotype ontologies are combined so that phenotypes observed in different species can be compared directly. Page 2 of 11 Page 2 of 11 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Lexical mappings h We use the AgreementMakerLight (AML) [21], released on 5 April 2016, to generate lexical mappings between ontologies. We used the automatic match mode of the AML with the default settings to generate the lexical map- pings that were used to extend the PhenomeNet ontology. The default settings of AML include use of the UBERON ontology, DO, and Wordnet as background knowledge, a lexical matcher, a word-based matcher that evaluates occurrence of the same words in class labels and syno- myms, and a string similarity measure (ISub). We find that our axiomatic approach can identify a large number of relations between classes that are not currently identified by other systems that do not uti- lize similar formal methods. However, our evaluation also shows that a large number of mappings can still be iden- tified through lexical and structural approaches, and that a purely axiomatic approach will miss many mappings that cannot currently be identified axiomatically due to incomplete and underspecified formalization of pheno- type classes. We illustrate how a combination of formal, lexical and structural approaches generates the most com- plete and comprehensive mappings between (phenotype) ontologies, and these mappings improve the application of phenotype ontologies in data analysis and translational research. In addition to mappings generated by the AML, we also incorporate mappings between the ontologies obtained from BioPortal [22]. For each mapping between classes from two ontologies, we add an equivalent class axiom to the PhenomeNET ontology. Methods The main application of PhenomeNET is the prioritiza- tion of candidate genes for human diseases by comparing human disease phenotypes to existing gene-phenotype associations derived from model organisms. In particular, human phenotypes associated with a disease can be com- pared to phenotypes observed in mouse or other model organisms using the integrated PhenomeNET ontology, and similarity between phenotypes can then be used to indicate the genetic basis of a disease. PhenomeNET has been successfully used to find candidate genes for diseases [3, 5], identify novel pathways [6], and repurpose drugs using mouse model phenotypes [7, 8]. Semantic similarity and evaluation data For additional external evaluation of our generated map- pings, we apply the PhenomeNET ontology to the priori- tization of candidate genes of human disease [3]. We use the phenotypes associated with knockout mice available Page 3 of 11 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Page 3 of 11 saliva secretion is a class from the biological process branch of the Gene Ontology (GO) [10]. from the Mouse Genome Informatics (MGI) database [23] and the phenotypes associated with human diseases from the Human Phenotype Ontology database [12]. We apply Resnik’s semantic similarity measure [24] together with the Best Matching Average strategy [25] to combine class similarities. The general pattern for defining a phenotype class in both the HP and MP ontologies, given Entity E and Qual- ity Q, is to declare them equivalent to ‘has part’ some (Q and ‘inheres in’ some E). In some cases, the Entity E is further constrained, e.g., by a loca- tion in which a certain process may happen. The “E” classes are generally taken either from the UBERON cross-species anatomy ontology [9] or from the GO. As the use of anatomy and physiology ontologies (UBERON and GO) is shared between MP and HP, it is possible to integrate both ontologies directly, based on the axiom pat- terns used to constrain their classes. However, the type of axiom pattern used in both ontologies results in a clas- sification that is primarily based on the PATO ontology, as the Quality Q is the main feature that distinguishes different classes. We evaluate the results using a list of gene–disease associations provided by the Human Phenotype Ontology database [12] as well as a set of mouse models of human disease provided by the MGI database [23]. Source code and experiments Source code for the PhenomeNET matching system, including parameter files, and the generated alignments, are available at http://github.com/bio-ontology-research- group/OAEI2016. Code to generate the PhenomeNET ontology is available at https://github.com/bio-ontology- research-group/phenomeblast/tree/master/fixphenotypes. In the PhenomeNET ontology, we rewrite all axioms in HP and MP using a pattern-based approach that allows us to utilize axioms from anatomy and physiology ontologies and enrich the classification of phenotype classes [11, 27]. In general, we declare phenotype classes defined using an Entity E and Quality Q as equivalent to ‘has part’ some (E and has-quality some Q) and we fur- ther add grouping classes that are defined as equivalent to ‘has part’ some ((‘part of’ some E)and has-quality some Quality). For example, based on the axiom that defines macroglossia (HP:0000158) as equivalent to ‘has part’ some (‘increased size’ and (‘inheres in’ some tongue) and (‘has modifier’ some abnormal)), we gener- ate two new axioms: macroglossia Equivalent To: ‘has part’ some (tongue and has-quality some ‘increased size’) as well as ‘tongue abnormality’ EquivalentTo: ‘has part’some ((‘part of’ some tongue) and has-quality some Quality). These two axioms, together with the transitivity and reflexivity of the part of’ relation, ensure that macroglossia becomes a subclass of tongue abnormality, and that all phenotypes affecting the tongue or a part of the tongue also become a subclass of tongue abnormality. The aim of rewriting the axioms is to base the classification of phenotype classes primarily on anatomical or physiological entities instead of the quality, and to utilize the axioms involving parthood in anatomy and physiology ontologies [11, 28]. Crucially, all axioms we generate fall in the OWL 2 EL profile [29, 30] and allow efficient automated reasoning using optimized OWL 2 EL reasoners such as ELK [31]. The first version of the Phe- nomeNET ontology (PhenomeNET-Plain) consists only of these axioms and no additional mappings. Combining knowledge-based and lexical approaches for ontology integration We developed and extended the PhenomeNET ontology to integrate several species-specific phenotype ontolo- gies and identify mappings between phenotype classes. Here, we consider a mapping between two classes (in two ontologies) a formal relation between them, i.e., an axiomatic relation such as equivalence, sub- or super- class, or disjointness. An alignment between two ontolo- gies is created by a set of mappings. Ontology matching is the process of finding mappings between classes in two ontologies. Ontology integration goes beyond identifica- tion of an ontology alignment in that two or more ontolo- gies are merged into a single ontology that encompasses all classes in the original ontologies [14]. Phenotype classes in the HP and MP ontologies are formally defined using the Entity-Quality (EQ) pattern [4, 26]. Based on the EQ patterns, a phe- notype is decomposed into an affected entity and a quality that specifies how the entity is affected. The Entity will usually be a class taken either from an anatomy ontology or a physiology ontology. For exam- ple, the phenotype class macroglossia (HP:0000158) describes an anatomical abnormality and is defined as equivalent to ‘has part’ some (‘increased size’ and (‘inheres in’ some tongue)and (‘has modifier’ some abnormal)), relying on the entity tongue (from the UBERON anatomy ontol- ogy [9]) and the quality increased size (from PATO) in its definition. The class abnormality of salivation (HP:0100755) is a physiological abnormality and is defined as equivalent to ‘has part’ some (quality and (‘inheres in’ some ‘saliva secretion’) and (‘has modifier’ some abnormal)), where In addition to this knowledge-based approach to linking the HP and MP ontologies, we also add lexical mappings, Page 4 of 11 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Page 4 of 11 within them are included in the PhenomeNET ontology. The aim of this pre-processing step is to avoid unsatisfi- able classes due to different conceptualizations between anatomy and phenotype ontologies, or within anatomy ontologies (Zebrafish Anatomy and UBERON) [3]. mappings derived from cross-references in the ontologies [5], mappings between HP and MP from BioPortal [22], and mappings generated by the AgreementMaker Light (AML) [14] in its default settings with a score greater than 0.7. Each mapping is added as a single equivalent classes axiom to the PhenomeNET ontology (PhenomeNET- Plain) to generate a version of the PhenomeNET ontology with lexical mappings (PhenomeNET-Map). Combining knowledge-based and lexical approaches for ontology integration g y Mappings between ontologies included in Phe- nomeNET are generated using the ELK reasoner [31]. We use ELK to classify the PhenomeNET ontology and identify pairs of equivalent classes C1 and C2 that belong to the ontologies to be aligned. These constitute equivalent class mappings. Furthermore, we also use ELK to identify pairs of classes C1 and C2 such that C1 is a proper sub- or super-class of C2 to generate sub- and super-class mappings. A reasoner such as ELK is also required to explore and visualize the PhenomeNET ontology structures, and the PhenomeNET-Map ontology can be explored and visualized in the AberOWL ontology repository [38]. Neither HP nor MP contain mappings to the DO or ORDO ontologies, despite a significant overlap between the four ontologies. Moreover, since neither DO nor ORDO contain axioms that follow a similar pattern to the axioms in HP and MP, we have to rely exclusively on lexical mappings in order to integrate DO and ORDO. To achieve this, we use the AML [14] in its default set- tings to generate mappings between HP and DO, HP and ORDO, MP and DO, MP and ORDO, and DO and ORDO (see Table 1). We then add an equivalent class axiom for each mapping AML identifies with a score greater than 0.7. The resulting ontology (PhenomeNET-Full) contains HP, MP, ORDO, and DO, and can be used to generate further mappings between these ontologies. Figure 1 pro- vides an overview of the different data sources we used to generate the mappings for the three PhenomeNET ontologies. Evaluation of mappings: HP and MP We employ the PhenomeNET ontology primarily for inte- grating the HP and MP ontologies. Using the axioms in the ontology alone (PhenomeNET-Plain), we identify 745 equivalent classes between the HP and MP ontologies (see Table 2). Additionally, a large number of sub- and super-class mappings can be identified based on query- ing the ontology using the ELK reasoner [31] for sub- or super-classes in the two ontologies. All versions of the PhenomeNET ontology contain the classes from the HP and MP ontologies as well as the subclass axioms between named classes asserted in these ontologies. Furthermore, the PhenomeNET ontol- ogy imports the ChEBI [32] and Mouse Pathology [33] ontologies using an OWL import statement. Additionally, PhenomeNET includes all classes from the UBERON, the GO, the BioSpatial Ontology [34], the Zebrafish Anatomy ontology [35], the PATO ontology [4], the Cell Ontol- ogy [36], and the Neuro-Behavior Ontology [37]. How- ever, these ontologies are not directly imported but rather pre-processed so that all disjointness axioms from these ontologies are excluded while all other axioms contained The number of pairs of equivalent classes identified increases to 1536 when adding explicit mappings derived from AML. Of these, 370 are generated by automated rea- soning and are also included in AML, 791 are generated from the AML-derived equivalent classes axioms, and 375 could only be derived through the automated rea- soning. For example, using the PhenomeNET ontology, we infer an equivalence class mapping between Copper accumulation in brain HP:0012676) and Increased brain copper level (MP:0011214) based on their Table 1 Number of classes, axioms and mappings in the PhenomeNET and AML ontologies System Ontology Number of classes Number of axioms Mappings added PhenomeNet-Plain HP-MP 219,423 1,399,411 0 PhenomeNet-Map HP-MP+mappings 219,423 1,400,570 1,160(AML), 639(BioPortal) PhenomeNet-Full HP-MP+DO-ORDO 241,817 1,631,543 1,489(AML), 1,018(BioPortal) HP-MP: 1,160 (AML), 639(BioPortal); DO-MP: 423 (AML); DO-HP: 1,074 (AML); ORDO-MP: 151 (AML); ORDO-HP: 531 (AML); AML HP-MP mappings 32,509 229,337 1,160(AML) AML Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Page 5 of 11 Fig. 1 An overview of the data sources and strategies used to generate the PhenomeNET ontologies. On one side, we use mappings between HP, MP, DO, and ORDO, generated using the AML ontology matching system; on the other side, we use the axioms used to define classes in HP and MP together with the background knowledge in other ontologies to generate mappings formally. Evaluation of mappings: HP and MP Journal of Biomedical Semantics (2017) 8:58 Predicting gene–disease associations To determine the impact of the different mapping approaches in biomedical data analysis, we also apply the three ontologies in the task for which PhenomeNET was originally designed, predicting gene–disease associations based on semantic similarity between mouse model phe- notypes and human phenotypes [3, 5]. For this purpose, we use the PhenomeNET ontology as an integrated ver- sion of both HP and MP so that semantic similarity can be computed simultaneously over both ontologies. Seman- tic similarity establishes a measure of relatedness between classes, or sets of classes, within an ontology (or, in some cases, between classes from multiple ontologies) [25]. Evaluation of mappings: HP and MP Using the ELK reasoner, we generate a hierarchical ontology structure (i.e., a taxonomy) from which we derive equivalent class, sub-class, and super-class mappings. The PhenomeNET-Full ontology is based on a combination of all these mapping approaches, while PhenomeNET-Map uses only the AML-generated mappings between HP and MP. PhenomeNET-Plain does not use any of the AML-generated mappings but solely relies on the axioms and background knowledge Fig. 1 An overview of the data sources and strategies used to generate the PhenomeNET ontologies. On one side, we use mappings between HP, MP, DO, and ORDO, generated using the AML ontology matching system; on the other side, we use the axioms used to define classes in HP and MP together with the background knowledge in other ontologies to generate mappings formally. Using the ELK reasoner, we generate a hierarchical ontology structure (i.e., a taxonomy) from which we derive equivalent class, sub-class, and super-class mappings. The PhenomeNET-Full ontology is based on a combination of all these mapping approaches, while PhenomeNET-Map uses only the AML-generated mappings between HP and MP. PhenomeNET-Plain does not use any of the AML-generated mappings but solely relies on the axioms and background knowledge shared definition ‘has part’ some (‘increased amount’ and (‘inheres in’ some (‘copper atom’ and (‘part of’ some brain))) and (‘has modifier’ some abnormal)). Such map- pings are not easily identified by methods that do not consider the axioms constraining the ontology classes. result of additional inferences obtained from adding the mappings from HP and MP to ORDO and DO, and combining them with the axioms in the PhenomeNET ontology. For example, we infer a new mapping between decreased IgG level (MP:0001805) and agammaglob- ulinemia (HP:0004432) based on the equivalence axioms between both classes and agammaglobulinemia (DOID:2583) generated by AML (based on the asserted synonym “hypogammaglobulinemia” shared between the classes in DO and MP). Table 2 summarizes our results. Additionally, we observe an increase in the number of equivalent class mappings when adding the ORDO and DO ontologies to the PhenomeNET ontology. The increase in mappings (from 1536 to 1582 classes) is a Table 2 Equivalent and sub-equivalent classes identified. Numbers in parentheses represent inferred (subclass) mappings System Ontology HP-MP (≡) HP-MP (⊑) DO-ORDO (≡) DO-ORDO (⊑) PhenomeNET-Plain HP-MP 745 2707 (96,278) 0 0 PhenomeNET-Map HP-MP+mappings 1536 3999 (107,268) 0 0 PhenomeNET-Full HP-MP+DO-ORDO 1582 4144 (112,366) 1527 4576 (16,838) Page 6 of 11 Rodríguez-García et al. Evaluation of mappings: ORDO and DO DO and ORDO. We provide full evaluation for the OAEI as Additional file 1; results are also available at http://oaei. ontologymatching.org/2016/results/phenotype/. DO and ORDO. We provide full evaluation for the OAEI as Additional file 1; results are also available at http://oaei. ontologymatching.org/2016/results/phenotype/. PhenomeNET is primarily designed for ontologies that follow the Entity-Quality definition pattern based on the PATO ontology. Neither ORDO nor DO follow this pat- tern, and ORDO and DO are primarily included in the PhenomeNET ontology through equivalent class axioms based on lexical mappings generated by AML. Notably, the mappings we generate are increased by including HP and MP. For example, we identify a mapping between mandibulofacial dysostosis (ORPHANET:155899) and Treacher Collins syndrome (DOID:2908), based on com- mon AML-generated mappings to mandibulofacial dysos- tosis (HP:0005321). As PhenomeNET relies on generating a taxonomic structure in which classes from HP and MP are combined, PhenomeNET also generated a large number of subclass and superclass mappings. While these were not explicitly evaluated, PhenomeNET was the only system explicitly focusing on these kind of mappings, while other partici- pating systems primarily focused on identifying mappings represented class equivalence. OAEI evaluation PhenomeNET participated in the Ontology Alignment Evaluation Initiative (OAEI) 2016 challenge where several ontology alignment systems where evaluated according to the following criteria: • precision and recall with respect to a silver standard generated by voting (using either two or three votes) the outputs of the participating systems, • recall with respect to manually generated mappings, • and a manual assessment of the mappings that were unique to a particular system. To evaluate the success of the three ontologies in dis- ease gene prioritization, we obtain mouse model pheno- types associated with loss-of-function mutations in single genes from the MGI database [23] as well as human dis- ease phenotypes associated with Mendelian diseases from the HPO database [12], and apply a semantic similar- ity measure [24, 41] to compare the phenotypic similar- ity between phenotypes associated with mouse mutants and human disease. We systematically compute phe- notypic similarity between 9131 loss-of-function mouse mutants and 7066 diseases. We perform this experiment three times, once for each version of the PhenomeNET ontology (PhenomeNET-Plain, PhenomeNET-Map, and PhenomeNET-Full). Additionally, to determine the effect of PhenomeNET’s knowledge-based approach, we also generate an integrated ontology based only on an align- ment between HP and MP generated by AML. In the first dataset, a silver standard reference alignment was generated from the systems participating in the OAEI challenge, using a vote of two of the participating sys- tems. PhenomeNET-Full reached an F-measure of 0.829 in the HP-MP task and 0.886 in the DO-ORDO task. The LogMap system [39] achieved the highest F-measure in this evaluation of 0.925 for the HP-MP task, and the FCA_Map system [40] achieved the an F-measure of 0.962 in the DO-ORDO task. Results are similar when evaluat- ing with a silver standard reference alignment generated by three votes of systems participating in the challenge. In particular in the DO-ORDO evaluation, PhenomeNET- Full achieved the second-highest F-score of 0.935, while the LogMap system [39] achieved an F-measure of 0.937. In the first dataset, a silver standard reference alignment was generated from the systems participating in the OAEI challenge, using a vote of two of the participating sys- tems. PhenomeNET-Full reached an F-measure of 0.829 in the HP-MP task and 0.886 in the DO-ORDO task. OAEI evaluation The LogMap system [39] achieved the highest F-measure in this evaluation of 0.925 for the HP-MP task, and the FCA_Map system [40] achieved the an F-measure of 0.962 in the DO-ORDO task. Results are similar when evaluat- ing with a silver standard reference alignment generated by three votes of systems participating in the challenge. In particular in the DO-ORDO evaluation, PhenomeNET- Full achieved the second-highest F-score of 0.935, while the LogMap system [39] achieved an F-measure of 0.937. When evaluating against manually created mappings, PhenomeNET-Full achieved the highest recall of 0.897 in the HP-MP task but could not generate any of the manually created mappings between DO and ORDO. Fur- thermore, when evaluating mappings that were uniquely identified by individual systems, 89 mappings between HP and MP as well as 3 mappings between ORDO and DO were generated only by the PhenomeNET ontologies and no other participating system. These were manually assessed, and PhenomeNET obtained a precision of 1.0 both for the 89 unique mappings generated between HP and MP as well as for the 3 mappings generated between g y We test how well this approach recovers known gene– disease associations. We use two sets for this evaluation: human gene–disease associations observed in a clinical context and presented in the Online Mendelian Inheri- tance in Man (OMIM) database [42], and mutant mice identified by curators as models of a human disease rep- resented in the MGI database [23]. The receiver operating characteristic (ROC) curves [43] for this evaluation are shown in Fig. 2. We find that the PhenomeNET-Map ver- sion, which focuses specifically on generating mappings between MP and HP, performs best among our ontologies in this evaluation (AUROC 0.794 for human gene–disease Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Page 7 of 11 Fig. 2 ROC curves for predicting gene–disease associations using the three different ontologies Fig. 2 ROC curves for predicting gene–disease associations using the three different ontologies which provide background knowledge to improve the gen- eration of mappings, and AML further provides methods for identifying and repairing incoherent matches between two ontologies. Similarly, LogMap [39] utilizes multi- ple sources of background knowledge, including related ontologies from BioPortal, and utilizes lexical match- ing together with background knowledge to generate alignments. LogMap further uses a reasoner to identify and repair inconsistent or incoherent mappings. OAEI evaluation Similar ontology mapping systems include XMap [45] and the cross-lingual ontology matching system LYAM++ [46]. A distinct approach is FCA-Map [40], based on Formal Concept Analysis (FCA) [47], which constructs formal contexts of classes from its properties and relations, gen- erates lattice structures and matches these concept lattices at a lexical and structural level. associations and 0.930 for mouse associations), followed by PhenomeNET-Full (AUROC 0.791 for human 0.929 for mouse gene–disease associations) and PhenomeNET- Plain (AUROC 0.790 and 0.920 for human and mouse, respectively). An ontology generated only by mappings from AML, however, performs better than any of the PhenomeNET ontologies despite producing a fewer num- ber of mappings. Using an ontology based only on the AML-derived mappings we achieve a AUROC of 0.795 and 0.934 for the human and mouse evaluation sets, respectively. However, none of the differences between the ontologies is statistically significant (p > 0.05 for all 12 comparisons, Wilcoxon rank sum test, Bonferroni correction). Discussion Related work One of the key features of the PhenomeNET system is its ability to identify subclass mapping in additional to equivalent class mappings. While PhenomeNET uses a reasoner for this purpose and relies on axioms that have specifically been developed for phenotype ontolo- gies [4], alternative approaches can also identify sub- class mappings based on matching sub-structures in ontology hierarchies or based on supervised machine learning [48, 49]. Related work A large number of ontology matching and alignment systems have been developed [44]. Several of these approaches have been applied to the same ontologies we use here. Most ontology matching systems combine methods based on lexical matching of class labels and synonyms, structural matches based on graph represen- tations of ontologies, and background knowledge coming from a variety of sources. We make use of the AML system [14] since AML is one of the leading ontology alignment systems. AML implements a modular ontol- ogy matching framework based on lexical and structural matching methods. AML can further make use of exter- nal resources such as DO, UBERON, MeSH and Wordnet, Due to the importance of integrating species-specific phenotype ontologies for biomedical research [50], sev- eral methods have been developed that specifically focus on the integration of phenotype ontologies. For example, PhenoHM [51] uses the Unified Medical Language System Page 8 of 11 Page 8 of 11 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 axiom patterns we use in the PhenomeNET [57] or that are used elsewhere [26]. (UMLS) MetaMap service [52] to map classes from MP to UMLS concepts describing disorders and phenotypes. The Uberpheno ontology [53] as well as the Monarch knowledge graph [54] use a combination of lexical map- pings and ontology axioms to generate mappings between MP and HPO. The main difference to our work lies in the representation patterns that are used to represent pheno- type classes and the way in which lexical mappings are generated. Another limitation of PhenomeNET is the reliance on OWL 2 EL which limits the expressivity of axiom patterns. The choice is mainly due to the size of the PhenomeNET ontology and the complexity of reason- ing. However, more complex axiom patterns would enable more comprehensive classification of phenotypes involv- ing absences and abnormalities [27]; experiments with an updated ontology will likely require improvement in OWL reasoning technologies. An alternative approach to finding relations between biomedical ontologies is to identify mappings between classes from different ontologies not based on the seman- tics of a class itself but rather based on shared annotations of the classes. For example, orthologous gene annota- tions can be used to identify orthologous phenotypes and thereby establish orthology relations between phenotypes. Knowledge-based, structural, and lexical mappings Knowledge-based, structural, and lexical mappings PhenomeNET is one of very few systems that are pri- marily based on automated reasoning to generate map- pings and does not rely on identifying similarity between class labels, synonyms, or other associated meta-data. PhenomeNET is a system intended to match pheno- types and as such, it is not a framework that can be applied to match ontologies in general. The axiom-based approach in PhenomeNET can be applied to any ontolo- gies that utilize PATO and the Entity-Quality definition patterns [4, 11]. In particular, PhenomeNET can not only be used to integrate MP and HPO, but also has been used to further integrate yeast, fly, worm, slime mold, and fish phenotypes [3, 56]. Furthermore, the combina- tion of semantic matching (using automated reasoning) and lexical matching in PhenomeNET mitigates some of the limitations of using lexical approaches alone, and we demonstrate this by inferring severa; mappings between HP and MP that cannot be inferred by other ontology matching systems. y p Finally, one limitation of most mapping approaches is their failure to consider subtle differences in ontologi- cal categories that may be obvious to ontology devel- opers and users but are not always reflected in the labels. This issue is particularly prevalent in the domains of phenotypes and diseases where the same label may be used to specify different ontological categories. A phenotype such as ‘agammaglobulinemia’ is an observa- tional phenomenon related to levels of gamma-globulin in blood, while the disease ‘agammaglobulinemia’ is a more complex entity that may involve a particular eti- ology and several signs and symptoms (of which the phenotype ‘agammaglobulinemia’ may be one). Inspec- tion of the written definition of ‘agammaglubulinemia’ (HP:0004432) in HPO indicates that the class refers to a deficiency or absence of immunoglobulins in serum. How- ever, the DO defines a class with this label as an immun- odeficiency syndrome that includes agammaglobulinemia as part of its phenotype, and ORDO similarly implies that this is a disease but only because of its position as a child of ‘Immunodeficiency predominantly affecting antibody production’ (as there is no textual or formal defi- nition of the class in ORDO). In MedGen (UID:168), the UMLS code C0001768 references both to HP:0004432 and Orphanet (ORPHANET:183669), but classifies both under ‘Disease or Syndrome’. However, relying on manually-created axioms also has several limitations. Related work This approach has been used previously to identify yeast models for mammalian vasculature formation [55], a strik- ing discovery since yeast has no blood vessels. To the best of our knowledge, these approaches have not yet been combined with mappings based entirely on the seman- tics of ontology classes and may provide a complementary source for future work on creating ontology mappings. Relying on automated reasoning over integrated phe- notype ontologies can result in incoherencies due to dif- ferent conceptualizations in the integrated ontologies. We avoid the incoherencies by removing disjointness axioms when including ontologies in PhenomeNET; however, this approach does not remove but only hide the underlying problems. Generic ontology matching systems have faced similar issues for a long time, and several methods have been proposed to automatically repair incoherent ontol- ogy mappings [58–60]. However, aligning incompatible conceptualizations across multiple ontologies is not triv- ial and automated methods still have limitations [61] that may require human intervention. In the future, we plan to investigate whether these methods can be applied to auto- matically repair some of the incoherencies we identified. Funding g Funding for GVG was provided by the National Science Foundation (Grant Number: IOS-1340112), the BBSRC national capability in plant phenotyping (Grant Number: BB/J004464/1) and the FP7 European Plant Phenotyping Network (Grant Agreement No. 284443). RH and MARG were supported by funding from the King Abdullah University of Science and Technology (Grant Number: FCC/1/1976-08-01). 9. Mungall C, Torniai C, Gkoutos G, Lewis S, Haendel M. Uberon, an integrative multi-species anatomy ontology. Genome Biol. 2012;13(1):5. doi:10.1186/gb-2012-13-1-r5. 9. Mungall C, Torniai C, Gkoutos G, Lewis S, Haendel M. Uberon, an integrative multi-species anatomy ontology. Genome Biol. 2012;13(1):5. doi:10.1186/gb-2012-13-1-r5. 10. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry MJ, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Tarver LI, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25(1): 25–9. doi:10.1038/75556. 10. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry MJ, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Tarver LI, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25(1): 25–9. doi:10.1038/75556. Author details 1Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology, 4700 KAUST, 23955-6900 Thuwal, Saudi Arabia. 2Computer, Electrical and Mathematical Sciences & Engineering Division (CEMSE), King Abdullah University of Science and Technology, 4700 KAUST, PO Box 2882, 23955-6900 Thuwal, Saudi Arabia. 3College of Medical and Dental Sciences, Institute of Cancer and Genomic Sciences, Centre for Computational Biology, University of Birmingham, B15 2TT Birmingham, UK. 4Institute of Translational Medicine, University Hospitals Birmingham, NHS Foundation Trust, B15 2TT Birmingham, UK. 5Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, SY23 2AX Aberystwyth, UK. References 1. Schofield PN, Hoehndorf R, Gkoutos GV. Mouse genetic and phenotypic resources for human genetics. Hum Mutat. 2012;33(5):826–36. h d f h f ld k h l f l 1. Schofield PN, Hoehndorf R, Gkoutos GV. Mouse genetic and phenotypic resources for human genetics. Hum Mutat. 2012;33(5):826–36. 2. Hoehndorf R, Schofield PN, Gkoutos GV. The role of ontologies in biological and biomedical research: a functional perspective. Brief Bioinform. 2011. doi:10.1093/bib/bbv011. http://bib.oxfordjournals.org/ content/early/2015/04/10/bib.bbv011.full.pdf+html. 2. Hoehndorf R, Schofield PN, Gkoutos GV. The role of ontologies in biological and biomedical research: a functional perspective. Brief Bioinform. 2011. doi:10.1093/bib/bbv011. http://bib.oxfordjournals.org/ content/early/2015/04/10/bib.bbv011.full.pdf+html. y p 3. Hoehndorf R, Schofield PN, Gkoutos GV. Phenomenet: a whole-phenome approach to disease gene discovery. Nucleic Acids Res. 2011;39(18):119. y p 3. Hoehndorf R, Schofield PN, Gkoutos GV. Phenomenet: a whole-phenome approach to disease gene discovery. Nucleic Acids Res. 2011;39(18):119. 4. Gkoutos GV, Green EC, Mallon A-MM, Hancock JM, Davidson D. Using ontologies to describe mouse phenotypes. Genome Biol. 2005;6(1):5. doi:10 1186/gb-2004-6-1-r8 approach to disease gene discovery. Nucleic Acids Res. 2011;39(18):119. 4. Gkoutos GV, Green EC, Mallon A-MM, Hancock JM, Davidson D. Using ontologies to describe mouse phenotypes. Genome Biol. 2005;6(1):5. doi:10.1186/gb-2004-6-1-r8. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Conclusions 6Department of Physiology, Development & Neuroscience, University of Cambridge, Downing Street, CB2 3EG Cambridge, UK. We have developed an ontology matching system for disease and phenotype ontologies. We generated three different version of the PhenomeNet ontology, each with different information and ontologies included. PhenomeNET is primarily based on deductive infer- ence and automated reasoning, and while it can utilize lexically-derived mappings in the ontology generation process, it does not on its own include any lexical match- ing algorithms. Our results demonstrate that a combi- nation of lexical and semantic approaches may improve upon mappings between ontologies generated using only one of these methods. Received: 17 March 2017 Accepted: 22 November 2017 Abbreviations 8. Hoehndorf R, Hiebert T, Hardy NW, Schofield PN, Gkoutos GV, Dumontier M. Mouse model phenotypes provide information about human drug targets. Bioinformatics. 2014;30(5):719–25. doi:10.1093/bioinformatics/btt613. Additional file 5. Hoehndorf R, Schofield PN, Gkoutos GV. An integrative, translational approach to understanding rare and orphan genetically based diseases. Interface Focus. 2013;3(2):20120055. doi:10.1098/rsfs.2012.0055. 6. Hoehndorf R, Dumontier M, Gkoutos GV. Identifying aberrant pathways through integrated analysis of knowledge in pharmacogenomics. Bioinformatics. 2012;28(16):2169–75. doi:10.1093/bioinformatics/bts350. 7. Hoehndorf R,Oellrich A,Rebholz-Schuhmann D,Schofield PN,Gkoutos GV. Linking PharmGKB to phenotype studies and animal models of disease for drug repurposing. Pac Symp Biocomput (PSB). 2012;2012:388–99. 8. Hoehndorf R, Hiebert T, Hardy NW, Schofield PN, Gkoutos GV, Dumontier M. Mouse model phenotypes provide information about human drug targets. Bioinformatics. 2014;30(5):719–25. doi:10.1093/bioinformatics/btt613. 9 Mungall C Torniai C Gkoutos G Lewis S Haendel M Uberon an Additional file 1: Supplementary Information. The supplementary information contains the full evaluation results of the PhenomeNET system and other participating systems for the OAEI challenge 2016. (PDF 26 kb) ; ( ) 6. Hoehndorf R, Dumontier M, Gkoutos GV. Identifying aberrant pathways through integrated analysis of knowledge in pharmacogenomics. Bioinformatics. 2012;28(16):2169–75. doi:10.1093/bioinformatics/bts350. Abbreviations OWL: Web ontology language 7. Hoehndorf R,Oellrich A,Rebholz-Schuhmann D,Schofield PN,Gkoutos GV Linking PharmGKB to phenotype studies and animal models of disease for drug repurposing. Pac Symp Biocomput (PSB). 2012;2012:388–99. Knowledge-based, structural, and lexical mappings In particular, the axioms are created by domain experts, and only about half the classes in MP and HP are constrained by an Entity-Quality based axiom. Furthermore, the quality of the axioms is diffi- cult to assess, and there are distinct differences between HP and MP in how the classes are constrained. A pos- sible solution to these challenges could be to generate phenotype ontologies fully automatically using anatomy and physiology ontologies as templates and applying the There are similar issues with, for example, hypogly- caemia which occurs in DO as a child of glucose Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Page 9 of 11 Page 9 of 11 Page 9 of 11 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 metabolism disease (DOID:9993), therefore as a disease, and in HPO (HP:0001943) as a phenotype defined as ‘A decreased concentration of glucose in the blood’. HPO cross-references the UMLS (C0020615) which uses this concept in the phenomenological sense yet again classifies it under ‘Disease or Syndrome’. The ambiguity of whether two classes are equivalent, even if they use the same label, is therefore deeply embedded in the ontologies, their structure and general domain, and there is no clear way to disambiguate these types of classes without manual expert inspection or, indirectly, by their different uses for annotation and analysis of data [62]. The semantic ambiguity reflected in these examples is partly a consequence of clinical usage of language where differ- ent entities (such as a disease and phenotype) are referred to by the same name. Resolving these issues is a problem that can likely only be addressed through expert curation across a very wide range of ontologies. The impact of these issues is likely not severe for human ontology users, but it remains a problem for any semantic approach to knowl- edge capture, analysis, and integration, and not least in the area of ontology matching. Consent for publication Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. 10. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry MJ, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Tarver LI, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. Nat Genet. 2000;25(1): 25–9. doi:10.1038/75556. Authors’ contribution Authors contribution GVG, PNS, RH conceived of the experiments, MARG implemented the alignment workflow and performed the evaluation; all authors interpreted the results and contributed to writing the manuscript. All authors read and approved the final manuscript. Availability of data and materials All data and materials are available from http://github.com/bio-ontology- research-group/OAEI2016 and https://github.com/bio-ontology-research- group/phenomeblast/tree/master/fixphenotypes. Page 10 of 11 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 11. Gkoutos GV, Schofield PN, Hoehndorf R. The anatomy of phenotype ontologies: principles, properties and applications. Brief Bioinform. 2017. in press. 30. Hoehndorf R, Dumontier M, Oellrich A, Wimalaratne S, Rebholz-Schuhmann D, Schofield P, Gkoutos GV. A common layer of interoperability for biomedical ontologies based on OWL EL. Bioinformatics. 2011;27(7):1001–8. 12. Köhler S, Doelken SC, Mungall CJ, Bauer S, Firth HV, Bailleul-Forestier I, Black GCM, Brown DL, Brudno M, Campbell J, FitzPatrick DR, Eppig JT, Jackson AP, Freson K, Girdea M, Helbig I, Hurst JA, Jähn J, Jackson LG, Kelly AM, Ledbetter DH, Mansour S, Martin CL, Moss C, Mumford A, Ouwehand WH, Park SM, Riggs ER, Scott RH, Sisodiya S, Vooren SV, Wapner RJ, Wilkie AOM, Wright CF, Vulto-van Silfhout AT, Leeuw ND, de Vries BBA, Washingthon NL, Smith CL, Westerfield M, Schofield P, Ruef BJ, Gkoutos GV, Haendel M, Smedley D, Lewis SE, Robinson PN. The human phenotype ontology project: linking molecular biology and disease through phenotype data. Nucleic Acids Res. 2014;42(D1):966–74. S h CL G ld h C AW E JT Th l h 31. Kazakov Y, Krötzsch M, Simancik F. The incredible elk. J A 2014;53(1):1–61. doi:10.1007/s10817-013-9296-3. 32. Degtyarenko K, Matos P, Ennis M, Hastings J, Zbinden M, McNaught A, Alcantara R, Darsow M, Guedj M, Ashburner M. Nucleic Acids Res. 2007;36(Database issue):344–50. 33. Schofield PN, Sundberg JP, Sundberg BA, McKerlie C, Gkoutos GV. J Biomed Semant. 2013;4(1):1–8. doi:10.1186/2041-1480-4-18. 34. Balhoff JP, Mikó I, Yoder MJ, Mullins PL, Deans AR. A semantic model for species description applied to the ensign wasps (hymenoptera: Evaniidae) of new caledonia. Syst Biol. 2013;62(5):639–59. doi:10.1093/sysbio/syt028. http://sysbio.oxfordjournals.org/content/62/ 5/639.full.pdf+html. 13. Smith CL, Goldsmith C-AW, Eppig JT. The mammalian phenotype ontology as a tool for annotating, analyzing and comparing phenotypic information. Genome Biol. 2004;6(1):7. doi:10.1186/gb-2004-6-1-r7. 35. Dahdul WM, Balhoff JP, Engeman J, Grande T, Hilton EJ, Kothari C, Lapp H, Lundberg JG, Midford PE, Vision TJ, Westerfield M, Mabee PM. Evolutionary characters, phenotypes and ontologies: curating data from the systematic biology literature. PLoS ONE. 2010;5(5):10708. doi:10.1371/journal.pone.0010708. 14. Faria D, Pesquita C, Santos E, Palmonari M, Cruz IF, Couto FM. In: Meersman R, Panetto H, Dillon T, Eder J, Bellahsene Z, Ritter N, De Leenheer P, Dou D, editors. The AgreementMakerLight Ontology Matching System. Berlin, Heidelberg: Springer; 2013, pp. 527–41. Availability of data and materials 15. Kibbe WA, Arze C, Felix V, Mitraka E, Bolton E, Fu G, Mungall CJ, Binder JX, Malone J, Vasant D, Parkinson H, Schriml LM. Disease ontology 2015 update: an expanded and updated database of human diseases for linking biomedical knowledge through disease data. Nucleic Acids Res. 2014;43:1071–8. doi:10.1093/nar/gku1011. 36. Bard J, Rhee SY, Ashburner M. An ontology for cell types. Genome Biol. 2005;6(2). doi:10.1186/gb-2005-6-2-r21. 37. Hoehndorf R, Hancock JM, Hardy NW, Mallon AM, Schofield PN, Gkoutos GV. Analyzing gene expression data in mice with the Neuro Behavior Ontology. Mamm Genome. 2014;25(1-2):32–40. 16. Sarntivijai S, Vasant D, Jupp S, Saunders G, Bento AP, Gonzalez D, Betts J, Hasan S, Koscielny G, Dunham I, Parkinson H, Malone J. Linking rare and common disease: mapping clinical disease-phenotypes to ontologies in therapeutic target validation. J Biomed Semant. 2016;7(1):1–11. doi:10.1186/s13326-016-0051-7. 38. Hoehndorf R, Slater L, Schofield PN, Gkoutos GV. Aber-OWL: a framework for ontology-based data access in biology. BMC Bioinformatics. 2015;16:26. 39. Jiménez-Ruiz E, Grau BC. Logmap: Logic-based and scalable ontology matching. In: International Semantic Web Conference. Heidelberg: Springer; 2011. p. 273–88. 17. Vasant D, Chanas L, Malone J, Hanauer M, Olry A, Jupp S, Robinson PN, Parkinson H, Rath A. Ordo: An ontology connecting rare disease, epidemiology and genetic data. In: Phenoday @ ISMB2014. 2014. b hl l dl h 40. Zhao M, Zhang S. FCA-Map results for OAEI 2016. In: Shvaiko P, et al, editors. Proceedings of the 11th International Workshop on Ontology Matching. Aachen: CEUR-WS; 2016. p. 172–7. 18. Robinson PN, Köhler S, Bauer S, Seelow D, Horn D, Mundlos S. The human phenotype ontology: a tool for annotating and analyzing human hereditary disease. Am J Hum Genet. 2008;83(5):610–5. 41. Harispe S, Ranwez S, Janaqi S, Montmain J. The semantic measures library and toolkit: fast computation of semantic similarity and relatedness using biomedical ontologies. Bioinformatics. 2014;30(5): 740–2. doi:10.1093/bioinformatics/btt581. http://bioinformatics. oxfordjournals.org/content/30/5/740.full.pdf+html. 19. Smith CL, Eppig JT. The mammalian phenotype ontology: enabling robust annotation and comparative analysis. Wiley Interdiscip Rev Syst Biol Med. 2009;1(3):390–9. 20. Schriml LM, Arze C, Nadendla S, Chang Y-WW, Mazaitis M, Felix V, Feng G, Kibbe WA. Disease ontology: a backbone for disease semantic integration. Nucleic Acids Res. 2011;40(D1):940–6. 42. Amberger J, Bocchini C, Hamosh A. A new face and new challenges for Online Mendelian Inheritance in Man (OMIM). Hum Mutat. 2011;32:564–7. 21. Availability of data and materials Faria D, Martins C, Nanavaty A, Oliveira D, Sowkarthiga B, Taheri A, Pesquita C, Couto FM, Cruz IF. AML results for OAEI 2015. In: Shvaiko P, et al, editors. Proceedings of the 10th International Workshop on Ontology Matching. Aachen: CEUR-WS; 2015. p. 116–23. 43. Fawcett T. An introduction to ROC analysis. Pattern Recogn Lett. 2006;27(8):861–74. doi:10.1016/j.patrec.2005.10.010. 44. Lorena OC, Francisco JR-M, Alma GR. Ontology matching: A literature review. Expert Syst Appl. 2015;42(2):949–71. 22. Noy NF, Shah NH, Whetzel PL, Dai B, Dorf M, Griffith N, Jonquet C, Rubin DL, Storey M-AA, Chute CG, Musen MA. Bioportal: ontologies and integrated data resources at the click of a mouse. Nucleic Acids Res. 2009;37(Web Server issue):170–3. doi:10.1093/nar/gkp440. 45. Djeddi WE, Khadir MT, Yahia SB. Xmap: results for oaei 2015. In: Shvaiko P, et al, editors. Proceedings of the 10th International Workshop on Ontology Matching. Aachen: CEUR-WS; 2015. p. 216–21. 23. Bello SM, Richardson JE, Davis AP, Wiegers TC, Mattingly CJ, Dolan ME, Smith CL, Blake JA, Eppig JT. Disease model curation improvements at mouse genome informatics. Database. 2012;2012:063. 46. Tigrine AN, Bellahsene Z, Todorov K. Lyam++ results for oaei 2015. In: Shvaiko P, et al., editors. Proceedings of the 10th International Workshop on Ontology Matching. Aachen: CEUR-WS; 2015. p. 176–180. 24. Resnik P. Semantic similarity in a taxonomy: An Information-Based measure and its application to problems of ambiguity in natural language. J Artif Intell Res. 1999;11:95–130. 47. Wille R. In: Ganter B, Stumme G, Wille R, editors. Formal Concept Analysis as Mathematical Theory of Concepts and Concept Hierarchies. Berlin, Heidelberg: Springer; 2005, pp. 1–33. doi:10.1007/11528784_1. 25. Pesquita C, Faria D, Falcao AO, Lord P, Couto FM. Semantic similarity in biomedical ontologies. PLoS Comput Biol. 2009;5(7):1000443. 48. Spiliopoulos V, Vouros GA, Karkaletsis V. Web Semant Sci Serv Agents World Wide Web. 2010;8(1):69–88. 26. Mungall C, Gkoutos G, Smith C, Haendel M, Lewis S, Ashburner M. Integrating phenotype ontologies across multiple species. Genome Biol. 2010;11(1):2. 49. Spiliopoulos V, Valarakos AG, Vouros GA. In: Bechhofer S, Hauswirth M, Hoffmann J, Koubarakis M, editors. CSR: Discovering Subsumption Relations for the Alignment of Ontologies. Berlin, Heidelberg: Springer; 2008, pp. 418–31. 27. Hoehndorf R, Oellrich A, Rebholz-Schuhmann D. Interoperability between phenotype and anatomy ontologies. Bioinformatics. 2010;26(24):3112–8. 50. Gkoutos GV, Schofield PN, Hoehndorf R. Computational tools for comparative phenomics: the role and promise of ontologies. Mamm Genome. 2012;23(9-10):669–79. 28. Availability of data and materials Boudellioua I, Mahamad Razali RB, Kulmanov M, Hashish Y, Bajic VB, Goncalves-Serra E, Schoenmakers N, Gkoutos GV, Schofield PN, Hoehndorf R. Semantic prioritization of novel causative genomic variants. PLOS Comput Biol. 2017;13(4):1–21. doi:10.1371/journal.pcbi.1005500. 51. Sardana D, Vasa S, Vepachedu N, Chen J, Gudivada RC, Aronow BJ, Jegga AG. PhenoHM: human-mouse comparative phenome-genome server. Nucleic Acids Res. 2010;38(Web Server issue):W165–74. 29. Motik B, Grau BC, Horrocks I, Wu Z, Fokoue A, Lutz C. OWL 2 Web Ontology Language Profiles (Second Edition). World Wide Web Consortium. 2012. https://www.w3.org/TR/owl2-profiles/. Accessed 4 Dec 2017. 52. Bodenreider O. The Unified Medical Language System (UMLS): integrating biomedical terminology. Nucleic Acids Res. 2004;32(Database issue):267–70. Page 11 of 11 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 53. Köhler S, Doelken SC, Ruef BJ, Bauer S, Washington N, Westerfield M, Gkoutos G, Schofield P, Smedley D, Lewis SE, Robinson PN, Mungall CJ. Construction and accessibility of a cross-species phenotype ontology along with gene annotations for biomedical research. F1000Research. 2013;2. doi:10.12688/f1000research.2-30.v1. 54. Mungall CJ, McMurry JA, Koehler S, Balhoff JP, Borromeo C, Brush M, Carbon S, Conlin T, Dunn N, Engelstad M, Foster E, Gourdine JP, Jacobsen JOB, Keith D, Laraway B, Lewis SE, NguyenXuan J, Shefchek K, Vasilevsky N, Yuan Z, Washington N, Hochheiser H, Groza T, Smedley D, Robinson PN, Haendel MA. The monarch initiative: an integrative data and analytic platform connecting phenotypes to genotypes across species. Nucleic Acids Res. 2017;45(D1):712–22. 55. McGary KL, Park TJ, Woods JO, Cha HJ, Wallingford JB, Marcotte EM. Systematic discovery of nonobvious human disease models through orthologous phenotypes. Proc Natl Acad Sci. 2010;107(14):6544–9. doi:10.1073/pnas.0910200107. 56. Hoehndorf R, Hardy NW, Osumi-Sutherland D, Tweedie S, Schofield PN, Gkoutos GV. Systematic analysis of experimental phenotype data reveals gene functions. PLoS ONE. 2013;8(4):60847. doi:10.1371/journal.pone. 0060847. 57. Hoehndorf R, Harris MA, Herre H, Rustici G, Gkoutos GV. Semantic integration of physiology phenotypes with an application to the cellular phenotype ontology. Bioinformatics. 2012;28(13):1783–9. doi:10.1093/bioinformatics/bts250. http://bioinformatics.oxfordjournals. org/content/28/13/1783.full.pdf+html. 58. Jiménez-Ruiz E, Cuenca Grau B, Horrocks I, Berlanga R. In: Aroyo L, Traverso P, Ciravegna F, Cimiano P, Heath T, Hyvönen E, Mizoguchi R, Oren E, Sabou M, Simperl E, editors. Ontology Integration Using Mappings: Towards Getting the Right Logical Consequences. Berlin, Heidelberg: Springer; 2009, pp. 173–87. 59. Santos E, Faria D, Pesquita C, Couto FM. Ontology alignment repair through modularization and confidence-based heuristics. PLOS ONE. 2016;10(12):1–19. doi:10.1371/journal.pone.0144807. 60. Availability of data and materials Meilicke C, Stuckenschmidt H, Tamilin A. Repairing ontology mappings. In: Proceedings of the 22Nd National Conference on Artificial Intelligence - Volume 2, AAAI’07. AAAI Press; 2007. p. 1408–13. http://dl.acm.org/ citation.cfm?id=1619797.1619871. 61. Pesquita C, Faria D, Santos E, Couto FM. To repair or not to repair: Reconciling correctness and coherence in ontology reference alignments. In: Proceedings of the 8th International Conference on Ontology Matching - Volume 1111, OM’13. Aachen, Germany: CEUR-WS.org. 2013. p. 13–24. http://dl.acm.org/citation.cfm?id=2874493.2874495. 62. Goldfain A, Smith B, Cowell L. Dispositions and the infectious disease ontology. In: Proceedings of Formal Ontologies in Information Systems (FOIS). Amsterdam: IOS Press. 2010. p. 400–13. 62. Goldfain A, Smith B, Cowell L. Dispositions and the infectious disease ontology. In: Proceedings of Formal Ontologies in Information Systems (FOIS). Amsterdam: IOS Press. 2010. p. 400–13. Rodríguez-García et al. Journal of Biomedical Semantics (2017) 8:58 Submit your next manuscript to BioMed Central and we will help you at every step: Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit p y y p • We accept pre-submission inquiries
https://openalex.org/W2510926951	http://www.m-hikari.com/asb/asb2013/asb9-12-2013/tarroASB9-12-2013.pdf	English	null	Origin of the viruses and their evolutionary history	Advanced studies in biology	2,013	cc-by	2,986	Department of Biology, Center for Biotechnology, Sbarro institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA. Committee on Biotechnologies and VirusSphere, World Academy of Biomedical Technologies, UNESCO, Paris, France. Department of Biology, Center for Biotechnology, Sbarro institute for Cancer Research and Molecular Medicine, Temple University, Philadelphia, PA, USA. Committee on Biotechnologies and VirusSphere, World Academy of Biomedical Technologies, UNESCO, Paris, France. Advanced Studies in Biology, Vol. 5, 2013, no. 9, 423 - 430 HIKARI Ltd, www.m-hikari.com http://dx.doi.org/10.12988/asb.2013.3835 Advanced Studies in Biology, Vol. 5, 2013, no. 9, 423 - 430 HIKARI Ltd, www.m-hikari.com http://dx.doi.org/10.12988/asb.2013.3835 Advanced Studies in Biology, Vol. 5, 2013, no. 9, 423 - 430 HIKARI Ltd, www.m-hikari.com http://dx.doi.org/10.12988/asb.2013.3835 Origin of the HIV The evolution of the AIDS virus and its migratory phenomena can be considered as a good example of how several viruses originated. Although various hypotheses have been proposed during the last 25 years, it is now clear that the human immunodeficiency virus (HIV) was formed through a process of natural evolution. The theory on the origin of the HIV that has found the greatest consensus maintains that this virus is derived from the mutations of a virus that infects some species of African chimpanzee, the Simian immunodeficiency Virus (SIV). Through molecular biology studies, it has been possible to establish a relationship between the HIV and the SIV, identifying a 98% genetic homology between these two viruses, and building a solid viral genealogical tree. The infection from HIV would be therefore a zoonosis, that is, an infection transmitted to the man by other animal species: HIV probably migrated from the primate reservoir to humans by hunting or by tribal rites that implied contact with the blood of these animals. SIV would then have become HIV via various genetic mutations over many years. This hypothesis has been confirmed by the study of a group of researchers of the University of Alabama in Birmingham, presented at the Sixth Conference on Retroviruses and Opportunistic Infections held in Chicago in February 1999, where a particular kind of chimpanzee, Pantroglodytes troglodytes, has been recognized as the most probable source of infection in humans. HIV would therefore have probably existed for long time in the small tribal communities of Africa. Urbanization, especially during the colonial period, caused mass migrations and the spread of more liberal customs, with a consequent increase of sexual contact between individuals from different areas (as well as prostitution). These movements and trends may have favored the local spread of HIV, creating a "cluster" of infected individuals, on which the future expansion of the infection was based. Subsequently, various factors such as the contact with the West, the use of unsterilized hypodermic syringes in vaccination campaigns, and the use of blood transfusions in cases of malaria, favored the wider spread of HIV, and its transmission to the West. From there, sexual liberation and drug addiction exacerbated this into the epidemic that became apparent in the '80s and '90s. Giulio Tarro Keywords: HIV, Herpesvirus, HPV, Smallpox Keywords: HIV, Herpesvirus, HPV, Smallpox Correspondence to: Prof. Dr. Giulio Tarro, Via Posillipo 286, 80123 Naples, Italy e-mail: gitarro@tin.it, giuliotarro@gmail.com Correspondence to: Prof. Dr. Giulio Tarro, Via Posillipo 286, 80123 Naples, Italy e-mail: gitarro@tin.it, giuliotarro@gmail.com Copyright © 2013 Giulio Tarro. 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. Abstract. The human immunodeficiency virus (HIV) originated via a process of natural evolution, probably emerging from the primate SIV reservoir into the human population via hunting or other behavior involving contact with the blood of these animals. A particular subspecies of chimpanzee, the Pantroglodytes troglodytes, has been recognized as the most probable original source of human infection. Analysis of viral genetic sequences has allowed researchers to estimate that the native strain of HIV originated in 1931. In the West, sexual behavior patterns and injecting drug use subsequently began the epidemic. Polymerase Chain reaction (PCR), is a technique in molecular biology that amplifies a specific region of deoxyribonucleic acid (DNA), and has been useful in the molecular characterization of viruses. The Variola major, the virus that causes the smallpox, lethal virus in the 30% of the cases, was eradicated in 1979 in the human species, thanks to a capillary vaccination on global scale. It has now become a “historical footprint” in two known laboratories, one in the USA and another in Russia, leaving no obvious source for its often-theorized use as a bioterrorist weapon. Nevertheless, mass vaccination against smallpox continues to be a leading initiative in Western countries to guard against bioterrorist attack. 424 Giulio Tarro Evolutionary history of the viruses When studying such a major component of the biosphere as the viruses, it is important to apply a molecular approach that allows their isolation and the determination of individuality among the various strains, types and subtypes of the same family. PCR, is a key technique for this, and its utilization with new and existing diagnostic methods for environmental and medical surveillance is a powerful approach to national health security. It creates an opportunity to: detect unusual microbiological events in the environment; assess the medical and public health significance of these events; position the public health and emergency response teams to respond quickly and appropriately to biothreats; and provide real-time health benefits to the population while maximizing early detection and appropriate responses to potential bioterrorist acts. Origin of the HIV An article published in the journal Nature from a group led by David Ho (director of the Aaron Diamond AIDS Research Center in New York), announced the discovery of traces of the genome of HIV in a blood sample belonging to a man who lived in Kinshasa (Congo) and died in 1959. By molecular analysis of this virus, compared Origin of the viruses and their evolutionary history 425 with more recently isolated viral strains, it has been possible to estimate the origin of the HIV as being before 1940, thereby suggesting the hypothesis that the transmission of the virus from chimpanzee to man first occurred approximately 70 years ago. In a follow-up paper, published in the journal Science, further analysis of the genetic sequence of the virus, aided by sophisticated statistical models, has allowed researchers to estimate that the native strain of HIV originated after 1931. Interaction between viruses and host The main strategy to evade the immune response is the genetic variation faced by the viral genome. The consequence of the heterogeneity of HCV gene expression and its ability for genetic and then phenotypic mutation, are therefore at the base of such a high rate of chronic infections, of the not efficacy of the therapies and also of the difficulty of preparing vaccines. Interaction between viruses and host Having entered the host cell, viruses can give rise to acute, latent or persistent infection. In the former case , the virus enters the body, replicates over a limited period of time and is then completely eliminated by the host (or causes its death). The latter types of infection are characterized by alternating replication of the virus and its latent infection, or the onset of chronic and continuous replication. The site of latency is different for each subfamily of herpes viruses, but these are usually located in areas of the body where they are protected from the immune system [9]. There are molecular mechanisms that allow the viral genome to remain in a latent state [11], and those leading to exit from latency and resumption of the lytic cycle of viral replication [6]. HSV1 and 2 infect epithelial cells and yield latent infections in the neurons [14]. HSV 1 is classically associated with oropharyngeal lesions, while HSV 2 primarily infects the genital mucosa. The Varicella Zoster Virus (VZV) causes the disease known as chickenpox in primary rash and establishes a latent infection in neurons, which [2], if reactivated, 426 Giulio Tarro causes herpes zoster (shingles). The Polyomaviridae family includes, among others, JC Virus (JCV), BK Virus (BKV) and SV40. The primary infection caused by these viruses is asymptomatic and occurs during childhood, followed by latency [7];[10]. The Polyomaviridae family includes, among others, JC Virus (JCV), BK Virus (BKV) and SV40. The primary infection caused by these viruses is asymptomatic and occurs during childhood, followed by latency [7];[10]. Reactivation of Polyomavirus depends on the function of the host immune system: when it is no longer competent or undergoes immunosuppression, the virus reactivates its replication and causes disease. Hepatitis viruses B and C are associated with hepatocellular carcinoma. Altogether, over the 50% of all liver cancer worldwide are attributable to hepatitis B infection, for which an effective preventative vaccine is available. Human Papilloma Viruses (HPV) have been correlated with cervical cancer, with genotypes 16 and 18 being considered particularly carcinogenic in humans [15]. In 2006 the FDA released the first vaccine against HPV. The viral proteins E6 and E7 are able to inhibit oncosuppressors during the process of malignant transformation [1];[13]. HCV belongs to the Flaviviridae family. Its infection can remain stable and cause mild hepatitis, liver cirrhosis or it may evolve in hepatocellular carcinoma [4]. Smallpox virus and vaccination From the " Malignancy" of the oncogenic viruses now we move to that one of the Smallpox virus and the possibility for such viruses to be used as weapons in bioterrorism even if they were eradicated by a global vaccination, real pacemaker of other Viral Vaccines. As it is now known, the "Variola major" virus that causes smallpox, which was lethal in 30% of cases, was eradicated, thanks to a capillary vaccination on a global scale in 1979 . It has now become a "historical footprint", guarded under maximum security and the superintendence of the World Health Organization, in two known laboratories, one in the USA and another in Russia. Since there are, in theory, no more strains of human smallpox elsewhere on the planet, a major question is where the new strains required to buil a bioterrorist weapon could possibly come from. Despite this, the debate on the vaccinations in recent times is closely bound to the Origin of the viruses and their evolutionary history 427 threat of a bioterrorist attack; these arguments define lurid, apocalyptic scenerios that have received disproportionate coverage in the mass media, and have already resulted in a whole series of "exercises", such as those held in England in December 2002, to face a hypothetical attack with smallpox virus. Meanwhile, it must be said that, despite innumerable articles, novels and films based on the scenario, an isolated bioterroristic attack would not seem to have a very large chance of instigating a devastating epidemic. Unlike biological attack conducted by an army (prepared for by using conventional bombardments to destroy command infrastructure, sanitary systems and buildings, and causing refugee crowding before launching an attack with pathogenic germs or toxins), bioterrorism would presumably launch an attack on a focused target, with an entire region able to react to the threat. This hypothesis is supported by declassified reports, such as the epidemiological studies on people successfully hospitalized following the experimental dissemination of a non-harmful bacterial agent by the US Department to the Defense in the New York subway in 1956. A less deliberate example occurred in England in 1962 when a researcher, George Bacon, became infected with a modified strain of Yersinia pestis (bubonic plague) at the biological warfare facility of Porton Down, UK, and exposed the outside world to this infection before dying. Smallpox virus and vaccination Continuing on this theme, another example is the accidental release of smallpox from the University of Birmingham, UK in 1978, which killed three people. Why did two dangerous microorganisms, Yersinia pestis (made more lethal by modification at Porton Down) and Variola major, both transmissible through the respiratory route not produce a catastrophic epidemic? Numerous studies have been compiled to address the incidents, and all have described the initial sanitary measures as being insufficient. Regarding the Birmingham incident, the role played by antivariola vaccination, obligatory for all in Europe at that point but poorly practiced in England, was also pointed out. But then, why was there no catastrophe? The ‘why’ to seem would be, (aside from the still-unclear mechanisms governing epidemic dynamics), the existence of a solid health infrastructure did not collapse at the announcement of the infection. In this sense, perhaps the element that could transform a bioterrorist attack in a catastrophe is in fact the irresponsible emphasis the mass media devote to this threat at present, which might be able to instigate a panic, with a consequent mass exodus from the area, spreading the epidemic. The use of mass-vaccination to protect against a terrorist attack using the smallpox virus has clearly been discarded, as indicated by a document published in the New York Times, "Supplemental Recommendation of the ACIP on Use of Smallpox" compiled June 20 2002 by the Advisory Committee on the practices of immunization and submitted to the Department of Human Health of the United States (HHS) and to the Center for the Control of the Epidemics (CDC), which approved in Despite this 428 Giulio Tarro the proposal of a vaccination of mass against the smallpox (of 500.000 people in the United States only) continues to be in the front among the "initiatives" of the Western countries to face a bioterroristic attack of which (beyond the case of the "letters at the anthrax" that allowes to glimpse responsibility not surely referable to some fanatical person) no comparison is found. Although the threat of these infectious diseases, and therefore the problem of administering vaccinations, seem to be relatively unimportant issues in Western countries, in many areas of the ‘Third World’ extremely poor sanitary conditions and the unattainably high cost of vaccines could result in a death sentence for millions of people. The author declares no conflict of interests. The author declares no conflict of interests. Evolution of man and genomic mutation. Future perspectives During the eight-million-year evolution from the state of monkey to man, the accumulation of genomic mutations amounted to only 2%; some viruses, however, can accumulate a similar proportion of genomic mutations during just 5 days of replicative activity. Type A influenza viruses have a strong tendency to mutate, changing their own structure rapidly, and this genetic variability can be divided into antigenic drift, with minor changes that are associated with sporadic cases or small outbreaks, and antigenic shift, with more significant changes and the creation of new subtypes, which is responsible for large epidemics and pandemics [3];[5];[8]. Methodologies for extracting virus and non viral antigens, and cancer vaccine development techniques allow further steps in understanding the role of viruses and the strategies of the immune system to produce humoral and cellular antibodies. Peptide search in the tumor liberated protein and cancer proteomics represent the most advanced discovery in anticancer peptide vaccines [12]. References [1] A. Baldwin, K. Munger. Molecular events associated with human papillomavirus-induced human cancers in Viral Oncology Khalili K., Jeang K-T. (Eds), Wiley – Blackwell, Hoboken, New Jersey, USA, 23-55. 2010. 429 Origin of the viruses and their evolutionary history Origin of the viruses and their evolutionary history [2] J. Chen, AA. Gershon, Z Li, RA. Cowls, MD. Gershon. Varicella zoster virus (VZV) infects and established latency in enteric neurons. J of NeuroVirol 17:578-589. 2011. [3] C. Esposito, A. Di Spirito, N Cuomo, G. Di Nicuolo, G. Flaminio, f. Altamura, D. Ambrosino, F. Cantalupo, C. Costa, F. Pentimalli, G. Tarro. Tracking the 2009 H1N1 influenza virus in the Italian region Campania. Journal of Cellular Physiology 227:2813-2817. 2012. [4] MA. Feitelson, H. Reis, J. Pan, B. Sun. Pathogenesis of acute abd chronic hepatitis C virus infection in Viral Oncology Khalili K., Jeang K-T. (Eds), Wiley – Blackwell, Hoboken, New Jersey, USA, 243-266. 2010. [5] DN. Fisman, R. Savage, J. Gubbay et al. Older age and a reduced likelihood of 2009 H1N1 virus infection. N Engl J Med 361, 2000-1. 2009. [6] GS. Hayward, JA. Donald, R. Arav-Boger. The role of KSHV in pathogenesis of Kaposi’s sarcoma in Viral Oncology Khalili K., Jeang K-T. (Eds), Wiley – Blackwell, Hoboken, New Jersey, USA, 377-407. 2010. [7] MJ. Imperiale, D. Das. Possible association of BK virus with prostate cancer in Viral Oncology Khalili K., Jeang K-T. (Eds), Wiley – Blackwell, Hoboken, New Jersey, USA, 129-148. 2010. [8] S. Jain, L. Kamimoto, AM. Brambley et al. Hospitalized patients with 2009 H1N1 influenza in the United States, April-June 2009. N Engl J Med 361, 1935-44. 2009. [9] JS. Pagano. Molecular pathobiology of EBV infection in Viral Oncology Khalili K., Jeang K-T. (Eds), Wiley – Blackwell, Hoboken, New Jersey, USA, 409-423. 2010. [10] K. Reiss, K. Khalili, L. Del Valle. JC virus association with brain tumors: the role of T antigen and insulin-like growth factor 1 in DNA repair fidelity in Viral Oncology Khalili K., Jeang K-T. (Eds), Wiley – Blackwell, Hoboken, New Jersey, USA, 89-111, 2010. 430 Giulio Tarro [11] B. Roizman, G. Zhou, T. Du. Checkpoint in productive and latent infections with herpes simplex virus 1: conceptualization of the issues. J of Neuro Virol 17:512-517. 2012. [11] B. Roizman, G. Zhou, T. Du. Checkpoint in productive and latent infections with herpes simplex virus 1: conceptualization of the issues. J of Neuro Virol 17:512-517. 2012. [12] G. Tarro. TLP sequenced and studies for its functions with targets diagnostic and therapeutic. Advanced Studies in Biology 5:191-198. 2013. [13] M. Thomas, D. Pim, L. Banks. The role of the human papillomavirus E6 oncoprotein in malignant progression in Viral Oncology Khalili K., Jeang K-T. Origin of the viruses and their evolutionary history (Eds), Wiley – Blackwell, Hoboken, New Jersey, USA, 57-58. 2010. [14] RL. Thompson, NM. Sawtell. The herpes simplex virus type 1 latency associated transcript locus is required for the maintenance of reactivation competent latent infections. J of NeuroVirol 17:552-558. 2011. [15] H. Zur Hausen. Infections causing human cancer. Wiley – VCH: Weinheim, Germany, p 532. 2007. Received: August 15, 2013 Received: August 15, 2013
https://openalex.org/W2081458437	https://bmcgastroenterol.biomedcentral.com/counter/pdf/10.1186/1471-230X-13-139	English	null	Klebsiella pneumoniae bacteremia and renosplenic abscesses without intestinal symptoms as the initial manifestations of non-steroidal anti-inflammatory drug-induced colitis: a rare case report	BMC gastroenterology	2,013	cc-by	4,272	* Correspondence: 940268@ms.kmuh.org.tw 1Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100, Tzyou 1st Road, Kaohsiung 807, Taiwan Full list of author information is available at the end of the article Abstract Background: Non-steroidal anti-inflammatory drugs (NSAIDs), the most widely prescribed drugs in the world, can cause gastrointestinal damage, including colitis. However, the prevalence of NSAID-induced colitis is unknown because the disease is often asymptomatic. Case presentation: We report the case of a 64-year-old female patient with a history of long-term NSAID use, who was hospitalized with septic shock caused by Klebsiella pneumoniae bacteremia. Computed tomography revealed multiple renal and splenic abscesses with diffuse colon wall thickening. A colonoscopy confirmed colitis with diffuse ulcers. NSAIDs were discontinued after this hospitalization. The abscesses improved after antibiotic treatment. A short course of balsalazide treatment was given under the suspicion of ulcerative colitis. Balsalazide was discontinued four months later due to a non-compatible clinical course. A follow-up colonoscopy two years later revealed a normal colon mucosa, and NSAID-induced colitis was diagnosed. Conclusion: This is the first reported case of combined bacterial splenic and renal abscesses without intestinal manifestations as the initial presentation of NSAID-induced colitis. In contrast to cases of K. pneumoniae bacteremia with primary liver abscesses in patients with diabetes mellitus in Taiwan, we presented the first case with abscesses caused by community-acquired K. pneumoniae in the kidneys and spleen without liver invasion. In conclusion, our case report alerts clinicians to the possibility that K. pneumoniae bacteremia combined with multiple abscesses can be associated with severe NSAID-induced colitis. Keywords: Non-steroidal anti-inflammatory drugs, Colitis, Bacteremia, Klebsiella pneumoniae, Renosplenic abscesses steroidal anti-inflammatory drugs, Colitis, Bacteremia, Klebsiella pneumoniae, Renosplenic abscesses Keywords: Non-steroidal anti-inflammatory drugs, Colitis, Bacteremia, Klebsiella pneumoniae, Ren Klebsiella pneumoniae bacteremia and renosplenic abscesses without intestinal symptoms as the initial manifestations of non-steroidal anti-inflammatory drug-induced colitis: a rare case report Klebsiella pneumoniae bacteremia and renosplenic abscesses without intestinal symptoms as the initial manifestations of non-steroidal anti-inflammatory drug-induced colitis: a rare case report Hung-Ling Huang3, Po-Liang Lu1,4, Chun-Yu Lin1,4, Yen-Hsu Chen1,3,4,5, Chao-Hung Kuo2 and Wei-Ru Lin1* Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 CASE REPORT Open Access © 2013 Huang 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. © 2013 Huang 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. Background found that the prevalence of NSAID enteropathy was around 44% [1]. Studies evaluating the colonic side effects of NSAIDs have suggested that NSAID-induced colitis is common; however, symptomatic NSAID- induced colitis is rare [2,3]. People taking NSAIDs are two to five times more likely to develop colonic inflam- mation than the general population. NSAIDs affect the large intestine by causing colonic ulceration and stric- ture formation. Approximately 10% of newly diagnosed colitis cases may be related to NSAID administration [4,5]. However, the prevalence of NSAID-induced colitis is unknown because the disease is often asymptomatic. Non-steroidal anti-inflammatory drugs (NSAIDs), despite their well-known adverse effects on the gastrointestinal (GI) tract, are widely prescribed worldwide. NSAIDs dam- age different regions of the GI tract, including the distal small bowel and colon can be the target of NSAIDs. A study using fecal calprotectin (a non-degraded neutro- phil cytosolic protein) to diagnose NSAID enteropathy * Correspondence: 940268@ms.kmuh.org.tw 1Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100, Tzyou 1st Road, Kaohsiung 807, Taiwan Full list of author information is available at the end of the article Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 Page 2 of 6 Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 Page 2 of 6 in the submucosal layer over the terminal ileum, cecum, ascending colon, and descending colon with perifocal fat stranding. The hyperemic change was compatible with the inflammatory reaction in the bowel. The CT also revealed more intense wall enhancement at the ter- minal ileum (Figure 2). A colonoscopy revealed diffuse ulceration with exudation in the distal terminal ileum and segmental ulceration in the colon, with a reduction in the lumen caliber in the sigmoid colon (Figure 3). The pathological analysis of colon biopsy specimens re- vealed ulcers, necrotic debris, and the infiltration of lym- phocytes into the edematous lamina propria (Figure 4). Three weeks later, a follow-up abdominal ultrasonography demonstrated partial resolution of the renal and splenic abscesses. NSAID-induced colitis usually has nonspecific histo- logical findings. The diagnosis of NSAID-induced co- lonic ulceration has been made based on a history of NSAID use and the exclusion of other causes [2]. The temporal relationship between NSAID use and symp- toms that resolve after cessation confirm the diagnosis of NSAID-induced colitis. Intra-abdominal abscesses as the initial presentation of NSAID-induced colitis has not been reported previ- ously. Case presentation The patient was treated in the outpatient department with 500 mg of oral cephradine, four times daily; 500 mg of metronidazole, three times daily; and balsalazide (2.25 g daily) for two months. The follow-up abdominal CT scan revealed complete resolution of the abscesses two months later, and the colonoscopy revealed multiple segmental longitudinal ulceration with skip lesions in the rectum, sigmoid, and terminal ileum. A colon biopsy showed necrotizing inflammation with the infiltration of mixed inflammatory cells ,including neutrophils, histio- cytes, and lymphocytes. Moreover, focal ulcerated colonic mucosa with inflammatory infiltration was found in the edematous, fibrous lamina propria. Some distorted glands were observed, but no crypt abscesses were noted. Balsalazide was discontinued after a course of four months. She had an uneventful course in the out-patient depart- ment, and a follow-up colonoscopy two years later revealed melanosis coli and internal hemorrhoid only, without ul- cers or stenosis over the entire colon. A 64-year-old Taiwanese woman presented with a his- tory of well-controlled type 2 diabetes mellitus and hypertension. She had been self-treating regularly with NSAIDs from pharmacy (diclofenac, 50 mg three times daily) for more than two years because of chronic low back pain caused by intervertebral disc herniation. Gen- eral malaise, poor appetite, intermittent postprandial nausea, urinary urgency and frequency were present one month before admission. She visited local clinics and re- ceived no specific diagnosis. Due to progressive shortness of breath and drowsiness for one week, she was sent to a medical center. She appeared acutely ill but afebrile, with a blood pressure of 74/34 mmHg, a pulse rate of 86/min, a respiratory rate of 22/min, and an oxygen saturation level of 93% in ambient air. Physical examination revealed no specific local findings. A blood assay revealed the following findings: leukocyte count, 44,400/μL with 85% neutrophils; hemoglobin, 12.3 g/dL; platelet count, 466, 000/μL; C-reactive protein, 235.76 mg/L; total bilirubin, 1.17 mg/dL; direct bilirubin, 0.17 mg/dL; aspartate amino- transferase, 38 IU/L; alanine aminotransferase, 21 IU/L; blood urea nitrogen, 96.77 mg/dL; creatinine, 7.8 mg/dL; sodium, 140 mmol/L; potassium, 8.6 mmol/L; and lactate, 20.3 mmol/L. The arterial blood gas analysis revealed se- vere metabolic acidosis (pH, 7.02; HCO3, 7.1 mmol/L). A chest radiograph revealed no pulmonary lesions. Urine analysis via catheterization revealed only mild hematuria. Background Herein, we report the unusual case of a patient with renal and splenic abscesses but without intestinal symptom as the initial manifestations of NSAID- induced colitis. Discussion Our case is unique due to the presentation of severe col- itis without an initial manifestation of intestinal symp- toms. NSAID-induced colitis was diagnosed based on the history of long-term NSAID use after excluding other etiologies. To the best of our knowledge, this is the first reported case of NSAID-induced colitis with renal and splenic abscesses caused by community- acquired K. pneumoniae without liver involvement. She was admitted to the intensive care unit due to septic shock, acute respiratory failure, acute renal failure, and hyperkalemia. She received mechanical ventilation, intravenous vasopressors, continuous veno- venous hemofiltration, and cefmetazole (2 g/day) was prescribed empirically. All blood cultures yielded K. pneumoniae. The urine culture was sterile. Contrast- enhanced abdominal computed tomography (CT) re- vealed multiple abscesses in the spleen and bilateral kidneys, with the largest lesion (approximately 5.2 cm in diameter) located in the right kidney (Figure 1). In addition, CT also revealed segmental bowel wall edema K. pneumoniae, a gram-negative encapsulated aerobic bacterium, is part of the normal flora of the human mouth and intestines. Human carrier rates in the com- munity range from 5 to 38% in the stool and from 1 to 6% in the nasopharynx [6,7]. The infection rate is higher for individuals with impaired host defenses, such as indi- viduals with diabetes mellitus and hepatobiliary tract dis- ease [8,9]. K. pneumoniae has been reported to be the most common pathogen associated with pyogenic hep- atic abscesses and splenic abscesses, especially in Taiwan Page 3 of 6 Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 Figure 1 Computed tomography scans of the abdomen revealing several rim-enhancing hypodense lesions in bilateral kidneys and the spleen. Figure 1 Computed tomography scans of the abdomen revealing several rim-enhancing hypodense lesions in bilateral kidneys and the spleen. ted tomography scans of the abdomen revealing several rim-enhancing hypodense lesions in bilateral kidneys and and countries in Eastern and Southeast Asia [10-12], and this bacterium accounts for 10–16% of cases of splenic abscess and approximately 25% of cases of renal abscess [13,14]. 19 years and found that K. pneumoniae was the most frequently encountered pathogen in blood cultures or abscess cultures, similar to observations from other Asian countries [11]. Ferraioli G. et al. found a different result; they observed that the most common pathogens were polymicrobial pathogens in pus cultures and Staphylococcus species in blood cultures [16]. Intra-abdominal abscesses have diverse symptoms. Discussion Fever and abdominal pain are the most frequent symp- toms, but a few cases have vague symptoms, as in the case presented herein. Imaging analysis, including ultra- sonography and computed tomography (CT), are neces- sary to diagnose intra-abdominal infections [15]. The pathogenesis of intra-abdominal abscesses caused by K. pneumoniae remains unclear. One hypothesis for the pathogenesis of pyogenic intra-abdominal abscesses involves hematogenous bacterial seeding from the GI tract. An animal study demonstrated that K. pneumoniae strains with genetic regulatory networks for translocation Splenic abscesses are rare, with a reported frequency of 0.14–0.7% in case reports [16]. Chang et al. analyzed 67 splenic abscess cases which occurred over a period of Figure 2 Computed axial tomography scans of the abdomen showing marked thickening of the wall of the terminal ileum, cecum, ascending colon, descending colon, and rectum with perifocal fat stranding. These finding are compatible with the findings of the colonoscopy. Figure 2 Computed axial tomography scans of the abdomen showing marked thickening of the wall of the terminal ileum, cecum, ascending colon, descending colon, and rectum with perifocal fat stranding. These finding are compatible with the findings of the colonoscopy. Page 4 of 6 Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 Figure 3 A colonoscopy revealed extensive segmental longitudinal ulceration of the mucosa extending over the rectum, sigmoid colon, and terminal ileum. revealed extensive segmental longitudinal ulceration of the mucosa extending over the rectum, sigmoid Figure 3 A colonoscopy revealed extensive segmental longitudinal ulceration of the mucosa extending over the rectum, sigmoid colon and terminal ileum Figure 3 A colonoscopy revealed extensive segmental longitudinal ulceration of the mucosa extending over the rectum, sigmoid colon, and terminal ileum. have the ability to cross the intestinal barrier [17]. A care- ful search for the source of K. pneumoniae bacteremia other than primary bacteremia should be considered, and which should include colon lesions survey [18]. In our case, the finding of thickened colonic wall prompted us to consider the colon as the possible source of K. pneumoniae bacteremia. have the ability to cross the intestinal barrier [17]. A care- ful search for the source of K. pneumoniae bacteremia other than primary bacteremia should be considered, and which should include colon lesions survey [18]. In our case, the finding of thickened colonic wall prompted us to consider the colon as the possible source of K. pneumoniae bacteremia. Discussion involved in the maintenance of microcirculation and blood flow to modulate the mucosal immune system. NSAIDs inhibit colonic prostaglandin synthesis, leading to the development of colitis and the aggravation of preexisting intestinal diseases. The ileocecal region is the most common site of NSAID-induced colonic injury. Pathological examination of colon specimens from patients with NSAID-induced colitis usually reveals sharply demarcated, semilunar or Prostaglandins represent one of the most important components of mucosal defense in the colon and are Figure 4 Photomicrograph of a sigmoid colon biopsy specimen displaying an ulcer, necrotic debris, and lymphocyte infiltration into the edematous lamina propria (hematoxylin and eosin, original magnification ×100). Figure 4 Photomicrograph of a sigmoid colon biopsy specimen displaying an ulcer, necrotic debris, and lymphocyte infiltration into the edematous lamina propria (hematoxylin and eosin, original magnification ×100). Page 5 of 6 Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 Page 5 of 6 Page 5 of 6 Conclusions We present a unique case of NSAID-induced colitis with- out any GI symptoms at initial presentation, that was as- sociated with septic shock caused by K. pneumoniae bacteremia with multiple renal and splenic abscesses. The colitis was found incidentally by CT and then iden- tified as NSAID-induced colitis. This is also the first reported case of disseminated abscess formation caused by community-acquired K. pneumoniae in the kidneys and spleen without liver involvement in an NSAID- induced colitis patient. 6. Fung CP, Lin YT, Lin JC, Chen TL, Yeh KM, Chang FY, Chuang HC, Wu HS, Tseng CP, Siu LK: Klebsiella pneumoniae in gastrointestinal tract and pyogenic liver abscess. Emerg Infect Dis 2012, 18:1322–1325. py g g 7. Podschun R, Ullmann U: Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998, 11:589–603. 8. Kang CI, Kim SH, Bang JW, Kim HB, Kim NJ, Kim EC, Oh MD, Choe KW: Community-acquired versus nosocomial Klebsiella pneumoniae bacteremia: clinical features, treatment outcomes, and clinical implication of antimicrobial resistance. J Korean Med Sci 2006, 21:816–822 8. Kang CI, Kim SH, Bang JW, Kim HB, Kim NJ, Kim EC, Oh MD, Choe KW: Community-acquired versus nosocomial Klebsiella pneumoniae bacteremia: clinical features, treatment outcomes, and clinical implication of antimicrobial resistance. J Korean Med Sci 2006, 21:816–822. 9. Wu HS, Wang FD, Tseng CP, Wu TH, Lin YT, Fung CP: Characteristics of healthcare-associated and community-acquired Klebsiella pneumoniae bacteremia in Taiwan. J Infect 2012, 64:162–168. 9. Wu HS, Wang FD, Tseng CP, Wu TH, Lin YT, Fung CP: Characteristics of healthcare-associated and community-acquired Klebsiella pneumoniae bacteremia in Taiwan. J Infect 2012, 64:162–168. This case is a reminder that hematogenous intra- abdominal abscesses caused by K. pneumoniae necessi- tate a thorough investigation of the GI tract. Assessing the presence of colitis should be considered even in the absence of preceding gastrointestinal symptoms, espe- cially for those who have previously used NSAIDs. 10. Lederman ER, Crum NF: Pyogenic liver abscess with a focus on Klebsiella pneumoniae as a primary pathogen: an emerging disease with unique clinical characteristics. Am J Gastroenterol 2005, 100:322–331. 10. Lederman ER, Crum NF: Pyogenic liver abscess with a focus on Klebsiella pneumoniae as a primary pathogen: an emerging disease with unique clinical characteristics. Am J Gastroenterol 2005, 100:322–331. 11. Authors’ contributions Authors contributions HLH, WRL, and PLL prepared the manuscript. YHC and CHK provided laboratory support. WRL and CHK cared for the patient and provided advice on the clinical aspects of the case report. All authors read and approved the final version of the manuscript. References D h 1. Deshpande V, Hsu M, Kumarasinghe MP, Lauwers GY: The clinical significance of incidental chronic colitis: a study of 17 cases. Am J Surg Pathol 2010, 34:463–469. 1. Deshpande V, Hsu M, Kumarasinghe MP, Lauwers GY: The clinical significance of incidental chronic colitis: a study of 17 cases. Am J Surg Pathol 2010, 34:463–469. The discontinuation of NSAIDs is the cornerstone of the treatment of NSAID-induced colitis. Symptoms usu- ally resolve within days to weeks of the withdrawal of NSAIDs, with the restoration of normal histology. Our patient received a colonoscopy two years later that re- vealed complete restoration of the colon mucosa; thus, this patient was diagnosed with NSAID-induced colitis. 2. Kurahara K, Matsumoto T, Iida M, Honda K, Yao T, Fujishima M: Clinical and endoscopic features of nonsteroidal anti-inflammatory drug-induced colonic ulcerations. Am J Gastroenterol 2001, 96:473–480. 2. Kurahara K, Matsumoto T, Iida M, Honda K, Yao T, Fujishima M: Clinical and endoscopic features of nonsteroidal anti-inflammatory drug-induced colonic ulcerations. Am J Gastroenterol 2001, 96:473–480. 3. Puspok A, Kiener HP, Oberhuber G: Clinical, endoscopic, and histologic spectrum of nonsteroidal anti-inflammatory drug-induced lesions in the colon. Dis Colon Rectum 2000, 43:685–691. 4. Tanner AR, Raghunath AS: Colonic inflammation and nonsteroidal anti- inflammatory drug administration. An assessment of the frequency of the problem. Digestion 1988, 41:116–120. p g 5. Katsinelos P, Christodoulou K, Pilpilidis I, Xiarchos P, Papagiannis A, Dimiropoulos S, Amperiadis P, Vasiliadis T, Tarpagos A, Katsos I, Eugenidis N Colopathy associated with the systemic use of nonsteroidal antiinflammatory medications. An underestimated entity. Hepatogastroenterology 2002, 49:345–348. Abbreviations NSAID N t Competing interests The authors declared that they have no competing interests. Author details 1 1Division of Infectious Diseases, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100, Tzyou 1st Road, Kaohsiung 807, Taiwan. 2Division of Gastroenterology, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. 3Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan. 4School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. 5Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan In this case, the colonoscopy findings and pathological findings were not able to determine the etiology of the colitis. Due to evidence of a possible association between the use of NSAIDs and the exacerbation or relapse of in- flammatory bowel diseases [26,27], NSAID-exacerbated inflammatory bowel disease was initially suspected. This patient was initially treated with balsalazide, but she be- came stable despite the discontinuation of balsalazide. Received: 26 April 2013 Accepted: 20 September 2013 Published: 22 September 2013 Competing interests The authors declared that they have no competing interests. There have been no studies to determine the risk of colitis related to different types of NSAIDs; however, many reports have stated that diclofenac, mefenemic acid, piroxicam, and ibuprofen could cause colorectal ulcers or colitis [4,20-22]. According to one case-controlled study, the long-term use of NSAIDs increased the risk of colonic mucosal lesions, suggesting that NSAIDs may contribute to the pathogenesis of colonic ulcers or colitis [23]. In addition, naproxen has been implicated in eosinophilic colitis [24], and diclofenac may be associated with pseudo- membranous colitis [25]. Consent circumferential, superficial ulcerations with normal sur- rounding mucosa. There are no specific histopatho- logical findings for NSAID-induced colitis except for the diaphragm-like stricture. Cellular infiltration into the lamina propria and epithelium may involve predom- inantly neutrophils or lymphocytes, depending on the chronicity of the lesion [19]. Symptomatic patients may have watery or bloody diarrhea, weight loss, fatigue, abdominal pain, anorexia, iron-deficiency anemia, and hypoalbuminemia. Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for re- view by the Editor of this journal. Abbreviations NSAIDs: Non-steroidal anti-inflammatory drugs; GI: Gastrointestinal; CT: Computed tomography. 11. Chang KC, Chuah SK, Changchien CS, Tsai TL, Lu SN, Chiu YC, Chen YS, Wang CC, Lin JW, Lee CM, Hu TH: Clinical characteristics and prognostic y g g 7. Podschun R, Ullmann U: Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998, 11:589–603. 8. Kang CI, Kim SH, Bang JW, Kim HB, Kim NJ, Kim EC, Oh MD, Choe KW: Community-acquired versus nosocomial Klebsiella pneumoniae bacteremia: clinical features, treatment outcomes, and clinical implication of antimicrobial resistance. J Korean Med Sci 2006, 21:816–82 Conclusions Chang KC, Chuah SK, Changchien CS, Tsai TL, Lu SN, Chiu YC, Chen YS, Wang CC, Lin JW, Lee CM, Hu TH: Clinical characteristics and prognostic Page 6 of 6 Page 6 of 6 Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 Huang et al. BMC Gastroenterology 2013, 13:139 http://www.biomedcentral.com/1471-230X/13/139 factors of splenic abscess: a review of 67 cases in a single medical center of Taiwan. World J Gastroenterol 2006, 12:460–464. factors of splenic abscess: a review of 67 cases in a single medical center of Taiwan. World J Gastroenterol 2006, 12:460–464. 12. Siu LK, Yeh KM, Lin JC, Fung CP, Chang FY: Klebsiella pneumoniae liver abscess: a new invasive syndrome. Lancet Infect Dis 2012, 12:881–887. 13. Lee CH, Leu HS, Hu TH, Liu JW: Splenic abscess in southern Taiwan. J Microbiol Immunol Infect 2004, 37:39–44. 13. Lee CH, Leu HS, Hu TH, Liu JW: Splenic absc J Microbiol Immunol Infect 2004, 37:39–44. 13. Lee CH, Leu HS, Hu TH, Liu JW: Splenic abscess in southe J Microbiol Immunol Infect 2004, 37:39–44. 14. Lee BE, Seol HY, Kim TK, Seong EY, Song SH, Lee DW, Lee SB, Kwak IS: Recent clinical overview of renal and perirenal abscesses in 56 consecutive cases. Korean J Intern Med 2008, 23:140–148. 15. Solomkin JS, Mazuski JE, Bradley JS, Rodvold KA, Goldstein EJ, Baron EJ, O’Neill PJ, Chow AW, Dellinger EP, Eachempati SR, et al: Diagnosis and management of complicated intra-abdominal infection in adults and children: guidelines by the Surgical Infection Society and the Infectious Diseases Society of America. Clin Infect Dis 2010, 50:133–164. Diseases Society of America. Clin Infect Dis 2010, 50:133–164. 16. Ferraioli G, Brunetti E, Gulizia R, Mariani G, Marone P, Filice C: Management of splenic abscess: report on 16 cases from a single center. Int J Infect Dis 2009, 13:524–530. 17. Tu YC, Lu MC, Chiang MK, Huang SP, Peng HL, Chang HY, Jan MS, Lai YC: Genetic requirements for Klebsiella pneumoniae-induced liver abscess in an oral infection model. Infect Immun 2009, 77:2657–2671. 18. Jeong SW, Jang JY, Lee TH, Kim HG, Hong SW, Park SH, Kim SG, Cheon YK, Kim YS, Cho YD, et al: Cryptogenic pyogenic liver abscess as the herald of colon cancer. J Gastroenterol Hepatol 2012, 27:248–255. 19. Goldstein NS, Cinenza AN: The histopathology of nonsteroidal anti- inflammatory drug-associated colitis. Am J Clin Pathol 1998, 110:622–628. y g 20. Conclusions Ravi S, Keat A, Keat E: Colitis caused by non-steroidal anti-inflammatory drugs. Postgrad Med J 1986, 62:773–776. 21. Witham R: Voltaren (diclofenac sodium)-induced ileocolitis 21. Witham R: Voltaren (diclofenac sodium)-induced ileocolitis. Am J Gastroenterol 1991, 86:246–247. Am J Gastroenterol 1991, 86:246–247. 22. Pan YS, Chen LT, Tseng CA, Su YC, Jan CM, Wang WM, Tsai KB: Clinical and endoscopic features of non-steroidal anti-inflammatory drug-induced colorectal ulcerations. J Formos Med Assoc 2005, 104:804–810. 22. Pan YS, Chen LT, Tseng CA, Su YC, Jan CM, Wang WM, Tsai KB: Clinical and endoscopic features of non-steroidal anti-inflammatory drug-induced colorectal ulcerations. J Formos Med Assoc 2005, 104:804–810. 23. Shibuya T, Ohkusa T, Yokoyama T, Harada A, Beppu K, Sakamoto N, Osada T, Nagahara A, Terai T, Otaka M: Colonic mucosal lesions associated with long‐term or short‐term administration of nonsteroidal anti‐ inflammatory drugs. Colorectal Dis 2010, 12:1113–1121. inflammatory drugs. Colorectal Dis 2010, 12:1113–1121. 24. Alfadda AA, Storr MA, Shaffer EA: Eosinophilic colitis: epidemiology, clinical features, and current management. Therap Adv Gastroenterol 2011, 4:301–309. 24. Alfadda AA, Storr MA, Shaffer EA: Eosinophilic colitis: epidemiology, clinical features, and current management. Therap Adv Gastroenterol 2011, 4:301–309. 25. Gentric A, Pennec Y: Diclofenac-induced pseudomembranous colitis. Lancet 1992, 340:126–127. 26. Ballinger A: Adverse effects of nonsteroidal anti-inflammatory drugs on the colon. Curr Gastroenterol Rep 2008, 10:485–489. 26. Ballinger A: Adverse effects of nonsteroidal anti-inflammatory drugs on the colon. Curr Gastroenterol Rep 2008, 10:485–489. 27. Kefalakes H, Stylianides TJ, Amanakis G, Kolios G: Exacerbation of inflammatory bowel diseases associated with the use of nonsteroidal anti-inflammatory drugs: myth or reality? Eur J Clin Pharmacol 2009, 65:963–970. 27. Kefalakes H, Stylianides TJ, Amanakis G, Kolios G: Exacerbation of inflammatory bowel diseases associated with the use of nonsteroidal anti-inflammatory drugs: myth or reality? Eur J Clin Pharmacol 2009, 65:963–970. doi:10.1186/1471-230X-13-139 Cite this article as: Huang et al.: Klebsiella pneumoniae bacteremia and renosplenic abscesses without intestinal symptoms as the initial manifestations of non-steroidal anti-inflammatory drug-induced colitis: a rare case report. BMC Gastroenterology 2013 13:139. Conclusions Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission
https://openalex.org/W2062755486	https://zenodo.org/records/2453621/files/article.pdf	English	null	TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY.	JAMA	1,901	public-domain	6,336	DISCUSSION. Statistics show an entirely different condition in the production of tuber- culosis of the abdominal organs from that in tuberculosis of the lungs; an indication that as tuberculosis in cattle has in- creased, tuberculosis in children has also increased. y g Dr. R. Harvey Cook, Oxford, Ohio—I would like to ask Dr. Salmon to give more in detail the reason why an animal during parturition is not good for food, other than from an esthetic idea and also from the fact that an animal is at this time more suspectible to disease. , In regard to the effect of gestation upon the flesh of animals, I believe that the physiologic changes which occur in the last stages of the period of gestation do affect the general health. I think that the waste products from a fetus which are thrown into the circulation of the mother have a certain effect upon the flesh of the animal. I think also that the physiologic preparations for parturition must have great effect. We know the effect upon the composition of the milk and it must have almost equal effect upon the composition of the flesh. Yet, as I have said, we have very little data to show what bad effects the meat has upon the consumer. I would myself rather eat horse or mule flesh than eat the flesh of animals in the last two weeks of the period of gestation, and I think most people would feel as I do. The sale of the meat, therefore, is just as properly prohibited as would be the flesh of other animals not usually used for food. p Dr. Salmon, in reply—I regret to say that there are many questions connected with the subject that I am unable to answer, especially with regard to the effect of exhaustion and fright on the flesh of the animal. The effect on deer chased by dogs is something of the same sort as that which happens to animals shipped long distances to market. We have a Federal law in this country that animals should be unloaded as often as once in 28 hours for feed, water and rest. Like many other statutes, it is honored chiefly by paying no atten- tion to it. I have been for several years sending out men with stock trains and getting evidence against the railroad com- panies and bringing prosecutions. DISCUSSION. Dr. Charles M. Hazen—I wish Dr. Salmon in closing might say a few words about the disturbances not amounting to pathologic conditions. For instance, the question of venison killed after a long chase and without bleeding being fit for food. Such questions would be interesting in this discussion. There has been some difference of opinion as to whether the milk was infectious when the mammary glands were free from tubercular deposit. A number of experiments have been made showing that a considerable proportion of tuberculous cows give infectious milk even when the udders are apparently healthy. The proportion of cows which have reacted to tuberculin and in which infection has been found in the milk varied from 5 to 66 per cent. When you have herds of cows of a hundred animals with 75 per cent, of them tuberculous, as is often the case, and a third giving infectious milk, there is a tremendous quantity of infection spread through the milk of these herds. q g Dr. Winfiei.d S. Hall, Chicago—I think this paper opens up an exceedingly wide field for research. How wide a field do the experiments now being conducted cover and are the questions asked in line for being solved in the Washington laboratories, and especially, how far can tuberculous milk be taken without endangering the health? g g Dr. David Paulson, Chicago—I have become acquainted, to a certain extent, with the benefits which are derived from the efficient work that is performed under the direction of the gov- ernment, as outlined by Dr. Salmon. . It is unfortunate that as far as Chicago is concerned it is still possible to dispose of diseased cattle to smaller slaughter houses outside of the regular packing-house district. The fact that animals are being continually killed in these smaller slaughter houses without any suitable examination and their flesh placed upon the market in Chicago, should certainly cause us not to relax our efforts in this direction until some definite provision is secured that fully covers this ground. You may be aware that many physicians have denied, with more or less positiveness, the danger of tuberculosis from in- fected cows, either from the meat or the milk. Postmortem examinations of adults seem to confirm their views so far as they go. However, they are not as complete as we should like to see them. With children the case is otherwise. y Read in a Symposium on Tuberculosis at the Fifty-second An- nual Meeting of the American Medical Association, in the Section on Hygiene and Sanitary Science, and approved for publication by the Executive Committee of the Section. DISCUSSION. A number of convictions have been made, but they have not had as much effect as would be supposed. Persons who ship stock to marked want to get it through as soon as possible. There is surprising barbarity exhibited in the shipping of food-producing animals to market. I have seen cases in which animals in hot weather have been kept in stock-cars without food, water or rest for 96 hours. When unloaded in stockyards they distend themselves enor- mously with water, which puts them in a worse condition than when they were unloaded. I believe that that kind of meat is not in the most wholesome condition for those who consume it. Just what effect it may have on the consumer I do not know. I have no data, but believe that many of the minor troubles which occur are due to such cases. Every physician knows that there are many illnesses for which he can not ac- count. When we consider the amount of meat consumed which is shipped in that way, I think we will agree that there must be a considerable effect on the health of the consumer. There is an opportunity for a great deal of close observation and experimentation to show the importance of correcting such conditions. by the men in the active practice of medicine. Would it not be well to have more co-operation and a more fre- quent exchange of views? which the whole gland was practically a mass of liquid caseous material having free connection with the milk ducts and was passing out in tremendous quantities with the milk. This only happens in a few cases. When we consider that, in large numbers of tests, 20 to 50 per cent, of the animals are found infected and that one to two in every hundred have tubercu- losis of the udder we see that in a great many cases the milk must be infected. which the whole gland was practically a mass of liquid caseous material having free connection with the milk ducts and was passing out in tremendous quantities with the milk. This only happens in a few cases. When we consider that, in large numbers of tests, 20 to 50 per cent, of the animals are found infected and that one to two in every hundred have tubercu- losis of the udder we see that in a great many cases the milk must be infected. Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. do not occur suddenly, although a great deal of snow falls in the north. The winters are equable and the climate is bracing. The summers are short and hot, while the autumn is delightful. As a whole the climate is healthy and invigorating. The surface of the state, for the most part, is a great plain, varied only by the cliffs bordering the rivers and lakes, and elevated from 600 to 1500 feet above the sea. The northern part of -the state is covered with immense forests of pine and hemlock. mountains on the west, the Mississippi river draining the valley which slopes toward its shores. The alti- tude of this valley varies from 400 to the highest mountain tops of 3000 feet. But the greater portion of the valley is free from mountains or hills of any great altitude. In order to understand the conditions that might influence the course of disease in tuber- culous patients in this region, the following is a brief description of the climates and physical characteristics of the various states: Minnesota's salubrity of climate is well known. The purity of the air and the dryness of the winters has given the state a well-deserved reputation as a resort for pulmonary invalids. If I mistake not, the summers are warm, with breezy nights; the winters are cold, clear and dry, with a light snowfall. The surface of the state is an undulating plain with an average eleva- tion of 1000 feet above the sea, but in the northeast there is a group of low sand hills, which rise about 60u feet higher. One thirty-fifth of the entire area is cov- ered by lakes, which are in the region of heavy forests. That the state is free from influences that tend to develop consumption is proved by the fact that it stands low in the group of states having a large number of deaths from this disease. Michigan, owing to its wide extent of latitude, has a varying climate, being quite mild in the southern part, but cold and rigorous in the winter in the northern part. There is a difference of about seven degrees be- tween the temperature any given day in the northern and southern part, but the climate is healthful and the death-rate low. TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. The northern part of the state as a rule is level and undulating, while the southern part is broken by hills, ranging from 500 to 1000 feet in height, and mountain ranges, the Iron mountains in the southeast, and Ozark moun- tains in the southwest. The bottom lands of the Mis- sissippi are extensive, having many lakes and lagoons with islands which are never submerged by the severest floods. cou t y y The climate of upper Illinois is healthful, its prox- imity to the lake securing purity of the air, and the country is swept by bracing lake breezes. The lower part of the state suffers along the river bottoms and the swamp lands from malaria. The lowest altitude is 340 feet, and the highest 1150 above the Gulf of Mexico, the state being generally level, the soil very rich, especially along the river bottoms where a soil of vege- table mold forty feet in depth is found and heavy crops of corn have been raised many years without rotation or fertilizing. Illinois stands fifth in the list of mor- tality from consumption, which must be due to the fact that Chicago furnishes so large a number of cases as to increase the state's death-rate. ll d North Dakota has a dry and salubrious climate. The winters are cold and there are heavy snowfalls, and the; winter is prolonged but not so severely felt as in the same latitude in Minnesota. The summers are hot and dry. The temperature ranges from 20 F. below to 100 above zero during the year. Consumption is almost unknown in the state. The greater portion of North Dakota is a rolling prairie in which are many streams and lakes, but no swamps or marshes. The altitude varies from 670 feet on the eastern line to 1900 en the western line, as at Ft. Buford. Kentucky affords a climate which is generally de- lightful, the mean average being 57 degrees F. The greatest rainfall is in the spring and winter, the sum- mer and fall being usually dry. It has a splendid record for healthfulness. It is a tableland sloping gradually from the southeast to the northwest, the spurs of the Cumberland mountains breaking the southeast- ern part. None of the summits of this spur reach over 3000 feet altitude. TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. The upper peninsula of Michigan is tugged and rocky, broken up by hills in the western section which rise to a height of 2000 feet, while the lower peninsula is composed of plains and tablelands, with occasional prairie and considerable timber. f prairie The annual mean temperature of Ohio is from 50 to 54 degrees, the warmest section being the southwest, on the Ohio river. The climate is mild and healthful, although sudden changes are liable to occur. Ohio has no mountains, although the interior of the state is 1200 feet above the sea level. From this elevated re- gion the surface slopes to Lake Erie on the north 565 feet above the sea, and the Ohio river on the south 430 feet above the sea level. The great divide which makes the watershed passes diagonally across the state from the northeast to the west. The soil is fertile and ex- tensive forests of hard wood abound. Iowa is one of the healthiest states in the union; malarial, endemic and epidemic diseases are rare. The winters are severe on account of the north and north- west winds, but not unhealthful, while the heat of summer is relieved by the breezes. The surface of the state is undulating and beautiful, with alternate prairies and forests, while the rivers are lined by bold bluffs and charming ravines. In the northern portion of the state there are small lakes which form part of the group extending into Minnesota, Dakota and Wisconsin. The highest point of the state is in Dickinson County, 1650 feet above the sea, and there is a gradual slope to the southeast until at the mouth of the Des Moines river the altitude is only 444 feet. Passing west into Indiana we find a variable climate, especially in the winter when the winds prevail from the west and northwest from the lakes. The state, however, is generally healthful, and the death-rate is low. The state has few hills, but is principally com- posed of level or undulating land, the highest eleva- tion being 540 feet on the Ohio river at the mouth of the Wabash. The country near the lake is sandy. h lthf l it y Missouri has the most rigorous climate of all the states in the middle group, as the winters are extremely severe, and the summers hot. The state, however, is healthful, notwithstanding its climate. TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. JOHN A. ROBISON, A.M., M.D. Attending Physician to the Cook County and Presbyterian Hospi- tals; President Chicago Society of Internal Medicine ; Secretary Illinois Society for the Prevention of Consumption. CHICAGO. JOHN A. ROBISON, A.M., M.D. JOHN ROBISON, A.M., M.D. Attending Physician to the Cook County and Presbyterian Hospi- tals; President Chicago Society of Internal Medicine ; Secretary Illinois Society for the Prevention of Consumption. CHICAGO. When asked to present a paper on this subject I con- sented willingly, but it was willingness born of ignor- ance; I had no idea of the task before me. On inves- tigating the literature on the subject of tuberculosis in the Middle States, I found that the mass of in- formation concerning the climatology of these states and the factors which might cause or cure consumption was very meager. The subject soon outran the bounds of a twenty-minute paper. Therefore, I have ar- bitrarily selected the following states for consideration: Michigan, Ohio, Indiana, Illinois, Kentucky, Wisconsin, Minnesota, Iowa, Missouri, North and South Dakota, Nebraska, and Kansas. They lie between the Appa- lachian system of mountains on the east and the Rocky In regard to tuberculous milk, I had a paper before another section in which I took up the subject more in detail than 1 could here. I am a firm believer in the danger of milk from a tuberculous cow. There are a number of udders in the Patho- logic Exhibit which show to what extent tuberculosis may in- vade the mammary glands. This morning the specimens were so solidly frozen that I could not trace the connection of the tubercles with the milk ducts. I have, however, seen cases in Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 do not occur suddenly, although a great deal of snow falls in the north. The winters are equable and the climate is bracing. The summers are short and hot, while the autumn is delightful. As a whole the climate is healthy and invigorating. The surface of the state, for the most part, is a great plain, varied only by the cliffs bordering the rivers and lakes, and elevated from 600 to 1500 feet above the sea. The northern part of -the state is covered with immense forests of pine and hemlock. Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. Many of these sanatoria are established at points which do not present so many advantages as many places in the middle states, and yet they are doing good work. y In this brief review of the climates found in the middle states we fail to find any reason why tuber- culosis should be more prevalent in any of these states than in the older states of the east or along the coasts of the Atlantic and Pacific oceans. In fact, there are many reasons why tuberculosis should be less prevalent, one fact alone being prominent, that is, in this vast region there are only fourteen cities having a population exceeding 100,000, and only fifty-three cities exceed- ing 25.000 in population. Crowded cities favor the development and spread of tuberculosis, which undoubt- edly accounts for the fact that Illinois ranks fifth in the list of mortality. g The majority of these sanatoria have been established by private enterprise, individuals, or societies, inde- pendent of the various governments. But the good work done in these semi-private institutions has aroused the interest of governments, the crowned heads, and is now arousing a great interest among the legislators of the United States. The list given by Knopf demonstrates the following facts: There are in Europe 127 sanatoria devoted to the consumptive, 35 in the United States, 2 in Canada, and 2 in Australia and Asia and 3 in Mexico; 15 of the European sanatoria are under municipal or govern- mental control, and 5 in the United States under municipal, county, state or government control. y In the list of states which have the greatest mor- tality from tuberculosis the following states in the mid- dle region stand in this relation: Ohio third, Illinois fifth, Tennessee sixth, Missouri seventh, Kentucky eighth, Indiana ninth, Michigan thirteenth, Wisconsin eighteenth, Texas nineteenth, Iowa twentieth, Minne- sota twenty-first, and Mississippi twenty-fourth. The reason Ohio, Illinois and Indiana rank so high is be- cause they are states which contain large cities; Ohio has four cities of a population exceeding 100.000, and several which approximate this figure; Indiana has several large cities, and Illinois includes Chicago with its population of nearly two millions, and several well- populated cities. The southern states have a high death-rate owing to the prevalence of consumption among the colored people. TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. many authorities who claim that these result dependent on climate so much as on the s of the patient's daily life. If this be true, t need be no argument that the disease may in this region. The question would be rather method can the cure be effected? This problem seems to be working its ow in a practical way, and that is by the esta of sanatoria. Many of these sanatoria are e at points which do not present so many adva many places in the middle states, and yet they good work. The majority of these sanatoria have been e by private enterprise, individuals, or societ pendent of the various governments. But work done in these semi-private institutions ha the interest of governments, the crowned hea now arousing a great interest among the leg the United States. The list given by Knopf demonstrates the facts: There are in Europe 127 sanatoria d the consumptive, 35 in the United States, 2 in and 2 in Australia and Asia and 3 in Mexi the European sanatoria are under municipal o mental control, and 5 in the United Stat municipal, county, state or government cont The states have been slow to respond to th movement until within the last three years, states have been waking up to the fact that th make provision for this large class of depe tients. The first state to erect a sanatorium fo sumptive is Massachusetts, which completed torium in January, 1898. It has been in nearly three years with results encouraging to t degree. Cook County has a hospital at Dunning i tion with the infirmary for advanced cases sumption, and while the percentage of rec necessarily low, there has been an improveme condition of many patients, owing to the b of their surroundings and appropriate treatm institution is doing a great deal of good by d d f h d l d i peaks reach 7000 feet above the sea. The altitude of the state varies, as is well illustrated when you look on the map and note that Huron has an altitude of 753 feet, Yankton 1222, and Deadwood 4600. If there are no factors to cause the disease, except the increase of population, what are the possibilities for the cure of the disease in the middle states ? TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. The answer to this question must hinge on the ex- perience of observers who have treated consumption in the lowlands and the lower altitudes, and a searching analysis of the value of the higher altitudes in the tteatment of the disease, and whether the home treat- ment of consumption with all the comforts the word home suggests, without the so-called climate, will pro- duce as good results as the desired climate without the home comforts. , , Over the wide prairies of Nebraska sweep the moun- tain breezes and owing to the splendid drainage, the dry exhilarating atmosphere is untainted by malaria. Along its western boundary are the foothills of the Rocky mountains, and the commencement of the Black Hills, the drainage being toward the Missouri river. North Platte, about the middle of the state, has an altitude of 2841 feet, and the altitude rises as you journey westward. As to the results of treatment in the lowlands and lower altitudes we cam only take the statistics as fur- nished by those who have observed the results at re- sorts situated in the respective levels. The results of treatment demonstrate the curability of consumption in the lower altitudes in percentages varying from 38 to 75, according to the stage of the disease. There are many authorities who claim that these results are not dependent on climate so much as on the supervision of the patient's daily life. If this be true, then there need be no argument that the disease may be cured in this region. The question would be rather, by what method can the cure be effected? j y Passing down into Kansas we find that the winters are comparatively mild, the summers hot but not op- pressive, and the atmosphere extraordinarily pure and clear at all seasons. The pure atmosphere offers to persons suffering from pulmonary complaints great re- lief, and a chance for a continuous outdoor life. It has an elevation of 750 at the mouth of the Kansas river, but slopes upward to an elevation of 3800 feet on the western border. You can find cities where good ac- commodations can be obtained at various altitudes, a? for instance, Wichita, 1366 feet; Hutchinson, 1540 feet; and Garden City, 3000 feet. This problem seems to be working its own solution in a practical way, and that is by the establishment of sanatoria. Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. The eastern half of the tableland has an average altitude of 1000 with ridges 500 feet higher. Wi i h , South Dakota has a milder climate, the spring being earlier even than in the states further east. The climate is healthy and salubrious. Like its northern neighbor it is composed principally of prairie, rolling and inter- spersed with water-courses. In the southwestern por- tion commence the famous Black Hills, some of whose higher. Going to the extreme north we find Wisconsin, whose climate is tempered by the great lakes. Great changes Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 peaks reach 7000 feet above the sea. The altitude of the state varies, as is well illustrated when you look on the map and note that Huron has an altitude of 753 feet, Yankton 1222, and Deadwood 4600. Over the wide prairies of Nebraska sweep the moun- tain breezes and owing to the splendid drainage, the dry exhilarating atmosphere is untainted by malaria. Along its western boundary are the foothills of the Rocky mountains, and the commencement of the Black Hills, the drainage being toward the Missouri river. North Platte, about the middle of the state, has an altitude of 2841 feet, and the altitude rises as you journey westward. Passing down into Kansas we find that the winters are comparatively mild, the summers hot but not op- pressive, and the atmosphere extraordinarily pure and clear at all seasons. The pure atmosphere offers to persons suffering from pulmonary complaints great re- lief, and a chance for a continuous outdoor life. It has an elevation of 750 at the mouth of the Kansas river, but slopes upward to an elevation of 3800 feet on the western border. You can find cities where good ac- commodations can be obtained at various altitudes, a? for instance, Wichita, 1366 feet; Hutchinson, 1540 feet; and Garden City, 3000 feet. In this brief review of the climates found in the middle states we fail to find any reason why tuber- culosis should be more prevalent in any of these states than in the older states of the east or along the coasts of the Atlantic and Pacific oceans. TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. In fact, there are many reasons why tuberculosis should be less prevalent, one fact alone being prominent, that is, in this vast region there are only fourteen cities having a population exceeding 100,000, and only fifty-three cities exceed- ing 25.000 in population. Crowded cities favor the development and spread of tuberculosis, which undoubt- edly accounts for the fact that Illinois ranks fifth in the list of mortality. In the list of states which have the greatest mor- tality from tuberculosis the following states in the mid- dle region stand in this relation: Ohio third, Illinois fifth, Tennessee sixth, Missouri seventh, Kentucky eighth, Indiana ninth, Michigan thirteenth, Wisconsin eighteenth, Texas nineteenth, Iowa twentieth, Minne- sota twenty-first, and Mississippi twenty-fourth. The reason Ohio, Illinois and Indiana rank so high is be- cause they are states which contain large cities; Ohio has four cities of a population exceeding 100.000, and several which approximate this figure; Indiana has several large cities, and Illinois includes Chicago with its population of nearly two millions, and several well- populated cities. The southern states have a high death-rate owing to the prevalence of consumption among the colored people. The average death-rate from consumption during the year 1890 throughout the United States was 268.81 per 100,000. But the average of the cities about the great lakes was much below this, while the average of the cities in the south was higher for reasons given above. On account of the fertility of the Mississippi valley If there are no factors to cause the disea the increase of population, what are the p for the cure of the disease in the middle states The answer to this question must hinge o perience of observers who have treated consu the lowlands and the lower altitudes, and a analysis of the value of the higher altitude tteatment of the disease, and whether the ho ment of consumption with all the comforts home suggests, without the so-called climate, duce as good results as the desired climate w home comforts. As to the results of treatment in the low lower altitudes we cam only take the statistic nished by those who have observed the resu sorts situated in the respective levels. The treatment demonstrate the curability of co in the lower altitudes in percentages varying to 75, according to the stage of the disease. THE BIOLOGIC TEST FOR SEMEN. C. G. FARNUM, M.D. working women, married young women and children at Lake Kushaqua in the Adirondack mountains. The Erie County (N. Y.) Almhouse is to build a $50,000 hospital for their consumptive inmates. Bellevue Hos- pital has established a ward to accommodate 200 pa- tients. , (From the Pathological Laboratory of Rush Medical College.) CHICAGO. In this work I have applied to semen the biologic test for blood. This test is based upon the fact that when certain substances, such as bacterial filtrates, peptone, milk, egg albumin, albuminous urine, blood serum, etc., are injected into animals at intervals for a period of time they produce in the blood of the animals sub- stances which form specific precipitates with the sub- stances injected A bill was introduced in the legislature of Massa- chusetts to establish a second state institution in the mountains, to accommodate the patients who apply for treatment. A bill was introduced January 2 in the legislature of Minnesota providing $150,000 for the erection and $50,000 for the maintenance of a state sanatorium for consumptives. injected. Rabbits have been injected intraperitoneally with either semen or testicular emulsions of the dog, bull or man, at intervals of from two to six days and in quanti- ties of from 5 to 10 c.c. at each injection. They received from five to eight injections each. The blood serum for the tests was diluted from twelve to eighty times and the semen or testicular emulsions from four to twenty\x=req-\ five times in physiologic salt solution. consumptives. California has appointed a committee to investigate the feasibility of establishing a state institution in the southern part of California. p Texas has a farm for consumptives two miles from the Hiuntville penitentiary. Men who went to the farm in the last stages of the disease are now hearty and stout, and evince no evidence of the disease. The plantation is self-supporting through the labor of the invalids. physiologic The blood serum of the rabbits injected with dog semen and with emulsion of dog's testicle gives distinct precipitates with clear filtrates of these substances, but no precipitate occurs with human semen or with the emulsion of bull's testicle. Normal rabbit's serum and the emulsion of dog's testicle give no reactions. The amniotic fluid from one of the rabbits gives the same reactions as her blood serum. THE BIOLOGIC TEST FOR SEMEN. C. G. FARNUM, M.D. I have not been able as yet to determine whether the serum of these rabbits gives precipitates with dog's serum; nor have I tested the agglutinating power on dog's spermatozoa of the serum of the treated rabbits. pp g g Louisiana is to establish a consumptive camp at Covington, 50 acres of land having been procured for that purpose. Michigan is considering a bill for the establishment of a state hospital. New Jersey is urg- ing an appropriation for the same purpose. The Penn- sylvania legislature is asked for $60,000 to establish free homes for consumptives near White Haven. Bos- ton has four hospitals for the treatment of consump- tives, and is about to erect another to cost $80,000. New Hampshire is about to investigate the question. The serum from a rabbit treated with emulsion of bull's testicle gives a precipitate both with the emulsion used for injection and with a salt solution emulsion of spermatozoa taken from the epididymis. No precipi- tate occurs with human semen, nor with the emulsion of dog's testicle, nor with an emulsion of goat sperma- tozoa. Hampshire g q The government sanatorium at Fort Stanton, N. M., is a success. It has been in operation 18 months, and has treated 92 patients, 12 of whom were discharged as recovered, 15 improved, and 11 died. The cases' received were nearly all advanced. y Look over this list and you will perceive that these proposed institutions will be located in different sec- tions of the country, many of which can lay no claim to advantages as health resorts. Cast your eye over the land between the Appalachian range and the Rocky mountains and you can pick out hundreds of places which present advantageous points for the location of sanatoria. Minnesota has a dry bracing air which sus- tains its reputation for healing it gained many years ago; the region of the great lakes proves by statistics that consumptives may live there; the pines of Wiscon- sin offer healing balms to the consumptive, and the Ozark mountains, hitherto neglected, are readily avail- able for the invalids of Chicago, St. Louis, Indian- apolis, and other great cities in the Mississippi valley. If you go further south into Kansas, approaching the Rocky mountain chain, you secure the desired altitude, and for the equable sedative climate you go down into Texas. TUBERCULOSIS IN THE MIDDLE STATES AND ITS CURABILITY. f p , y, g The states have been slow to respond to the onward movement until within the last three years, when the states have been waking up to the fact that they should make provision for this large class of dependent pa- tients. The first state to erect a sanatorium for the con- sumptive is Massachusetts, which completed a sana- torium in January, 1898. It has been in operation nearly three years with results encouraging to the highest degree. g Cook County has a hospital at Dunning in connec- tion with the infirmary for advanced cases of con- sumption, and while the percentage of recoveries is necessarily low, there has been an improvement in the condition of many patients, owing to the betterment of their surroundings and appropriate treatment. The institution is doing a great deal of good by removing advanced cases from homes and lodgings where they are sources of contagion. among p p The average death-rate from consumption during the year 1890 throughout the United States was 268.81 per 100,000. But the average of the cities about the great lakes was much below this, while the average of the cities in the south was higher for reasons given above. On account of the fertility of the Mississippi valley the country is rapidly being thickly settled, especially in the north, and this fact alone should increase the prevalence of consumption, but I am confident that the 1900 census will prove that there has been no in- crease. Therefore, there can be no intrinsic causes in the valley to increase the prevalence of the disease. g The sentiment in favor of state institutions is grow- ing throughout the United States. New York will build a state hospital as soon as a site can be selected, to cost $200,000 and to accommodate 200 patients. A movement is also on foot to establish a sanatorium for Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015 THE BIOLOGIC TEST FOR SEMEN. C. G. FARNUM, M.D. The serum of the rabbit treated with human semen gives a. distinct reaction with both recent and old emul- sions of human semen in salt solution; also with human semen which had been dried and preserved thirty-four days on filter paper and on cloth, which were soaked in salt solution and the fluid carefully filtered. But with emulsions of bull, of dog and of goat testicles no reac- tion takes place. With human blood serum, human semen gives no precipitate. g p ec p tate While the experiments should be extended much fur- ther and modified in various ways, I think it safe to con- clude that blood sera of animals treated with different semens and testicular emulsions contain precipitins, which probably are specific. Th h b p y p That the substance which gives rise to the precipi- tin is contained in the semen itself and not in the blood serum or its direct derivatives seems probable from the absence of precipitate in human serum to which is added the serum of rabbit treated with human semen. Here there is also room for much further experimenta- tion. For one, I would answer the question of the curability of consumption in the middle states in the affirmative; hence, I would urge the physicians who are present from these states to return to their homes and agitate for the erection of state sanatoria, or private sanatoria if the state will not give a helping hand. I am sorry to say my own state, Illinois, which stands fifth in the mor- tality list, refused to take high rank in providing a state sanatorium. The excuse is, no money, but a glance at the appropriation list will tell where the money went. Since dried semen of considerable age (34 days) gives the reaction it would seem that the test may be of practi- cal value for the detection of the nature of suspected' seminal spots. Havana's Sanitary Condition.—During the month of November there were no cases and no deaths from yellow fever. The city has had no such record for any preceding; November since 1762. Downloaded From: http://jama.jamanetwork.com/ by a New York University User on 05/14/2015
https://openalex.org/W3174242164	https://opus.bibliothek.uni-wuerzburg.de/files/26118/s12862-021-01858-x.pdf	English	null	Is mRNA decapping by ApaH like phosphatases present in eukaryotes beyond the Kinetoplastida?	BMC ecology and evolution	2,021	cc-by	12,890	BMC Ecology and Evolution BMC Ecology and Evolution Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 https://doi.org/10.1186/s12862-021-01858-x Open Access Abstract Background: ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and have been identified in all eukaryotic super-groups. Only two of these proteins have been functionally characterised. We have shown that the ApaH like phosphatase ALPH1 from the Kinetoplastid Trypanosoma brucei is the mRNA decapping enzyme of the parasite. In eukaryotes, Dcp2 is the major mRNA decapping enzyme and mRNA decapping by ALPHs is unprec- edented, but the bacterial ApaH protein was recently found decapping non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or could be more widespread among eukaryotes. Results: We screened 827 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to characterize the phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of this protein family. For most organisms, we found ALPH proteins to be either absent (495/827 organisms) or to have non-cytoplasmic localisation predictions (73% of all ALPHs), excluding a function in mRNA decapping. Although, non-cytoplasmic ALPH proteins had in vitro mRNA decapping activity. Only 71 non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisations. However, in contrast to Kineto- plastida, these organisms also possess a homologue of Dcp2 and in contrast to ALPH1 of Kinetoplastida, these ALPH proteins are very short and consist of the catalytic domain only. Conclusions: ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme, or use it exclusively outside the cytoplasm. The acceptance of mRNA as a substrate indicates that ALPHs, like bacterial ApaH, have a wide substrate range: the need to protect mRNAs from unregulated degradation is one possible explanation for the selection against the presence of cytoplasmic ALPH proteins in most eukaryotes. Kineto- plastida succeeded to exploit ALPH as their only or major mRNA decapping enzyme. 71 eukaryotic organisms outside the Kinetoplastid lineage have short ALPH proteins with cytoplasmic localisation predictions: whether these pro- teins are used as decapping enzymes in addition to Dcp2 or else have adapted to not accept mRNAs as a substrate, remains to be explored. Keywords: ApaH like phosphatase, ApaH, ALPH, Trypanosoma brucei, mRNA decapping, m7G cap, mRNA cap, ALPH1, Kinetoplastida Keywords: ApaH like phosphatase, ApaH, ALPH, Trypanosoma brucei, mRNA decapping, m7G cap, mRNA cap, ALPH1, Kinetoplastida Is mRNA decapping by ApaH like phosphatases present in eukaryotes beyond the Kinetoplastida? Paula Andrea Castañeda Londoño1, Nicole Banholzer1, Bridget Bannermann2 and Susanne Kramer1* Background Eukaryotic phosphatases play essential roles in regulating many cellular processes and can be classified in several ways, based on catalytic mechanism, substrate speci- ficity, ion requirements and structure. One four-group Correspondence: susanne.kramer@uni-wuerzburg.de 1 Zell- Und Entwicklungsbiologie, Biozentrum, Universität Würzburg, Würzburg, Germany Full list of author information is available at the end of the article Correspondence: susanne.kramer@uni-wuerzburg.de 1 Zell- Und Entwicklungsbiologie, Biozentrum, Universität Würzburg, Würzburg, Germany Full list of author information is available at the end of the article g, y ull list of author information is available at the end of the article © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://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. Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 2 of 19 capping of bacterial mRNAs [24] mostly or entirely caused by usage of Np4A as non-canonical transcrip- tion initiation nucleotide [25]. Many other dinucleoside polyphosphates can be used for co-transcriptional cap- ping even in the absence of stress, including methylated versions [26]. ApaH is the major decapping enzyme for all dinucleoside polyphosphate caps [24, 26], suggesting that its main function is to regulate gene expression by modulating the mRNA decapping process. Intriguingly, the enzyme can both enhance decapping (by decapping nucleoside-tetraphosphate capped RNA) and inhibit decapping (by cleaving Np4A and preventing its incorpo- ration to the mRNA). classification distinguishes phosphoprotein phosphatases (PPPs), metal-dependent protein phosphatases (PPM, sometimes classified as a subgroup of PPP), protein tyrosine phosphatases and aspartic acid-based phos- phatases [1]. The eukaryotic PPP group includes the Ser/ Thr phosphatases PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7 [1, 2] but has been extended to include three families of bacterial origin: Shewanella-like SLP phosphatases, Rhizobiales-like (RLPH) phosphatases and ApaH-like (ALPH) phosphatases [3–6]. ( ) p p [ ] ApaH like phosphatases (ALPHs) evolved from the bacterial ApaH protein and were present in the last com- mon ancestor of eukaryotes [3, 5]. They are widespread throughout the entire eukaryotic domain, although they have been lost in certain sub-branches such as land plants and chordates [6]. To the best of our knowl- edge, only two ALPHs have been functionally charac- terised to date. One is the S. cerevisiae ALPH protein (YNL217W), a Zn2+ dependent endopolyphosphatase of the vacuolar lumen [7] that is also active with Co2+ and possibly Mg2+ [8]. The enzyme’s main function is the cleavage of vacuolar poly(P). A function of Ppn2 in stress response is suggested by the fact that the increase in short polyphosphate upon Ppn2 overexpression corre- lates with an increased resistance to peroxide and alkali [9]. The second characterised ALPH protein is ALPH1 of the Kinetoplastida Trypanosoma brucei: we recently found that ALPH1 is the only or major trypanosome mRNA decapping enzyme [10], the enzyme that removes the m7 methylguanosine (m7G) cap present at the 5´end of most eukaryotic mRNAs. This finding was surprising, as all other known eukaryotic mRNA decapping enzymes belong to a different enzyme family, the nudix hydro- lases (the prototype is Dcp2). Trypanosomes lack ortho- logues to Dcp2 and all decapping enhancers associated with Dcp2, but use the ApaH like phosphatase ALPH1 instead. T. brucei ALPH1 consists of the catalytic domain with N- and C- terminal extensions of about equal sizes, that appear unique to Kinetoplastida and bear no simi- larities to any known domains. Puzzled by these novel functions of bacterial ApaH and trypanosome ALPH1 in mRNA decapping, we here set out to investigate whether mRNA decapping is a major function of eukaryotic ALPHs, or whether this function is restricted to trypanosomes. We developed a Python algorithm for the identification of ALPHs in all avail- able eukaryotic reference proteomes and identified 441 ALPHs in 332/827 proteomes. We show that most ALPH proteins consist exclusively of the catalytic domain and many have predicted transmembrane regions or signal peptides and predicted non-cytoplasmic localisation, indicating functions distinct from mRNA decapping. We show in vitro mRNA decapping activity for three of these non-cytoplasmic ALPH proteins, indicating that, similar to bacterial ApaH, eukaryotic ALPHs accept non-physiological substrates and thus are likely to have a rather wide substrate range. The data indicate that there may be selection against the presence of cytoplas- mic ALPH proteins in eukaryotes, possibly to protect mRNAs from unregulated degradation. Apart from the kinetoplastids, only 71 organisms possess ALPH proteins with cytoplasmic localisation predictions. Results d ifi Interestingly, recent data indicate, that the bacterial precursor protein of ALPH, ApaH, may have an analo- gous function to Trypanosome ALPH1 in decapping of bacterial mRNAs. In vitro, ApaH cleaves diadenosine tetraphosphate (Ap4A) into two molecules of ADP [11, 12], but is also active towards other NpnN nucleotides (with n ≥ 3) [13–15]. Deletion of the ApaH gene causes a marked increase in Np4A levels and a wide range of phe- notypes [16–23]. Np4A was therefore suggested to act as an alarmone (a signalling molecule involved in stress response), but no Np4A receptor has yet been identified. Instead, recent data show that stress induced increase in Np4A levels cause massive nucleoside-tetraphosphate In contrast to the Kinetoplastida, these ALPH proteins consisted (with 2 exceptions) of the catalytic domain only and all organ- isms have an orthologue to Dcp2 (with 3 exceptions likely caused by genome incompleteness). General features of ApaH‑like phosphataseshi p p p The dataset was used to refine the characteristics of ALPH proteins (Fig. 2). 40% of all analysed organisms have at least one ALPH isoform. The highest percent- age is found in Discoba with 94%, the lowest in Diapho- retickes with 24% (Fig. 2A). Of the 332 APLH-positive organisms, 25% have more than one ALPH isoform: most (81%) have two and with one exception no organism has more than four (Fig. 2B). Organisms with multiple ALPHs were mostly enriched among the Discoba (94% of organisms with ALPH had at least 2 ALPHs) (Fig. 2B). The vast majority of all ALPH proteins are very short and consist mainly of the catalytic domain with short N- and C-terminal extensions (Fig. 2C). The median size of the C-terminal extensions is 26 amino acids: only 52 of all 441 ALPH proteins have C-termini extending 100 amino acids and most of these (31) are ALPHs of Discoba. The size of the N-terminus is slightly more variable and has a median of 87 amino acids. Only 61 of all 441 ALPH proteins have N-termini extending 200 amino acids and many of these (28) belong to ALPHs of Discoba. The largest variance in the size of ALPH N- and C-termini is found in Discoba, reflecting the presence of two different ALPH variants in the Kinetoplastida (discussed below). Figure 1 summarises all organisms of this study in phy- logenetic groups based on the latest eukaryotic classifi- cation suggested by [30]. For each group, the fraction of organisms with and without ALPH is indicated in orange and blue, respectively. 332 of all 827 organisms included in this study have at least one ALPH, and these organ- isms are distributed in a patchy way throughout all eukaryotes. Most Euglenozoa have ALPHs (32 of 33), many Stramenopiles (25 of 28) and Fungi (238 of 298), but also Rhodophyceae (3 of 4), Chlorophyta (11 of 18), Haptista (2 of 2) and some Metazoa (17 of 296). ALPHs are absent from land plants (100 proteomes tested), Api- complexa (20 proteomes tested), Ciliata (3 proteomes tested) and Dinoflagellates (1 proteome tested). They are largely absent from Chordata (134 proteomes tested, only Branchiostoma floridae has ALPH) and from Ecdysozoa (144 proteomes tested, only 4 Arachnida have ALPH) and fully absent from the few available proteomes of Amoebozoa (9 proteomes tested) and Metamonada (3 proteomes tested). Identification of ApaH like phosphatases in available eukaryotic proteomes ALPHs belong to the PPP family of phosphatases and possess the four conserved signature motifs (motif 1–4) of this family, GDxHG, GDxxDRG, GNHE, and HGG, sometimes with conservative substitutions [2]. One distinctive feature of ALPHs are two changes in the GDxxDRG motif: The second Asp is replaced by a neu- tral amino acid and the Arg residue is replaced by Lys. In addition, ALPHs have two C-terminal motifs [3, 6] that we here call motif 5 and 6. We screened 827 complete ukaryotic proteomes (Additional file 3: Table S1a) for the Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 3 of 19 presence of ApaH like phosphatases with a home-made Python algorithm; these included all reference proteomes available on UniProt [27] and all available Kinetoplastida proteomes available on TriTrypDB [28, 29]. The algo- rithm is based on recognising the six sequence-motifs that are characteristic for ALPHs. The matrices used to define these motifs were optimised stepwise using yeast and Kinetoplastida proteomes to not miss any ALPH (controlled by BLAST) while on the other hand not to recognise PPPs, and in particular not the closely related phosphatases SLP, RLPH and ApaH (sequences taken from [6]). The final algorithm also included restrictions on distances between the motifs 1 to 2, 2 to 3 and 5 to 6 that we found highly conserved. More details are in mate- rial and methods. BLAST screens on selected proteomes without ALPH proteins revealed that ALPHs were either fully absent or, in rare cases, present in a truncated ver- sion and missing at least one of the motifs, mostly the N- or C-terminal one. ALPH proteins with missing N- or C-terminal motifs may have arisen from sequencing or annotation errors and were not included in the final list (Additional file 4: Table S2c) (11 proteins in total, all belong to phylogenetic groups with ALPH proteins pre- sent). Only for the Kinetoplastida, ALPHs with wrongly annotated start codons were manually included (based on comparison with related Kinetoplastida and available genome information). presence of ApaH like phosphatases with a home-made Python algorithm; these included all reference proteomes available on UniProt [27] and all available Kinetoplastida proteomes available on TriTrypDB [28, 29]. The algo- rithm is based on recognising the six sequence-motifs that are characteristic for ALPHs. Identification of ApaH like phosphatases in available eukaryotic proteomes The matrices used to define these motifs were optimised stepwise using yeast and Kinetoplastida proteomes to not miss any ALPH (controlled by BLAST) while on the other hand not to recognise PPPs, and in particular not the closely related phosphatases SLP, RLPH and ApaH (sequences taken from [6]). The final algorithm also included restrictions on distances between the motifs 1 to 2, 2 to 3 and 5 to 6 that we found highly conserved. More details are in mate- rial and methods. BLAST screens on selected proteomes without ALPH proteins revealed that ALPHs were either fully absent or, in rare cases, present in a truncated ver- sion and missing at least one of the motifs, mostly the N- or C-terminal one. ALPH proteins with missing N- or C-terminal motifs may have arisen from sequencing or annotation errors and were not included in the final list (Additional file 4: Table S2c) (11 proteins in total, all belong to phylogenetic groups with ALPH proteins pre- sent). Only for the Kinetoplastida, ALPHs with wrongly annotated start codons were manually included (based on comparison with related Kinetoplastida and available genome information). proteins in eukaryotic subgroups. We extend the availa- ble dataset from 52 ALPHs (38 organisms) to 441 ALPHs (332 organisms), this way providing a better resolution of ALPH distribution across eukaryotes.h The aim of this work was not to analyse horizontal gene-transfer between eukaryotes, bacteria and archaea; however, we could confirm the presence of ALPHs in a subgroup (11/25) of Myxococcales, as described in [6] (Additional file 5: Table S3A). We detected ALPH in 1 of 285 archaean proteomes (OX = 1906665 GN = EON65_52185, UniProt: UP000292173) (Addi- tional file 5: Table S3B); it is possible that this protein is a contamination. All prokaryotic ALPHs consisted mostly of the catalytic domain with almost no N- or C terminal extensions. General features of ApaH‑like phosphataseshi A phylogenetic tree built from the catalytic domains of ALPHs mostly reflects the eukary- otic tree (Additional file 1: Figure S1). Taken together, the data indicate that ALPH was present in the last common ancestor of all eukaryotes and was then selectively lost in certain sub-branches. Our data fully agree with the data of [6], regarding absences and presences of ALPH The amino acid distances between some of the ALPH motifs are highly conserved (Fig. 2D): 93.7% of all ALPHs have between 28 and 30 amino acids between motif 1 and 2 (72% have exactly 29). The distance between motif 2 and 3 is exactly 26 amino acids for 83.0% of ALPHs and between 25 and 33 for 98.0% of ALPHs. The distance between the two C-terminal motifs 5 and 6 is exactly 19 amino acids for 92.5% of ALPHs. The distances between motif 3 and 4 are well conserved in ALPHs of Diapho- retickes and Discoba (94% are between 55 and 64 amino acids) but less well within Amorphea. The distances between motif 4 and 5 are poorly conserved. Sequence motifs were created for all six motifs (Fig. 2E) [31]. Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 4 of 19 Mostly, these are conserved with some group-specific preferences at certain positions indicated with orange bars (Fig. 2E). The ALPH sequence of S. cerevisiae is shown as an example, to illustrate the definitions of al features discussed above (Fig. 2F). Fig. 1 Presence and absence of ALPH in the different eukaryotic subgroups. 827 eukaryotic proteomes were screened for the presence of ALPH. Absence or presence of ALPH is shown in pie diagrams in blue and orange, respectively, for each phylogenetic group. The diameter of a circle roughly reflects the number of available proteomes. The phylogenetic tree was taken from [30]. All organisms are listed in Additional file 3: Table S1 Mostly, these are conserved with some group-specific preferences at certain positions indicated with orange bars (Fig. 2E). The ALPH sequence of S. cerevisiae is shown as an example, to illustrate the definitions of all features discussed above (Fig. 2F). shown as an example, to illustrate the definitions of all features discussed above (Fig. 2F). Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 5 of 19 Fig. 2 General Features of ALPHs. A Proteomes with (orange) and without (blue) ALPHs. General features of ApaH‑like phosphataseshi B Number of ALPH proteins per organism. C Sizes of the different ALPH ‘domains’ (N-terminus, catalytic domain, C-terminus) are presented as box plot (waist is median; box is IQR; whiskers are ± 1.5 IQR) and in addition as individual data points (transparent blue circles). The catalytic domain is defined as the range between the first and last motif, with an additional six N-terminal amino acids and an additional eight C-terminal amino acids (compare F). D Distances between the six different motifs (in amino acids) are presented as box plot (waist is median; box is IQR; whiskers are ± 1.5 IQR) and in addition as individual data points (transparent blue circles). E Sequence motifs were created with WebLogo [31]. The most obvious differences between the three groups are marked with orange bars. F The sequence of ALPH from S. cerevisiae is shown as a typical example, to illustrate the position of the catalytic domain (blue), the N- and C-termini (grey) as well as all six motifs (red) and the distances between the motifs that we found most conserved (green). Details on all data presented in Fig. 2 can be found in Additional file 3: Table S1 Fig. 2 General Features of ALPHs. A Proteomes with (orange) and without (blue) ALPHs. B Number of ALPH proteins per organism. C Sizes of the different ALPH ‘domains’ (N-terminus, catalytic domain, C-terminus) are presented as box plot (waist is median; box is IQR; whiskers are ± 1.5 IQR) and in addition as individual data points (transparent blue circles). The catalytic domain is defined as the range between the first and last motif, with an additional six N-terminal amino acids and an additional eight C-terminal amino acids (compare F). D Distances between the six different motifs (in amino acids) are presented as box plot (waist is median; box is IQR; whiskers are ± 1.5 IQR) and in addition as individual data points (transparent blue circles). E Sequence motifs were created with WebLogo [31]. The most obvious differences between the three groups are marked with orange bars. F The sequence of ALPH from S. cerevisiae is shown as a typical example, to illustrate the position of the catalytic domain (blue), the N- and C-termini (grey) as well as all six motifs (red) and the distances between the motifs that we found most conserved (green). Details on all data presented in Fig. ALPHs of Opisthokonts (fungi and holozoa)h evidence for the majority of fungal APLH proteins being non-cytoplasmic. Experimental data confirm non-cyto- plasmic localisation for the ALPH protein of S. cerevisiae (YNL217w, Ppn2): two high-throughput studies indicate vacuolar localisation [37, 38] and recent data show that Ppn2 is delivered to the vacuolar lumen via the multive- sicular body pathway, where it functions as an endopoly- phosphatase [7]. The majority of available ALPH sequences (292) are from Fungi, because the number of available proteomes is high (298) and 80% of these proteomes contain at least one ALPH. We investigated these ALPH sequences further by looking for predicted domains (Interpro [32]), signal peptides and trans-membrane helices (Phobius [33] and Target P [34]) and predicted localisation (DeepLoc [35] and WoLF PSORT [36]) (Fig. 3A and Additional file 3: Table S1b). ALPHs of Fungi have very short C-termini (median = 26 amino acids) and only slightly larger N-ter- mini (median = 97 amino acids). Most fungal ALPHs (95.2%) do not contain any predictable domain in addi- tion to the catalytic ALPH domain. Of the 14 ALPHs that have a further domain, three have domains with func- tions in cytochrome c complex assembly (IPR021150, IPR018793) indicating mitochondrial functions and five ALPHs have a THIF-type NAD/FAD binding fold, usu- ally found in the ubiquitin activating E1 family, indicat- ing a possible function in protein degradation. The ALPH of Lentinula edodes has a Peroxin-3 domain, indicating a peroxisomal function. Two ALPHs of Rachicladosporium have Pectate lyase domains, indicating a possible function in degradation of cell wall material. ALPH of Phycomyces blakesleeanus has a spore coat protein CotH (IPR014867) domain, a domain of bacterial origin with unknown func- tion in eukaryotes. The ALPH of Rhizopogon vesiculosus has a second ALPH domain.hi ALPHs are underrepresented in Holozoans (Fig. 1). In particular, all 130 vertebrate proteomes lack ALPHs and of the three available non-vertebrate proteomes of Chor- data, only the Lancelet Branchiostoma floridae is ALPH- positive. Out of the 140 available Ecdysozoan proteomes, only four organisms (Arachnida species) contain ALPH. ALPHs are present in subgroups of Cnidarians (2/3), Echinodermatans (1/2), Lophotrochozoens (8/11), Pla- cozoans (1/2) and Ichthyosporeans (1/2). 7 of these 18 Holozoan ALPH proteins have a predicted signal pep- tide or transmembrane helix at their C-termini and with the exception of ALPH from Lottia gigantea, all proteins have non-cytoplasmic localisation predictions, mostly to the mitochondria (13/18) (Fig. 3C, Additional file 3: Table S1c). Fig. 3 ALPHs of Opisthokonts. A ALPH proteins of Fungi are presented as schematics with their catalytic domain and additional predicted features (further domains, transmembrane helices, signal peptides) (right). Localisation predictions (Target P, DeepLoc) are shown on the left. The organism names and more details can be found in Additional file 3: Table S1b. B Pie charts summarizing localisation predictions (DeepLoc) (left) and predicted transmembrane helix or signal peptides (right) for ALPH proteins from all Fungi. C ALPH proteins from Holozoa are presented as in (A). More details are listed in Additional file 3: Table S1c (see figure on next page.) General features of ApaH‑like phosphataseshi 2 can be found in Additional file 3: Table S1 Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 6 of 19 ALPHs of Opisthokonts (fungi and holozoa)h Two proteins have an additional domain: ALPH of Pomacea canaliculata has a domain of the FAD/NAD(P)-binding superfamily N- terminal of the catalytic domain and ALPH of Leptotrombidium deliense may be interacting with actin, as it has a Kaptin domain C-terminal of its ALPH domain. All the 42 organisms containing an ALPH protein with a cytoplasmic localisation prediction also have a Dcp2 homologue (Blast, Additional file 3: Table S1b and c), indicating that a function of the ALPH protein in mRNA decapping, if present, is not exclusive. The most interesting finding was the presence of pre- dicted trans-membrane regions and/or signal pep- tides within the C-terminal region in 78.1% of all fungal ALPH proteins: 184 ALPH proteins have predicted membrane helices (mostly one), a further 41 ALPH proteins have predicted signal peptides, 3 ALPH pro- teins have both predicted and only 64 ALPH proteins have neither predicted (Fig. 3A and B, Additional file 3: Table S1b). In agreement with these data, only 49 ALPH proteins (16.8%) have predicted cytoplasmic localisa- tion (mostly the ones without predicted membrane heli- ces and signal peptides) (Fig. 3A and B, Additional file 3: Table S1b). The remaining proteins are predicted to be in the golgi (52.1%), endoplasmatic reticulum (19.5%), mitochondrion (6.5%), lysosome/vacuole (3.8%), nucleus (2 proteins, 0.7%), peroxisome (1 protein, 0.3%) and extracellular (1 protein, 0.3%). Prediction data need to be considered with care in the absence of experimen- tal evidence, but taken together, the data provide strong ALPHs of diaphoretickes We found no ALPHs in land plants (100 proteomes), Apicomplexa (20 proteomes), Ciliata (3 proteomes) or Dinoflagellata (1 proteome). ALPH is present in 3 of 4 species of red algae, 11 of 18 species of green algae (Chlo- rophyta), in the filamentous green algae Klebsormidium nitens, in the photosynthetic Alveolate Vitrella brassica- formis, in the cryptophyte algae Guillardia theta and in the two available Haptista proteomes. 25 of 28 Strameno- piles have ALPHs: These are mostly (non-photosyn- thetic) Oomycetes, including for example Phytophthora Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 7 of 19 parasitica: all the strains of this plant pathogen have three ALPH isoforms. Predicted signal peptides or trans- membrane helices are present at the C-termini of many Chloroplastida ALPHs (11/19), as well as in ALPH pro- teins of the Alveolata Vitrella brassicaformis, the diatom Phaeodactylum tricornutum and the Haptista Emiliania huxleyi; in all cases the presence of transmembrane helices or signal peptides correlates with a predicted non-cytoplasmic localisation (Fig. 4). With two excep- tions, all 19 ALPH proteins from Chloroplastida have non-cytoplasmic localisation predictions (11 mit chondrion, 4 chloroplast, 2 endoplasmic reticulum In contrast, the remaining 74 ALPH proteins fro Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 8 of 19 Fig. 3 continued Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 9 of 19 Fig. 4 ALPHs of Diaphoretickes. ALPHs of Diaphoretickes are presented as schematics with their catalytic domain (dark blue) and additional predicted features (further domains, transmembrane helices and signal peptides) (right) and localisation predictions (Target P and DeepLoc) (left). More details are listed in Additional file 3: Table S1d Fig. 4 ALPHs of Diaphoretickes. ALPHs of Diaphoretickes are presented as schematics with their catalytic domain (dark blue) and additional predicted features (further domains, transmembrane helices and signal peptides) (right) and localisation predictions (Target P and DeepLoc) (left). More details are listed in Additional file 3: Table S1d identifiable homologue to Dcp2 of Arabidopsis thaliana (in some cases only as a fragment as proteomes were not complete) (Additional file 3: Table S1d). The three remaining organisms with no identifiable Dcp2 homo- logue are Chrysochromulina tobinii, Hyaloperonospora arabidopsidis and Pythium insidiosum. However, closely related species to all three organisms have Dcp2 and it is likely that the absence of a Dcp2 is caused by genome incompleteness: the proteome of C. ALPHs of diaphoretickes tobinii is 37% incom- plete and the proteomes of H. arabidopsidis and P. insid- iosum are 7% and 1% incomplete, respectively. Thus, any function of an ALPH protein in mRNA decapping is likely not exclusive but occurs in addition to Dcp2. non-Chloroplastida have mostly cytoplasmic localisation predictions, with only 7 exceptions (6 mitochondrion, 1 chloroplast).h The majority of ALPH proteins from Diaphoretickes is very short and, in particular ALPHs of Oomycetes, consist almost exclusively of the catalytic domain with very short N- and C-terminal extensions. Of all 63 Dia- phoretickes ALPH proteins, only five have additional domains: the ALPH of Chlamydomonas reinhardtii has a predicted Transposase IS605 domain (a transposon of bacterial origin), the ALPH of Helicosporidium has a coatomer delta subunit domain and the three large and almost identical ALPH proteins of Chlorella sorokini- ana have a PsbQ-like domain (IPR023222), an Arm-like repeat and a second ALPH domain. A PsbQ-like domain is typical for proteins of the photosystem II, consistent with localisation predictions of the three Chlorella soro- kiniana ALPH proteins to the chloroplast. ALPHs of Euglenozoa ALPHs are present in all Kinetoplastida and in their close relative Euglena gracilis (Additional file 3: Table S1e). The only exception is the free-living, non-parasitic Bodo saltans. Of the 29 Diaphoretickes that have ALPH proteins with predicted cytoplasmic localisation, 26 have a readily Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 10 of 19 Fig. 5 ALPHs of Euglenozoa. A ALPH proteins of Euglenozoa are presented schematically along a maximum likelihood phylogenetic tree based on an alignment of the catalytic domains. All details on Euglenozoa ALPHs are listed in Table S1e. The localisation of T. brucei ALPH1 and ALPH2 was determined by expressing eYFP fusion proteins. One representative fluorescent image is shown for each protein. At least three different clonal cell lines showed identical localisation. B Sequence motifs of the ALPH1 orthologues and the non-ALPH1 orthologues. Major differences are marked by asterisks (See figure on next page.) ALPHs of Kinetoplastida fall into two groups (Fig. 5A): each organism has exactly one ALPH that is homologous to T. brucei ALPH1, the mRNA decapping enzyme [10]. These ALPHs all have a C-terminal extension of between 220 and 278 amino acids and, with two exceptions (Lep- tomonas pyrrhocoris and T. grayi), they all have N-ter- minal extensions of a similar size. The in vitro mRNA decapping activity of T. brucei ALPH1 does not require the N-terminus [10], and the two ALPHs that lack the N-terminus are therefore also likely active in mRNA decapping. The fact that no Kinetoplastida strain has lost its ALPH1 homologue, and the absence of homologues to the canonical mRNA decapping enzymes (DCP1/ DCP2), indicates that all Kinetoplastida rely on ALPH1 for mRNA decapping. in particular not with our experimental data. The decap- ping enzyme T. brucei ALPH1, fused to eYFP either C- or N- terminally, localises to the cytoplasm, P-bodies and to the posterior pole of the cell and this cytoplasmic locali- sation is consistent with the essential function of ALPH1 in mRNA decapping ([10] Fig. 5A). It is likely that ALPH1 orthologues of all other Kinetoplastida have predominant cytoplasmic localisations too. To investigate the locali- sation of an non-ALPH1 homologue, T. brucei ALPH2 was expressed as C-terminal eYFP fusion in procyclic trypanosomes using an inducible expression system [39]. ALPH2 showed a localisation pattern characteristic of mitochondrial proteins and co-localised with a mito- chondrial stain (MitoTracker™ Orange) (Fig. 5A). ALPHs of Euglenozoa Euglena is too distantly related to the Kinetoplastida to unequivocally assign its ALPH to the ALPH1 or non ALPH1 group, but its sequence motifs (GDIHG, GDLVGKG, GNHD, HAG, VFFGH, LDTG) are more similar to the non-ALPH1 group and this is also sug- gested by the absence of N- and C-terminal extensions (Fig. 5A). Interestingly, Euglena ALPH is the only Kine- toplastida ALPH with a predicted trans-membrane domain. Euglena ALPH was not enriched within purified mitochondria fractions [40] and also not within chlo- roplasts (Martin Zoltner, Charles University in Prague, personal communication). Like Kinetoplastida, Euglena has no recognisable homologue to the canonical mRNA decapping enzyme DCP1/DCP2 in its genome and the absence of an ALPH1 homologue raises the question of how mRNA decapping is achieved in this organism. In addition to the ALPH1 homologue, all but one Kinetoplastid have between one and three additional ALPH proteins that consist exclusively of the catalytic domain (Fig. 5A). These ALPH proteins of Leishmania, Leptomonas, Crithidia and Endotrypanum have exten- sions within the catalytic domain that are mainly caused by enlarged distances between motif 1 and 2 (up to 115 nucleotides in Leptomonas and Crithidia, more than in any other ALPH) and between motif 4 and 5 (up to 156 nucleotides). The only Kinetoplastida that has no ALPH apart from ALPH1 is Leishmania tarentolae; blast searches identified a protein at the expected position in the genome which lacked both motif 5 and motif 6, indi- cating a recent loss of the protein.ff Differences between the two different groups of Kineto- plastida ALPH proteins are also obvious within the phos- phatase and ALPH motifs: several positions show major differences; most pronounced is the difference in motif 1 (GDVHG/GDIHG for ALPH1/non-ALPH1) and in motif 6 (IDTG/LDSG for ALPH1/non-ALPH1) (Fig. 5B). When a phylogenetic tree is constructed from the sequences of the catalytic domains, the Kinetoplastida ALPH1 homo- logues form a separate group, distant to the group of the non-ALPH1 homologues, indicating that the ALPH1 decapping enzyme has evolved only once in the last com- mon ancestor of the Kinetoplastida (Fig. 5A).fi As previously reported, Blast searches cannot iden- tify a Dcp2 homologue in Kinetoplastida, with the one exception of Perkinsela (not examined in this study) [10]. Perkinsela is an obligate intracellular component of the Amoebozoa Neoparamoeba. ALPHs of Euglenozoa Its Dcp2 has closest simi- larity to Dcp2 of flowering plants and the gene may have been taken up by lateral gene transfer. ALPHs have in vitro mRNAs decapping activityh The decapping activity of the three active enzymes was tested in the presence of different bivalent ions (Mg2+, Mn2+, Co2+ and Zn2+) (Fig. 6B). All the enzymes had mRNA decapping activity, but the ion requirements for optimal activity differed: the ALPH of Sphaeroforma arctica showed the highest decapping activity with Mn2+ and some activity with Co2+, but none with Mg2+ or Zn2+. T. brucei ALPH2 had the best decapping activity with Co2+ and some with Mg2+ and possibly Mn2+, but none with Zn2+. Auxenochlorella protothecoides ALPH had the best activity with Mg2+ and some with Co2+, but none with Zn2+ and Mn2+. These differences in ion requirements indicate that the enzyme activities are likely specific and not caused by contaminating bacterial enzymes, which would be expected to require identical ions in all samples. Moreover, the catalytically inactive mutant ALPH of Sphaeroforma arctica had no decapping activity, when tested with its supportive ion (Mn2+), fur- ther strong evidence for the specificity of the decapping activity. differences in gel mobility on urea acrylamide gels com- plemented with acryloylaminophenyl boronic acid [42, 43]. The decapping activity of the three active enzymes was tested in the presence of different bivalent ions (Mg2+, Mn2+, Co2+ and Zn2+) (Fig. 6B). All the enzymes had mRNA decapping activity, but the ion requirements for optimal activity differed: the ALPH of Sphaeroforma arctica showed the highest decapping activity with Mn2+ and some activity with Co2+, but none with Mg2+ or Zn2+. T. brucei ALPH2 had the best decapping activity with Co2+ and some with Mg2+ and possibly Mn2+, but none with Zn2+. Auxenochlorella protothecoides ALPH had the best activity with Mg2+ and some with Co2+, but none with Zn2+ and Mn2+. These differences in ion requirements indicate that the enzyme activities are likely specific and not caused by contaminating bacterial enzymes, which would be expected to require identical ions in all samples. Moreover, the catalytically inactive mutant ALPH of Sphaeroforma arctica had no decapping activity, when tested with its supportive ion (Mn2+), fur- ther strong evidence for the specificity of the decapping activity. broad substrate specificity and cleaves for example both NpnN nucleotides [13–15] as well as mRNA capped with NpnN [24, 26]. The need to protect capped mRNAs from uncontrolled degradation may create selection for the loss or non-cytoplasmic localisation of eukaryotic ALPH proteins. ALPHs have in vitro mRNAs decapping activityh The phylogenetic data from us and others [6] show that ApaH like phosphatases were present in the last common ancestor of eukaryotes. Since then, 60% of eukaryotes have lost the enzyme and of those ALPH proteins that are still present, 73% have predicted non-cytoplasmic localisations. ALPH proteins are pyrophosphatases and at least the related bacterial ApaH protein has a rather Protein localisation predictions are difficult in Eugleno- zoa as these organisms have diverse and poorly defined targeting signals: localisation predictions from Phobius, DeepLoc and Target P are listed in Additional file 3: Table S1e but do not correlate well with each other, and, Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 11 of 19 Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 12 of 19 Fig. 6 ALPH proteins have mRNA decapping activity. A Recombinant ALPH proteins were produced in Arctic Express DE cells and purified via Nickel affinity chromatography. A Coomassie stained gel loaded with the purified proteins (red asterisk) is shown. Note that the Arctic Express DE cells contain chaperons (yellow asterisk). Chaperonin Cpn60 is often co-purified, in the case of TbALPH2 to a large extent. B The enzymes were tested for in vitro decapping activity in the presence of 1 mM of divalent ions, as indicated. A reaction without enzyme served as negative control. For each enzyme, an asterisk marks the conditions for best decapping activity. The gels are representative gels out of at least three replicates. An inactive mutant of Sphaeroforma arctica has no activity (last lane) Fig. 6 ALPH proteins have mRNA decapping activity. A Recombinant ALPH proteins were produced in Arctic Express DE cells and purified via Nickel affinity chromatography. A Coomassie stained gel loaded with the purified proteins (red asterisk) is shown. Note that the Arctic Express DE cells contain chaperons (yellow asterisk). Chaperonin Cpn60 is often co-purified, in the case of TbALPH2 to a large extent. B The enzymes were tested for in vitro decapping activity in the presence of 1 mM of divalent ions, as indicated. A reaction without enzyme served as negative control. For each enzyme, an asterisk marks the conditions for best decapping activity. The gels are representative gels out of at least three replicates. An inactive mutant of Sphaeroforma arctica has no activity (last lane) differences in gel mobility on urea acrylamide gels com- plemented with acryloylaminophenyl boronic acid [42, 43]. Discussion p g y y What is the likelihood for these 441 ALPH proteins to function in mRNA decapping? The most stringent criterion is intracellular localisation: mRNA decapping enzymes must be cytoplasmic to access their substrates. 321/441 ALPH proteins have non-cytoplasmic localisa- tion prediction and these predictions correlate in many cases with predicted transmembrane helices and signal peptides (Fig. 7). For two ALPH proteins, yeast Ppn2 and T. brucei ALPH2, non-cytoplasmic predictions were con- firmed experimentally [7, 37, 38], this work). This leaves 120 ALPH proteins with predicted cytoplasmic localisa- tion. A second essential feature of an mRNA decapping enzyme is the need to regulate its activity: For the nudix domain protein Dcp2, this regulation is done by the N-terminal domain, which mediates interactions with decapping enhancers [58]. The T. brucei mRNA decap- ping enzyme ALPH1 has unique N- and C-terminal extensions and the C-terminal extensions are conserved across all Kinetoplastida ALPH1 homologues and it is therefore tempting to speculate that they contribute to enzyme regulation. 87 of the 120 ALPH proteins with cytoplasmic predictions consist of the catalytic domain only (Fig. 7). The absence of additional sequences does not preclude the protein from being an mRNA decap- ping enzyme, but any interaction with putative decapping regulators must occur directly via the catalytic domain. The remaining 33 proteins that have sequences in addi- tion to the catalytic domain include the 31 Kinetoplastida homologues to the T. brucei mRNA decapping enzyme ALPH1, the ALPH protein of the fungi Moniliophthora roreri (Gamma-glutamyl cyclotransferase-like domain) and the ALPH protein of Ectocarpus siliculosus (disor- dered regions) (Fig. 7). A third criteria to evaluate the likelihood of an ALPH enzyme to act in decapping is to look for the presence of an alternative mRNA decapping enzyme. Orthologues to the mRNA decapping enzyme Dcp2 were readily detectable by Blast in all organisms with cytoplasmic ALPH proteins, with the marked excep- tion of the Kinetoplastida. To summarize, if any ALPH proteins from non-Kinetoplastida function in mRNA decapping, they are unlikely the exclusive mRNA decap- ping enzyme and their regulation will, with two possible exceptions, entirely depend on the catalytic domain. mRNAs are essential molecules in both eukaryotes and prokaryotes and need to be protected from unregu- lated exonucleolytic degradation. 5´-3´exoribonucleases require monophosphorylated RNA 5´ends and the major protection strategy is therefore to avoid such monophos- phates. ALPHs have in vitro mRNAs decapping activityh To test this hypothesis, we tested ALPH pro- teins with non-cytoplasmic localisation prediction for mRNA decapping activity in vitro. broad substrate specificity and cleaves for example both NpnN nucleotides [13–15] as well as mRNA capped with NpnN [24, 26]. The need to protect capped mRNAs from uncontrolled degradation may create selection for the loss or non-cytoplasmic localisation of eukaryotic ALPH proteins. To test this hypothesis, we tested ALPH pro- teins with non-cytoplasmic localisation prediction for mRNA decapping activity in vitro. We produced recombinant ALPH proteins from randomly selected organisms of the three eukaryotic kingdoms that have ALPHs: we used ALPH of the Ich- thyosporea Sphaeroforma arctica, ALPH of the green algae Auxenochlorella protothecoides (A0A087SQ73) and ALPH2 from Trypanosoma brucei (ALPH2 has mito- chondrial localisation and is not the mRNA decapping enzyme). As a control, ALPH of Sphaeroforma arctica was also produced as an inactive mutant by mutating a conserved amino acid in the metal ion binding motif (GDVIG:GNVIG [41]). All four proteins were purified from Arctic express cells (Fig. 6A) and subsequently tested in in vitro decapping assays using a 39 nucleotide long RNA oligo with a m7G cap structure as a substrate. Capped and uncapped oligos can be distinguished by The data show that all ALPH enzymes tested accept capped mRNA as a substrate in vitro. Importantly, the tested ALPH proteins have non-cytoplasmic localisation Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 13 of 19 Page 13 of 19 The major aim of this work was to find out how wide- spread mRNA decapping by ApaH like phosphatases is. To identify ALPH proteins in all available eukaryotic proteomes, we have developed a Python based algorithm based on recognising the six conserved motifs of ALPH. We detected 441 ALPH proteins in 332 of 827 organisms. The full details of these 441 ALPH proteins are summa- rized in Additional file 3: Table S1 and contribute to a more comprehensive knowledge of this enzyme family. predictions, indicating that mRNA cannot be their physi- ological substrate. Instead, the findings suggest that ALPH proteins may have a similar broad substrate speci- ficity as previously shown for bacterial ApaH. The data support the hypothesis that the preference for none or non-cytoplasmic ALPHs in many eukaryotes could serve to prevent uncontrolled mRNA degradation. predictions, indicating that mRNA cannot be their physi- ological substrate. ALPHs have in vitro mRNAs decapping activityh Instead, the findings suggest that ALPH proteins may have a similar broad substrate speci- ficity as previously shown for bacterial ApaH. The data support the hypothesis that the preference for none or non-cytoplasmic ALPHs in many eukaryotes could serve to prevent uncontrolled mRNA degradation. Discussion Most eukaryotic mRNAs are modified at their 5´end by an m7 methylguanosine (m7G) cap, linked via an inverted 5´-5´bridge [44]. A small fraction of eukary- otic RNAs carries non-canonical nucleotide metabolite caps, for example an NAD + cap [45, 46]. Bacteria were long believed to simply keep the triphosphate that is naturally present at their 5´end after transcription, but more recently were found to have diphosphate 5´ends [47], and also non-canonical metabolite caps, such as the NAD + cap [48], the 3-dephospho-coenzyme A cap (dpCoA) [49] or dinucleoside polyphosphate caps [24– 26]. Archaea mRNAs are not well studied but do carry triphosphates caps [50]. In common to the increasing number of different mRNA caps and mRNA 5´ end modifications discov- ered throughout all kingdoms of life is the pyrophosphate bond, and therefore the requirement of a pyrophos- phatase to remove the cap or the polyphosphate. Enzymes of four different families can cleave pyrophos- phate bonds at the 5´end of mRNAs [51]. DXO and his- tidine triad (HIT) proteins, act mainly on faulty RNAs or on cap remnants or remove non-canonical NAD + caps without cleaving the pyrophosphate bond [51]. This leaves two pyrophosphatase families that act on intact mRNAs. Nudix domain proteins include the prototypic eukaryotic mRNA decapping enzyme Dcp2 [52] and sev- eral further eukaryotic nudix hydrolases with activity to the m7G cap and non-canonical metabolite caps [53–55] as well as the bacterial RppH [56] and NudC proteins [57] that act on di- or triphosphorylated mRNAs and on NAD + caps [51]. Enzymes of the fourth group, the ApaH family only entered the field of mRNA decapping most recently. The ApaH like phosphatase ALPH1 from Trypa- nosoma brucei was shown to be the only or major mRNA decapping enzyme in these parasites [10]. Bacterial ApaH cleaves of non-canonical nucleoside-tetraphosphate caps [24–26] and likely regulates gene expression during stress. Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 14 of 19 Fig. 7 Summary of the major findings of this work. For the ALPH proteins of Kinetolastida, non-cytoplasmic localisation was assumed for the non-ALPH1 orthologues and cytoplasmic localisation for ALPH1 orthologues, based on localisation studies in Trypanosoma brucei (Fig. 5) Fig. 7 Summary of the major findings of this work. Identification of ApaH like phosphatases Identification of ApaH like phosphatases A novel algorithm was developed using Python 3 to iden- tify ALPHs in a set of proteome fasta files. The algorithm initially used narrow matrices to identify the 6 conserved motifs (4 common to all PPP and 5–6 unique to ALPHs) based on the data of [3, 6], these matrices were succes- sively extended manually using a set of yeast proteomes and euglenozoan proteomes as training sets, controlled by BLAST. These initial screens gave information about conserved and non-conserved distances between the 6 motifs. Proteins that had only one motif missing were manually inspected further. If they possessed the miss- ing motif in an only slightly different version (e.g. only one amino acid altered in a conservative way) and, at the same time, had the correct distances between the motifs (where these distances are conserved), the motif matrix was extended to include this motif. One excep- tion was made for R at position 6 in motif 2: this is not tolerated, as it prevented the clear distinction of ALPHs from the related ApaH and RLPH enzymes [6]. Several rounds of manual iterations and matrix improvements resulted in an algorithm that uses one narrow matrix for the initial screen and, if not all motifs were found, the algorithm tolerates up to two motifs from an extended matrix. Furthermore, we included restrictions to three of the distances between the motifs. This algorithm was run with all 827 proteomes and identified 430 ALPH pro- teins. Thereafter, we screened all 827 proteomes for ‘one motif missing’, successively at each position (data shown in Additional file 4: Table S2a). Manual analysis of these data resulted in three further changes to the matrix and identification of 11 additional ALPH proteins (details in Additional file 4: Table S2a). The final software was run again on all 827 proteomes but did not identify any addi- tional ALPH proteins. In the final version of the software, the narrow matrices are defined as [G, D, VILT, HQ, G], [G, DN, LMIFVT, EIVTLAC, NSATFGVQIDHM, KQN, GASHT], [G, N, HNWQ, ED], [H, AG, G], [VIT, IVY- FLAMT, FY, G, H], [LVIMT, DE, STG, GASRN] for motif 1, 2, 3, 4, 5 and 6, respectively. Identification of ApaH like phosphatases The extended matrices are defined as [GxX, DxX, VILTxX, HQxX, GxX], [GxX, DNxX, TIFLYVMxX, ETIALCVMxX, EQTIASDFGH- CVNMxX, KQNxX, GASHTxX], [GxX, NxX, HNWQxX, EDxX], [HxX, AGLVxX, GxX], [AVILCMTxX, One open question is whether all organisms outside the Kinetoplastida use DCP2 orthologues as their major mRNA decapping enzyme. While DCP2 has been func- tionally characterised in yeast [52], human [64] and plants [65], there are no experimental data on Meta- monada and Discoba. Metamonada species possibly have a DCP2 homologue: in Trichomonas this can be identi- fied by Blast and in Giardia by more sophisticated in silico methods [66, 67]. Whether Discoba have DCP2 is unclear. A possible DCP2 homologue is identifiable by blast in the Discoba Naegleria, but not in Euglena. The question whether DCP2 was present in the last com- mon ancestor and selectively lost in Kinetoplastida, or whether it evolved subsequently to the separation of the Discoba cannot be answered without experimental data. In the last few years, more and more mRNA cap vari- ants have been discovered in both prokaryotes and eukaryotes, and more and more enzymes that act in mRNA decapping, including ApaH like phosphatases and bacterial ApaH. Our analysis of eukaryotic ApaH like phosphatases is an important contribution towards a more comprehensive picture of mRNA decapping. Discussion Usually, nudix hydrolases were exploited for this task, but Kinetoplastida uniquely use one of their ALPH proteins. Some non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisation: if these ALPH proteins are indeed cytoplasmic, they could act in mRNA decapping in addition to Dcp2 or else, they may have been evolved to not accept capped mRNA as a substrate. common ancestor of all eukaryotes had ALPH. This assumption is supported by the presence of ALPH pro- teins in all eukaryotic kingdoms found in this and earlier [6] studies. With the evolution of the eukaryotic mRNA cap, eukaryotes faced two problems: first, the cap needed to be protected from unregulated degradation by pyroph- osphatases and second, an mRNA decapping enzyme was needed for regulated degradation. The first chal- lenge was likely addressed in multiple ways, including for example protection by cap binding proteins like the eIF4F complex. One important evolutionary strategy may have been to inactivate the bacterial-inherited ALPH proteins. Many eukaryotes fully lost these bacterial- derived enzymes or at least did not tolerate cytoplasmic localisation. Evolution has solved the second challenge by evolving pyrophosphatases as regulatable mRNA decap- ping enzymes. Usually, nudix hydrolases were exploited for this task, but Kinetoplastida uniquely use one of their ALPH proteins. Some non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisation: if these ALPH proteins are indeed cytoplasmic, they could act in mRNA decapping in addition to Dcp2 or else, they may have been evolved to not accept capped mRNA as a substrate. available reference proteomes. Trypanosomes and possi- bly a few other organisms use ALPH proteins for mRNA decapping; however, the physiological functions of the vast majority of eukaryotic ALPH proteins remain to be discovered. In vivo experiments are essential, as the wide substrate range of ALPH will impede the identification of physiological substrates in vitro. Discussion For the ALPH proteins of Kinetolastida, non-cytoplasmic localisation w non-ALPH1 orthologues and cytoplasmic localisation for ALPH1 orthologues, based on localisation studies in Trypanosoma All three ALPH proteins that we tested had mRNA decapping activity in vitro, even though none of these enzymes is likely to use mRNA as their physiological substrate as all three have (predicted or proven) mito- chondrial localisation. This broad substrate range of ALPHs is not unexpected: the related bacterial ApaH protein cleaves NpnN nucleotides [13–15] as well was mRNA capped with NpnN [24, 26] and even has phos- phatase and ATPase activity in addition to its pyrophos- phatase activity [59]. The only other characterised ALPH protein, the S. cerevisiae ALPH protein Ppn2 has, to our knowledge, not been tested for mRNA decapping activ- ity. Substrate unspecificity is not restricted to ApaH and ApaH like phosphatases, but also found in (decapping) enzymes of the nudix domain family [60]: the major bac- teria decapping enzyme RppH was initially identified as diadenosine polyphosphate hydrolase [61] and can also cleave NAD+ caps [55]. Recent in vitro studies of mam- malian nudix proteins shows that many have mRNA decapping activity towards a range of caps and some also towards free metabolites [53, 55, 62, 63]; how many of these nudix proteins have mRNAs as their natural sub- strates is still debated.h The broad substrate range of ALPHs that includes mRNA caps provides an explanation for its loss or non- cytoplasmic localisation in most eukaryotes. The last Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 15 of 19 common ancestor of all eukaryotes had ALPH. This assumption is supported by the presence of ALPH pro- teins in all eukaryotic kingdoms found in this and earlier [6] studies. With the evolution of the eukaryotic mRNA cap, eukaryotes faced two problems: first, the cap needed to be protected from unregulated degradation by pyroph- osphatases and second, an mRNA decapping enzyme was needed for regulated degradation. The first chal- lenge was likely addressed in multiple ways, including for example protection by cap binding proteins like the eIF4F complex. One important evolutionary strategy may have been to inactivate the bacterial-inherited ALPH proteins. Many eukaryotes fully lost these bacterial- derived enzymes or at least did not tolerate cytoplasmic localisation. Evolution has solved the second challenge by evolving pyrophosphatases as regulatable mRNA decap- ping enzymes. Conclusions Despite being present in all eukaryotic kingdoms, ApaH like phosphatases are poorly understood with only a few studies available. Here, we present a new algorithm for the identification of ALPH proteins and we provide a comprehensive dataset of ApaH like phosphatases of all Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 16 of 19 IVYFLAMTxX, FYxX, GxX, HxX], [LVIMTxX, DExX, LATGSVxX, LMGRASNExX] for motif 1, 2, 3, 4, 5 and 6, respectively. Furthermore, the algorithm restricts the distances between the motifs to 14–150 (motif 1 to 2), 25–80 (motif 2 and 3) and 17–40 (motif 5 to 6). As one further control to ensure that all ALPH proteins were identified, we screened all 827 proteomes again with matrices tolerating more amino acids at positions in the motifs that appeared less conserved (matrices shown in Additional file 4: Table S2b): again, no further ALPH pro- teins were identified. following the instructions of the company. The con- centration of all purified proteins was estimated from Coomassie gels using a titration of a protein with known concentration for calibration. Proteomes Proteomes were downloaded from Uniprot, using all available reference proteomes of UniProt_release 2019_02 (For Archaea: 2019_11). [27]. Additional file 3: Table S1a lists all proteomes used in this work. For Kine- toplastida, we downloaded all available proteomes from TriTrypDB (February 2019) [28, 29]. During the revision process, we screened UniProt_release 2021_01 selec- tively for all phylogenetic groups (Fig. 1) that had no ref- erence proteomes available in UniProt_release 2019_02: this search identified proteomes of two Haptista and one Dinoflagellate. MGSSHHHHHHSSGLVPRGSASMSDSEVNQEAK- PEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLR- RLMEAFAKRQGKEMDSLRFLYDGIRIQADQT- PEDLDMEDNDIIEAHREQIGGHMENLYFQGEASAT. For cloning reasons, all proteins had the extension GSGSGC at the C-terminus. For the inactive mutant of the Sphaeroforma arctica ALPH, the highly conserved GDVIG motif involved in metal ion binding was mutated to GNVIG [41]. The purification of the recombinant proteins was done via Nickel-agarose beads using stand- ard procedures as previously described [10], except that expression in Arctic Express DE cells was done at 10 °C, Cloning and expression of recombinant proteins All ALPH proteins were expressed either full length (T. brucei ALPH2 = Tb927.4.4330) or with minor N-termi- nal truncations to exclude the transmembrane regions (Sphaeroforma arctica: amino acids 21–316; Auxeno- chlorella protothecoides: amino acids 21–327) in Arctic Express DE competent cells (Agilent). At the N-terminus, the proteins were fused to a tag consisting of a 6xHis- Tag, a Thrombin cleavage site, a SUMO tag and a TEV cleavage site, resulting in the following additional protein sequence: Phylogeny Th The tree of the Euglenozoa ALPHs (Fig. 5) was done with all ALPH catalytic domains (defined as starting 6 amino acids upstream of motif 1 and ending 8 amino acids downstream of motif 6) using MEGA X [68, 69]. All the sequences were aligned using Muscle (gap open −2.90; gap extend: 0.00; Hydrophobicity Multiplier: 1.20; Max Iterations: 16; Cluster Method: UPGMA; Min Diag Length (Lambda): 24) [70] and gaps were manually removed. The best protein substitution model was calcu- lated: JTT + G + I (BIC = 23908). The tree was built using the Maximum Likelihood method (500 bootstrap cycles) and the JTT matrix-based model [71]. The tree with the highest log likelihood (−11295.89) is shown. The per- centage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the JTT model, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter = 1.0217). The rate variation model allowed for some sites to be evolutionary invariable (+ I, 6.38% of sites). i ALPHs with missing motifs due to wrongly annotated start codons or sequencing errors will not be detected, with the exception of an unidentified amino acid included in the extended matrix (x, X). For the Kine- toplastida, such ALPHs were manually included based on the available genome information from TriTrypDB [28, 29]; for all other organisms, such truncated ALPH proteins (11 in total) are listed in Additional file 4: Table S2c. Finally, all proteins were manually inspected: even though the extended sequence matrix would allow the presence of motifs with little homology to the consensus motif, we found that the extended matrix was used rather rarely (1.9% of all motifs, affecting 11.3% of all ALPH pro- tein; no ALPH protein had 2 motifs form the extended matrix) and if so, the extended motifs had still most amino acids from the narrow matrix. The Python pro- gramme is available in Additional file 2: Python Software. Different methods (minimal evolution, neighbour join- ing) gave slightly different trees, but, importantly, with all methods, the ALPH1 isoforms group together in one clade that never contains non-ALPH1 isoforms. Microscopy Z-stack images (100 stacks at 100 nm distance) were taken with a custom build TILL Photonics iMic micro- scope equipped with a sensicam camera (PCO), decon- volved using Huygens Essential software (Scientific Volume Imaging B. V., Hilversum, The Netherlands). All images are presented as Z-projections (method sum sliced) produced by ImageJ software. Funding g Open Access funding enabled and organized by Projekt DEAL. This work was funded by the Deutsche Forschungsgemeinschaft (Kr4017 4-1). Availability of data and materials All data generated or analysed during this study are included in this published article and its Additional files. Authors’ contributions SK wrote the manuscript and did the in silico work of this study, with the exception of the phylogenetic tree of Additional file 1: Figure S1 that was done by BB. PACL and NB did all wet work (protein expression, decapping assays). All authors read and approved the final manuscript. RNA Samples were separated on urea acrylamide gels that contained acryloylaminophenyl boronic acid [42, 43]: boronyl groups form stable complexes with the m7G cap structure, this way increasing the difference in gel mobility between capped and uncapped oligos [42, 43]. For 12% gels, a 10 ml gel solution was freshly pre- pared with distilled water, containing 12% acrylamide/ bis-acrylamide (19:1) (Sigma-Aldrich), 7 M urea (Sigma- Aldrich), 0.1% ammonium persulfate, 4 µl TEMED, 0.25% acryloylaminophenyl boronic acid (Sigma-Aldrich) and 100 mM Tris–acetate (pH 9.0). Gels were run in 0.5 × TBE buffer (45 mM Tris–HCl pH 8.3, 45 mM boric acid, 1 mM EDTA) using the Mini-PROTEAN Tetra Ver- tical Electrophoresis cell (gel size 10 × 8 cm, BioRad). Each run was preceded by a 30 min pre-run at 300 V to warm the gel. The purified RNA samples were prepared for gel electrophoresis by adding 1 µl loading dye (gel loading buffer II, Invitrogen) followed by incubation at Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12862-021-01858-x. Additional file 1: Figure S1. Phylogenetic tree of ALPH in eukaryotes. Additional file 2: Python Software. Additional file 3: Table S1. List of all organisms that were screened for the presence of ALPH and detailed information on all ALPH proteins that were identified in this study. Acknowledgements We like to thank Martin Zoltner (Charles University in Prague, Czech Republic), Mark Carrington (University of Cambridge, UK) and Mark Field (University of Dundee, UK) for sharing unpublished proteomes and data on Euglena and Bodo Saltans. A special thank you goes to both our reviewers, and in particular to reviewer 2, whose constructive and detailed criticism has helped to signifi- cantly improve both the data and the manuscript. Work with trypanosomes Trypanosoma brucei Lister 427 procyclic cells expressing a TET repressor (pSPR2.1) were used for all experiments [39]. Cells were cultured in SDM-79 [72] at 27 ℃ and 5% CO2. Transgenic trypanosomes were generated using Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 17 of 19 95 °C for 5 min. Samples were loaded immediately to the pre-rinsed wells of the pre-run gel and the gel was run at 200 V (after a 10 min run at 170 V) for 30–40 min, stained for 20 min in SYBR™ Green II RNA Gel Stain (1:10,000 in 0.5 × TBE, ThermoFisher Scientific) and imaged on the iBright™ (Invitrogen) with nucleic acid settings. standard procedures [73]. All experiments used logarith- mically growing trypanosomes. The mitochondria were stained by adding 1 µM MitoTracker™ Orange CMTM- Ros (Invitrogen) to living cells; imaging was done within 30 min. In vitro decapping assays Each decapping reaction was done in 10 µl volume (with RNAse-free water) containing 50 mM Tris–HCl (pH 7.9), 100 mM NaCl, 1 mM metal ions (variable), 40 units Ribolock RNAse inhibitor (ThermoFisher Scientific), 0.25 µM recombinantly-produced ALPH enzyme and 0.39 µM capped RNA oligo m7G-AACUAACGCUAU UAUUAGAACAGUUUCUGUACUAUAUUG (Bio-Syn- thesis, Texas, USA). The sample was incubated for 1 h at 37 °C. Control reactions were done without enzymes. The reaction was stopped by ethanol precipitation: 1 µl 3 M RNAse-free sodium acetate (pH 5.5) (Ambion), 1 µl RNAse-free glycogen (ThermoFisher Scientific) and 30 µl cold 100% ethanol was added, the sample incubated at −20 °C for 30 min, followed by centrifugation (15 min, 20,000 g). The pellet (containing the RNA oligo) was washed once in 80% ethanol, air-dried, resolved in 5 µl RNAse-free water (ThermoFisher Scientific) and either stored at −80 °C or directly prepared for loading to the gel. Additional file 4: Table S2. a) Documentation of the last step of software improvement: the ‘missing motif screen’ on the complete set of pro- teomes. b) extended control matrix. c) list of ALPH proteins with truncated N- or C-termini. Additional file 4: Table S2. a) Documentation of the last step of software improvement: the ‘missing motif screen’ on the complete set of pro- teomes. b) extended control matrix. c) list of ALPH proteins with truncated N- or C-termini. Additional file 5: Table S3. The 285 available reference proteomes for Archaea were downloaded from UniProt on 7th of January 2020. They were screened with the Python programme for the presence of ALPH. Only one proteome contained one ALPH. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Competing interests Competing interests The authors declare that they have no competing interests. References Warrenfeltz S, Basenko EY, Crouch K, Harb OS, Kissinger JC, Roos DS, et al. EuPathDB: the eukaryotic pathogen genomics database resource. Methods Mol Biol. 2018;1757:69–113. 8. Andreeva N, Ledova L, Ryazanova L, Tomashevsky A, Kulakovskaya T, Eldarov M. Ppn2 endopolyphosphatase overexpressed in Saccharo- myces cerevisiae: comparison with Ppn1, Ppx1, and Ddp1 polyphos- phatases. Biochimie. 2019;163:101–7. 30. Adl SM, Bass D, Lane CE, Lukes J, Schoch CL, Smirnov A, et al. Revisions to the classification, nomenclature, and diversity of eukaryotes. J Eukaryot Microbiol. 2019;66:4–119. 9. Ryazanova LP, Ledova LA, Andreeva NA, Zvonarev AN, Eldarov MA, Kulakovskaya TV. Inorganic polyphosphate and physiological proper- ties of Saccharomyces cerevisiae yeast overexpressing Ppn2. Biochemis- try Mosc. 2020;85:516–22. 31. Crooks GE, Hon G, Chandonia J-M, Brenner SE. WebLogo: a sequence logo generator. Genome Res. 2004;14:1188–90. 32. Mitchell AL, Attwood TK, Babbitt PC, Blum M, Bork P, Bridge A, et al. InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 2019;47:D351–60. 10. Kramer S. The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes. PLoS Pathog. 2017;13:e1006456. 33. Käll L, Krogh A, Sonnhammer ELL. Advantages of combined transmem- brane topology and signal peptide prediction–the Phobius web server. Nucleic Acids Res. 2007;35:W429–32. 11. Barton GJ, Cohen PT, Barford D. Conservation analysis and structure prediction of the protein serine/threonine phosphatases. Sequence similarity with diadenosine tetraphosphatase from Escherichia coli suggests homology to the protein phosphatases. Eur J Biochem. 1994;220:225–37. 34. Almagro Armenteros JJ, Salvatore M, Emanuelsson O, Winther O, Heijne von G, Elofsson A, et al. Detecting sequence signals in targeting peptides using deep learning. Life Sci Alliance. 2019;2. 12. Koonin EV. Bacterial and bacteriophage protein phosphatases. Mol Microbiol. 1993;8:785–6. 35. Almagro Armenteros JJ, Sønderby CK, Sønderby SK, Nielsen H, Winther O. DeepLoc: prediction of protein subcellular localization using deep learn- ing. Bioinformatics. 2017;33:3387–95. 13. Plateau P, Fromant M, Brevet A, Gesquière A, Blanquet S. Catabo- lism of bis(5’-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine- 5’,5’-P1, P4-tetraphosphate pyrophosphohydrolase. Biochemistry. 1985;24:914–22. g 36. Horton P, Park K-J, Obayashi T, Fujita N, Harada H, Adams-Collier CJ, et al. WoLF PSORT: protein localization predictor. Nucleic Acids Res. 2007;35:W585–7. 37. Huh W-K, Falvo JV, Gerke LC, Carroll AS, Howson RW, Weissman JS, et al. Global analysis of protein localization in budding yeast. Nature. 2003;425:686–91. 14. Guranowski A, Jakubowski H, Holler E. Catabolism of diadenosine 5’,5"’- P1, P4-tetraphosphate in procaryotes. References GMP-dependent pathways. J Bacteriol. 2010;192:3011–23. 1. Kerk D, Templeton G, Moorhead GBG. Evolutionary radiation pattern of novel protein phosphatases revealed by analysis of protein data from the completely sequenced genomes of humans, green algae, and higher plants. Plant Physiol. 2008;146:351–67. 22. Ji X, Zou J, Peng H, Stolle A-S, Xie R, Zhang H, et al. Alarmone Ap4A is elevated by aminoglycoside antibiotics and enhances their bacteri- cidal activity. Proc Natl Acad Sci USA. 2019;116:9578–85. 23. Farr SB, Arnosti DN, Chamberlin MJ, Ames BN. An apaH mutation causes AppppA to accumulate and affects motility and catabolite repression in Escherichia coli. Proc Natl Acad Sci USA. 1989;86:5010–4. y 2. Shi Y. Serine/threonine phosphatases: mechanism through structure. Cell. 2009;139:468–84. 2. Shi Y. Serine/threonine phosphatases: mechanism through structure. Cell. 2009;139:468–84. 3. Andreeva AV, Kutuzov MA. Widespread presence of “bacterial-like” PPP phosphatases in eukaryotes. BMC Evol Biol. 2004;4:47. 3. Andreeva AV, Kutuzov MA. Widespread presence of “bacterial-like” PPP phosphatases in eukaryotes. BMC Evol Biol. 2004;4:47. 24. Luciano DJ, Levenson-Palmer R, Belasco JG. Stresses that Raise Np4A Levels Induce Protective Nucleoside Tetraphosphate Capping of Bacte- rial RNA. Mol Cell. 2019;1–19. 4. Uhrig RG, Moorhead GB. Two ancient bacterial-like PPP family phos- phatases from Arabidopsis are highly conserved plant proteins that possess unique properties. Plant Physiol. 2011;157:1778–92. 25. Luciano DJ, Belasco JG. Np4A alarmones function in bacteria as precur- sors to RNA caps. Proc Natl Acad Sci USA. 2020;117:3560–7. y 5. Uhrig RG, Labandera A-M, Moorhead GB. Arabidopsis PPP family of serine/threonine protein phosphatases: many targets but few engines. Trends Plant Sci. 2013;18:505–13. 26. Hudeček O, Benoni R, Reyes-Gutierrez PE, Culka M, Šanderová H, Hubálek M, et al. Dinucleoside polyphosphates act as 5’-RNA caps in bacteria. Nat Commun. 2020;11:1052–111. 6. Uhrig RG, Kerk D, Moorhead GB. Evolution of bacterial-like phospho- protein phosphatases in photosynthetic eukaryotes features ancestral mitochondrial or archaeal origin and possible lateral gene transfer. Plant Physiol. 2013;163:1829–43. 27. UniProt Consortium. UniProt: a worldwide hub of protein knowledge. Nucleic Acids Res. 2019;47:D506–15. 28. Aslett M, Aurrecoechea C, Berriman M, Brestelli J, Brunk BP, Carrington M, et al. TriTrypDB: a functional genomic resource for the Trypanosomatidae. Nucleic Acids Res. 2010;38:D457–62. y 7. Gerasimaitė R, Mayer A. Ppn2, a novel Zn2+-dependent polyphos- phatase in the acidocalcisome-like yeast vacuole. J Cell Sci. 2017;130:1625–36. 7. Gerasimaitė R, Mayer A. Ppn2, a novel Zn2+-dependent polyphos- phatase in the acidocalcisome-like yeast vacuole. J Cell Sci. 2017;130:1625–36. 29. Received: 18 December 2020 Accepted: 10 June 2021 20. Johnstone DB, Farr SB. AppppA binds to several proteins in Escherichia coli, including the heat shock and oxidative stress proteins DnaK, GroEL, E89, C45 and C40. EMBO J. 1991;10:3897–904. 21. Monds RD, Newell PD, Wagner JC, Schwartzman JA, Lu W, Rabinowitz JD, et al. Di-adenosine tetraphosphate (Ap4A) metabolism impacts biofilm formation by Pseudomonas fluorescens via modulation of c-di- GMP-dependent pathways. J Bacteriol. 2010;192:3011–23. Author details 1 1 Zell- Und Entwicklungsbiologie, Biozentrum, Universität Würzburg, Würzburg, Germany. 2 Department of Medicine, University of Cambridge, Cambridge, UK. Page 18 of 19 Page 18 of 19 Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Received: 18 December 2020 Accepted: 10 June 2021 References Purification and properties of diadenosine 5‘,5"’-P1, P4-tetraphosphate (symmetrical) pyrophospho- hydrolase from Escherichia coli K12. J Biol Chem. 1983;258:14784–9. 38. Breitkreutz A, Choi H, Sharom JR, Boucher L, Neduva V, Larsen B, et al. A global protein kinase and phosphatase interaction network in yeast. Sci- ence. 2010;328:1043–6. 15. Guranowski A. Specific and nonspecific enzymes involved in the catabolism of mononucleoside and dinucleoside polyphosphates. Pharmacol Ther. 2000;87:117–39. 39. Sunter J, Wickstead B, Gull K, Carrington M. A new generation of T7 RNA polymerase-independent inducible expression plasmids for Trypanosoma brucei. PLoS ONE. 2012;7:e35167. 16. Kimura Y, Tanaka C, Sasaki K, Sasaki M. High concentrations of intracel- lular Ap4A and/or Ap5A in developing Myxococcus xanthus cells inhibit sporulation. Microbiology (Reading). 2017;163:86–93. 40. Hammond MJ, Nenarokova A, Butenko A, Zoltner M, Dobáková EL, Field MC, et al. A uniquely complex mitochondrial proteome from Euglena gracilis. Ãvila-Arcos MC, editor. Mol Biol Evol. 2020;66:4. 17. Ismail TM, Hart CA, McLennan AG. Regulation of dinucleoside polyphosphate pools by the YgdP and ApaH hydrolases is essential for the ability of Salmonella enterica serovar typhimurium to invade cultured mammalian cells. J Biol Chem. 2003;278:32602–7. 41. Ogris E, Mudrak I, Mak E, Gibson D, Pallas DC. Catalytically inactive protein phosphatase 2A can bind to polyomavirus middle tumor antigen and support complex formation with pp60(c-src). J Virol. 1999;73:7390–8. 18. Hansen S, Lewis K, Vulić M. Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli. Antimicrob Agents Chemother. 2008;52:2718–26. 42. Nübel G, Sorgenfrei FA, Jäschke A. Boronate affinity electrophoresis for the purification and analysis of cofactor-modified RNAs. Methods. 2017;117:14–20. 19. Nishimura A, Moriya S, Ukai H, Nagai K, Wachi M, Yamada Y. Diadeno- sine 5’,5’“-”P1, P4-tetraphosphate (Ap4A) controls the timing of cell division in Escherichia coli. Genes Cells. 1997;2:401–13. 43. Igloi GL, Kössel H. Affinity electrophoresis for monitoring terminal phosphorylation and the presence of queuosine in RNA. Application of polyacrylamide containing a covalently bound boronic acid. Nucleic Acids Res. 1985;13:6881–98. Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Castañeda Londoño et al. BMC Ecol Evo (2021) 21:131 Page 19 of 19 Page 19 of 19 44. Furuichi Y. Discovery of m(7)G-cap in eukaryotic mRNAs. Proc Jpn Acad Ser B Phys Biol Sci. 2015;91:394–409. 61. Bessman MJ, Walsh JD, Dunn CA, Swaminathan J, Weldon JE, Shen J. References Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evo- lutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547–9. 68. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: molecular evo- lutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35:1547–9. 53. Sharma S, Grudzien-Nogalska E, Hamilton K, Jiao X, Yang J, Tong L, et al. Mammalian Nudix proteins cleave nucleotide metabolite caps on RNAs. Nucleic Acids Res. 2020;48:6788–98. 69. Stecher G, Tamura K, Kumar S. Molecular Evolutionary Genetics Analysis (MEGA) for macOS. Mol Biol Evol. 2020;37:1237–9. 54. Grudzien-Nogalska E, Kiledjian M. New insights into decapping enzymes and selective mRNA decay. WIREs RNA. 2017;8:1379. 70. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32:1792–7. 55. Grudzien-Nogalska E, Wu Y, Jiao X, Cui H, Mateyak MK, Hart RP, et al. Structural and mechanistic basis of mammalian Nudt12 RNA deNADding. Nat Chem Biol. 2019;15:575–82. 71. Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992;8:275–82. 71. Jones DT, Taylor WR, Thornton JM. The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci. 1992;8:275–82. 56. Deana A, Celesnik H, Belasco JG. The bacterial enzyme RppH triggers messenger RNA degradation by 5’ pyrophosphate removal. Nature. 2008;451:355–8. 72. Brun R, Schönenberger. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979;36:289–92. 72. Brun R, Schönenberger. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979;36:289–92. 57. Cahová H, Winz M-L, Höfer K, Nübel G, Jäschke A. NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs. Nature. 2015;519:374–7. 73. McCulloch R, Vassella E, Burton P, Boshart M, Barry JD. Transformation of monomorphic and pleomorphic Trypanosoma brucei. Methods Mol Biol. 2004;262:53–86. 58. Wurm JP, Sprangers R. Dcp2: an mRNA decapping enzyme that adopts many different shapes and forms. Curr Opin Struct Biol. 2019;59:115–23. References The gene ygdP, associated with the invasiveness of Escherichia coli K1, desig- nates a Nudix hydrolase, Orf176, active on adenosine (5“)-pentaphospho- (5”)-adenosine (Ap5A). J Biol Chem. 2001;276:37834–8. y 45. Kiledjian M, Eukaryotic RNA. 5’-End NAD+ Capping and DeNADding. Trends Cell Biol. 2018;28:454–64. y (5”)-adenosine (Ap5A). J Biol Chem. 2001;276:37834–8. 62. Abdelraheim SR, Spiller DG, McLennan AG. Mammalian NADH diphos- phatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein. Biochem J. 2003;374:329–35. 46. Wang J, Alvin Chew BL, Lai Y, Dong H, Xu L, Balamkundu S, et al. Quantify- ing the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA. Nucleic Acids Res. 2019;47:e130. 47. Luciano DJ, Vasilyev N, Richards J, Serganov A, Belasco JG. A novel RNA phosphorylation state enables 5’; end-dependent degradation in Escheri- chia coli. Mol Cell. 2017;67:44–6. 63. Abdelraheim SR, Spiller DG, McLennan AG. Mouse Nudt13 is a mito- chondrial nudix hydrolase with NAD(P)H pyrophosphohydrolase activity. Protein J. 2017;36:425–32. 48. Chen YG, Kowtoniuk WE, Agarwal I, Shen Y, Liu DR. LC/MS analysis of cel- lular RNA reveals NAD-linked RNA. Nat Chem Biol. 2009;5:879–81. 64. van Dijk E, Cougot N, Meyer S, Babajko S, Wahle E, Séraphin B. Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures. EMBO J. 2002;21:6915–24. 49. Kowtoniuk WE, Shen Y, Heemstra JM, Agarwal I, Liu DR. A chemical screen for biological small molecule-RNA conjugates reveals CoA-linked RNA. Proc Natl Acad Sci USA. 2009;106:7768–73. 65. Xu J, Yang JY, Niu QW, Chua NH. Arabidopsis DCP2, DCP1, and varicose form a decapping complex required for postembryonic development. Plant Cell. 2006;18:3386–98. 50. Clouet-d’Orval B, Batista M, Bouvier M, Quentin Y, Fichant G, Marchfelder A, et al. Insights into RNA processing pathways and associated-RNA degrading enzymes in Archaea. FEMS Microbiol Rev. 2018;42:579–613. 66. Williams CW, Elmendorf HG. Identification and analysis of the RNA degrading complexes and machinery of Giardia lamblia using an in silico approach. BMC Genomics BioMed Central. 2011;12:586–615. y 51. Kramer S, McLennan AG. The complex enzymology of mRNA decap- ping: Enzymes of four classes cleave pyrophosphate bonds. WIREs RNA. 2019;10:1511. 67. Bannerman BP, Kramer S, Dorrell RG, Carrington M. Multispecies reconstructions uncover widespread conservation, and lineage-specific elaborations in eukaryotic mRNA metabolism. Elias MC, editor. PLoS ONE. 2018;13:e0192633–23. 52. Dunckley T, Parker R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J. 1999;18:5411–22. 68. Publisher’s Note 59. Sasaki M, Takegawa K, Kimura Y. Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus. FEBS Lett. 2014;588:3395–402. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. y 60. McLennan AG. Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both? Cell Mol Life Sci. 2012;70:373–85. 60. McLennan AG. Substrate ambiguity among the nudix hydrolases: biologically significant, evolutionary remnant, or both? 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https://openalex.org/W3047570836	https://iris.uniroma1.it/bitstream/11573/1487063/1/Collamati_Drop-in_2020.pdf	English	null	A DROP-IN beta probe for robot-assisted 68Ga-PSMA radioguided surgery: first ex vivo technology evaluation using prostate cancer specimens	EJNMMI research	2,020	cc-by	7,228	* Correspondence: Riccardo.faccini@uniroma1.it Francesco Collamati and Matthias N. van Oosterom are co-first authors. 1Istituto Nazionale di Fisica Nucleare, Sezione di Roma, Piazzale Aldo Moro 2, 00185 Rome, Italy 4Dipartimento di Fisica, Università di Roma Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy Full list of author information is available at the end of the article Collamati et al. EJNMMI Research (2020) 10:92 https://doi.org/10.1186/s13550-020-00682-6 Collamati et al. EJNMMI Research (2020) 10:92 https://doi.org/10.1186/s13550-020-00682-6 Open Access A DROP-IN beta probe for robot-assisted 68Ga-PSMA radioguided surgery: first ex vivo technology evaluation using prostate cancer specimens 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. A DROP-IN beta probe for robot-assisted 68Ga-PSMA radioguided surgery: first ex vivo technology evaluation using prostate cancer specimens Francesco Collamati1†, Matthias N. van Oosterom2,3†, Micol De Simoni1,4, Riccardo Faccini1,4* , Marta Fischetti1,5, Carlo Mancini Terracciano1,4, Riccardo Mirabelli1,4, Roberto Moretti4,6, Judith olde Heuvel7, Elena Solfaroli Camillocci1,8, Florian van Beurden2,3, Henk G. van der Poel3, Renato A. Valdes Olmos2,9, Pim J. van Leeuwen3, Fijs W. B. van Leeuwen2,3,10 and Silvio Morganti1 Background: Recently, a flexible DROP-IN gamma-probe was introduced for robot-assisted radioguided surgery, using traditional low-energy SPECT-isotopes. In parallel, a novel approach to achieve sensitive radioguidance using beta-emitting PET isotopes has been proposed. Integration of these two concepts would allow to exploit the use of PET tracers during robot-assisted tumor-receptor-targeted. In this study, we have engineered and validated the performance of a novel DROP-IN beta particle (DROP-INβ) detector. Methods: Seven prostate cancer patients with PSMA-PET positive tumors received an additional intraoperative injection of ~ 70 MBq 68Ga-PSMA-11, followed by robot-assisted prostatectomy and extended pelvic lymph node dissection. The surgical specimens from these procedures were used to validate the performance of our DROP-INβ probe prototype, which merged a scintillating detector with a housing optimized for a 12-mm trocar and prograsp instruments. Results: After optimization of the detector and probe housing via Monte Carlo simulations, the resulting DROP-INβ probe prototype was tested in a robotic setting. In the ex vivo setting, the probe—positioned by the robot—was able to identify 68Ga-PSMA-11 containing hot-spots in the surgical specimens: signal-to-background (S/B) was > 5 when pathology confirmed that the tumor was located < 1 mm below the specimen surface. 68Ga-PSMA-11 containing (and PET positive) lymph nodes, as found in two patients, were also confirmed with the DROP-INβ probe (S/B > 3). The rotational freedom of the DROP-IN design and the ability to manipulate the probe with the prograsp tool allowed the surgeon to perform autonomous beta-tracing. Conclusions: This study demonstrates the feasibility of beta-radioguided surgery in a robotic context by means of a DROP-INβ detector. When translated to an in vivo setting in the future, this technique could provide a valuable tool in detecting tumor remnants on the prostate surface and in confirmation of PSMA-PET positive lymph nodes. Keywords: Radioguided surgery, Beta particle detection, Robot-assisted surgery, Prostate cancer, PET, PSMA © The Author(s). © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. DROP-INβ probe development β The β-detection probe used in this study was based on a cylindrical scintillator (6 mm diameter and 3 mm height) made of mono-crystalline para-terphenyl (doped with 0, 1% in mass of (E,E)-1,4-Diphenyl-1,3-butadiene) [15]. Being a non-hygroscopic organic scintillator with high light yield (~ 140% of anthracene) and low density (1.23 g/cm3), this material provides a high sensitivity to β par- ticles and elevated transparency to photons (e.g., the 511 keV γ rays as induced by PET radiopharmaceuti- cals). To improve light collection from the scintillator, the detector was surrounded with a 2-mm-thick white diffusing Delrin ring and covered in front with two 4 μm layers of a reflective aluminized-Mylar film. The light tightness of this assembly was achieved by adding an ex- ternal black poly-vinyl-chloride ring of 2 mm, covered on the front by a 15-μm layer of aluminum. Light collec- tion efficiency was maximized using a 3 × 3 mm2 silicon photomultiplier (SiPM C-series 30035, SensL Ltd.). After a first Monte Carlo-based study of such a probe in a Ga-PSMA context [16], a dedicated laboratory characterization has been performed. A detection effi- ciency of ~ 90% for 68Ga β particles and ~ 2.5% for 511 keV γ rays has been found [17]. g y For many diagnostic evaluations (e.g., PSMA), PET ra- diopharmaceuticals are still preferred. These PET isotopes induce both gamma ray from annihilation (i.e., 511 keV photon) and β+ particle (i.e., positron) emissions, provid- ing two possible detection routes that can be exploited for radioguidance purposes. Since the intraoperative detection of 511 keV gamma rays requires heavily collimated ap- proaches, and thus cumbersome probes, direct detection of β+ particle emissions has been explored [4]. Recently, a sensitive β-probe detector-technology, appropriate for both β−(i.e. electron) and β+ radioguided surgery was in- troduced [12–14]. Due to intrinsic differences among the interaction with matter of beta and gamma particles, beta probes require a small active area and basically no colli- mation: as a result, such β-probes can be much smaller and lighter than γ-probes, especially when active materials are chosen that are insensitive to the 511 keV γ-ray back- ground [15]. Therefore, such a detector allows to exploit the unique spatial resolution achievable with beta emis- sion. In fact, tissue penetration of ~ 1 MeV β-particles is The β detector was placed at the tip of the DROP-IN probe housing. Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research (2020) 10:92 Page 2 of 10 Page 2 of 10 Background much smaller than that of γ-rays (~ millimeters vs ~ centi- meters) making it a unique “surface scanning” technique, much less limited by the “shine-through” of deeper lying tracer uptake [12]. Direct beta detection might thus be a very effective methodology to detect tumors nearby healthy organs characterized by elevated physiological up- take of the radiopharmaceutical (e.g., tumor nearby healthy prostate). Radioguided surgery (RGS) is an interventional nuclear medicine technique that enables surgeons to identify, during the surgical procedure, lesions that had been de- tected with non-invasive preoperative imaging. Such guidance is achieved using a combination of radioactive tracers (i.e., radiopharmaceuticals) and intraoperative de- tection modalities [1]. The direct correlation between preoperative tracer mapping using, e.g., PET/CT and in- traoperative detection, reduces the probability of missing a lesion that had been preoperatively identified using the imaging [2]. Applications of this approach include the localization of metastases or primary tumor margins [3]. In an effort to explore the use of the widely available PET radiopharmaceutical [68Ga]Ga-PSMA-11 (68Ga-la- beled Glu-urea-Lys (Ahx)-HBED-CC) for robot-assisted radioguided surgery purposes, we have developed a DROP-IN beta (DROP-INβ) probe that exploits both the high beta detection efficiency and the compactness of such a detector [12] with the maneuverability of the DROP-IN concept [11] (see Fig.1). In this paper, we present its engineering together with its first characterization on ex vivo surgical specimens (i.e., pros- tate and lymph nodes) of PSMA-PET positive patients that received an additional dose of [68Ga]Ga-PSMA-11 during surgery. Nowadays, a noticeable amount of commercially avail- able radiopharmaceuticals is used for radioguided sur- gery [4]. Radioguidance based on low-energy (< 150 keV) gamma ray emitting radiopharmaceuticals is most com- monly applied for sentinel lymph node (SN) biopsy pro- cedures using (indocyanine green-)99mTc-nanocolloid [5], radioguided occult lesion localization (ROLL) proce- dures using 99mTc-labeled macro-aggregates [6], radio- guided 125I-seed localization (RSL) procedures [7], and 99mTc-PSMA-guided resection of lymph node metasta- ses in prostate cancer patients [3]. Hence, the most fre- quently used detection modality for intraoperative localization is the gamma-detection probe, which pro- vides numerical and acoustical feedback proportional to the amount of radiopharmaceutical localized. Unique for this modality is that it supports relatively “deep” signal detection (i.e., tissue only provides marginal attenuation of gamma ray emissions). Recently, the introduction of the DROP-IN gamma (DROP-INγ) probe concept helped to make radioguidance compatible with robotic surgery [8–11]. DROP-INβ probe development Similarly to the previously optimized DROP-INγ probe [11], a 45° angle grip was incorporated at the end of longitudinal axis of the probe, tailored to the ProGrasp Forceps (Intuitive Surgical Inc.), an instru- ment that is often used during a prostatectomy and lymph node dissection. Maintaining its compatibility Page 3 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research Fig. 1 DROP-INβ probe design. a Schematic representation of the probe components. b Example of one of the Monte Carlo simulations optimizing β-particle detection and γ-photon transparency. c Overview of the probe application setup, showing its high maneuverability Fig. 1 DROP-INβ probe design. a Schematic representation of the probe components. b Example of one of the Monte Carlo simulations optimizing β-particle detection and γ-photon transparency. c Overview of the probe application setup, showing its high maneuverability Fig. 1 DROP-INβ probe design. a Schematic representation of the probe components. b Example of one of the Monte Carlo simulations optimizing β-particle detection and γ-photon transparency. c Overview of the probe application setup, showing its high maneuverability with the da Vinci (Intuitive Surgical Inc.) apparatus, this ensured the maneuverability needed to fully exploit the specificity of beta-RGS. In fact, differently from gamma probes, beta detection requires the probe to have full ac- cess to the surface to be examined, due to the significant signal attenuation in tissue. Optimization of the DROP INβ probe design In order to optimize the design of the β-probe, a dedi- cated Monte Carlo simulation was performed in Geant4 [19]. In this simulation, the whole detector was recon- structed, and all physical processes of interest were taken into account to effectively reproduce particle scat- tering, absorption, energy deposition, and secondary par- ticles generation. These simulations indicated that a cavity behind the β particle detector would result in a lower noise-background: additional layers of material could in fact promote β+ to γ conversion close to the detector, creating noise-background (Fig. 1b). This de- sign concept yielded a light-weight probe construction (Fig. 1a), mostly transparent to 511 keV γ-induced noise. The housing was printed using acrylonitril-butadieen- styreen plastics and a Dimension Elite 3D printer (Stra- tasys Ltd.). Final dimensions of the whole probe were a length of 55 mm and a diameter of 12 mm, due to the available detector prototype. In the future, however, this diameter could be reduced (e.g., to 8 mm) if necessary. DROP-INβ probe development Portable electronics based on an Arduino Due (Arduino AG) equipped with a custom analog shield providing signal conditioning and trigger logic were used for the readout [18]. Sampling time was 1 s. At the end of the chain, the output in terms of counts per second (CPS) was displayed on a tablet, via wire- less connection. First ex vivo probe evaluation Patient selection In total, 7 patients with primary diagnosed locally (ad- vanced) high-risk prostate cancer were included (see Page 4 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research (2020) 10:92 “background” on the surrounding tissue (i.e., fat tissue and negative lymph nodes). Table 1). Inclusion criteria consisted of a primary tumor ≥2 cm (based on MRI) with a minimal average PSMA tracer uptake of 1.7 kBq/mL (based on PSMA PET/CT). These patients were mostly redirected to our clinical in- stitute for prostate cancer treatment; initial diagnostics was performed at the referring hospital. Therefore, based on local availability and preferences, diagnostic PSMA- PET/CT was performed with 18F-DCFPyl. This should however provide comparable uptake as 6[68Ga]Ga- PSMA-11 [20]. SUVmean measurements were performed by manually defining a volume of interest in the prostate tumor, using OsiriX medical imaging software (Pixmeo SARL). All patients were scheduled for a robot-assisted radical prostatectomy and extended pelvic lymph node dissection. In order to minimize radioactive exposure to both patient and medical personnel, a limited dose of ~ 70 MBq (median 68, IQR 63.5–82) [68Ga]Ga-PSMA-11 for radioguidance was intravenously administered in the operating room (OR), after docking the da Vinci robot. The study was approved by the local ethics committee (NL66218.031.18, trial NL8256 at trialregister.nl) and all patients provided a written informed consent. Pathology Following analysis, all specimens were sent to pathology for assessment using standard histopathological proce- dures [21]. Additionally, distances between the tumor and the inked specimen borders were measured at marked locations. Monitoring of radioactive exposure in the operating room To investigate the feasibility of radioguided surgery using [68Ga]Ga-PSMA-11, radiation safety was consid- ered an important topic. Therefore, radiation dose, as re- ceived by the operating room staff, was carefully monitored [22]. The surgeon (located behind the robotic console), the scrub nurse (located next to the patient in the sterile field), the assisting nurse (moving around the operating room, outside the sterile field), the anesthetist (located at the head of the patient, outside the sterile field), and the researcher (located > 1 m away from the patient, outside the sterile field) all had their own elec- tronic radiation dosimeter (MGPInstruments DMC 2000; Mirion Technologies, Ltd.). Probe countings At the end of the surgical procedure, roughly 2.5 h after injection (median 150 min; IQR 120–172.5), the surgical specimens (prostate and lymph node packages if present) were rinsed with saline and scanned using the DROP-INβ probe mounted on a da Vinci robot using the ProGrasp forceps instrument. Rinsing of the ex vivo specimens was performed to remove possible urine contamination, since [68Ga]Ga-PSMA-11is known to undergo renal clearance [20]. For prostate samples, “signal” was defined as the highest counting area, as confirmed with preoperative imaging informa- tion. The “background” was defined as the area nearby the “signal” where the counting rate dropped to the plateau value that was found in the rest of the sample (thus representing tracer uptake in the healthy prostate tissue). For lymph node samples, the “signal” was acquired on the lymph node itself, and Probe usage The developed DROP-INβ probe easily fitted through standard 12 mm trocars and pick-up of the probe was fa- cile using the standard da Vinci instruments of the sur- gical robot. Being a tethered design, probe maneuverability allowed for positioning with 6 degrees of freedom, as inherited from the ProGrasp forceps, with an effective scanning range of 0–140o around the tip of the instrument. Scanning with the probe could be per- formed autonomously from the surgical robotic console, not requiring the help of an assistant. Pt # patient number, N.A. not applicable, LNs lymph nodes, ExR external iliac right, ObR obturator right, ObL obturator left Ex vivo probe evaluation Ex vivo probe evaluation The seven included patients displayed clear PSMA-PET positive primary tumors (see Table 1), with a SUVmean The seven included patients displayed clear PSMA-PET positive primary tumors (see Table 1), with a SUVmean Table 1 Preoperative patient characteristics Pt # Age PSA (ng/mL) Gleason score Prostate volume on MRI (cc) Tumor stage SUVmean in primary tumor focus on PET SUVmean positive LNs on PET 1 71 4.4 4 + 4 = 8 30 cT2aN0M0 13.8 N.A. 2 57 5.3 4 + 4 = 8 55 cT1cN0M0 3.3 N.A. 3 73 8.3 4 + 5 = 9 76 cT3aN1M0 17.8 5.6 (ExR), 3.1 (ObR) 4 66 2.7 4 + 4 = 8 47 cT3bN0M0 4.1 N.A. 5 63 6.4 4 + 5 = 9 41 cT2cN0M0 11.7 N.A. 6 55 9.3 4 + 4 = 8 28 cT2bN0M1 14.7 N.A. 7 48 4.4 4 + 5 = 9 62 cT3bN1M0 13.3 4.8 (ObL), 3.5 (ExR) Pt # patient number, N.A. not applicable, LNs lymph nodes, ExR external iliac right, ObR obturator right, ObL obturator left Table 1 Preoperative patient characteristics Collamati et al. EJNMMI Research (2020) 10:92 Page 5 of 10 Page 5 of 10 in the tumor > 3. Additionally, two patients had PSMA- PET positive lesions, suspected for lymph node metasta- ses (see Fig. 2 and Table 1). together with their smaller SUVmean with respect to other cases, ended up limiting as expected the possibility of beta-tracing. g Figure 3 illustrates usage of the DROP-IN beta probe on surgical specimens using the da Vinci robot. Table 2 shows a summary of the collected data. In general, probe background measurements without any tissue (i.e., “dark counts”) were in the order of 0–2 CPS, while uncovered tumor areas, cleaved if necessary, provided count rates between 130 and 250 CPS. Due to its normal (i.e., de- fault) PSMA expression levels, healthy prostate tissue yielded ~ 5–45 CPS. The primary tumor in patients 1, 3, 5, 6, and 7 provided a maximum S/B (signal to back- ground ratio) > 5, displaying a maximum count rate of ~ 247 CPS on the surface of the excised prostate speci- men. At pathology, only patients 1 and 7 harbored true positive resections margins (i.e., tumor cells were found in the inked borders of the prostate at pathology). Ex vivo probe evaluation How- ever, in patients 3, 5, and 6, tumor was found within 1 mm of the resection margin, confirming a superficial tumor location. The maximum S/B measured for the prostate specimens in patients 2 and 4 was much lower, < 2.5. In these cases, pathology indicated that the tumor was located > 1.5 mm below the specimen margin, indi- cating a negative surgical margin. This occurrence, g Interestingly, patients 3 and 7 both harbored 2 lymph nodes each that were positive on preoperative PSMA- PET. Using the DROP-INβ probe, these lymph nodes also showed elevated tracer uptake with respect to the other lymph nodes and surrounding fat tissue: S/B > 3. At pathology, metastasis was only found in three of these lymph nodes, suggesting a false-positive PSMA up- take in one lymph node. In this limited group of PET positive lymph nodes, the smallest metastasis the probe was capable to detect had a 7-mm diameter (SUVmean of 5.6 on preoperative PSMA-PET, time between injection and measurement 3 h). All PET negative lymph nodes that were excised and analyzed yielded the same count- ing rates as nearby background tissue. Monitoring of radioactive exposure in the operating room The average radiation dose per surgery performed, as measured for the operating room staff, was 0.005 mSv for the surgeon, 0.016 mSv for the scrub nurse, 0.002 mSv for the assisting nurse, 0.001 mSv for the anesthetist, and 0.001 mSv for the researcher. Taking in Fig. 2 Preoperative tumor mapping using PSMA-PET. a Example of total body PET maximum intensity projection with tumor focus in prostate (blue, upwards arrow) and lymph node metastasis (green, downwards arrow). b PET/CT slice of the same patient illustrating a clear tumor focus within the prostate (blue arrow; SUVmean = 17.8). c PET/CT slice of the same patient displaying a lymph node metastasis (green arrow; SUVmean = 5.6) Fig. 2 Preoperative tumor mapping using PSMA-PET. a Example of total body PET maximum intensity projection with tumor focus in prostate (blue, upwards arrow) and lymph node metastasis (green, downwards arrow). b PET/CT slice of the same patient illustrating a clear tumor focus within the prostate (blue arrow; SUVmean = 17.8). c PET/CT slice of the same patient displaying a lymph node metastasis (green arrow; SUVmean = 5.6) Page 6 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research Fig. 3 DROP-INβ probe evaluation in relation to pathology. a Overview of the robot-assisted OR setup. b Example of robot-assisted beta-tracing with the DROP-INβ probe on the surface of a resected prostate sample. c Histopathology slide displaying tumor spread within the prostate with respect to the specimen surface. d Example of robot-assisted beta-tracing on the surface of a resected lymph node package. e Histopathology slide showing tumor spread within a PSMA-PET positive lymph node Fig. 3 DROP-INβ probe evaluation in relation to pathology. a Overview of the robot-assisted OR setup. b Example of robot-assisted beta-tracing with the DROP-INβ probe on the surface of a resected prostate sample. c Histopathology slide displaying tumor spread within the prostate with respect to the specimen surface. d Example of robot-assisted beta-tracing on the surface of a resected lymph node package. e Histopathology slide showing tumor spread within a PSMA-PET positive lymph node This initial ex vivo validation of the DROP-INβ probe concept showed a high signal to background (> 5) for tu- mors located < 1 mm from the resected surface, suggest- ing that the technique has the potential to support robotic surface scanning of primary tumor margins in prostate cancer. Monitoring of radioactive exposure in the operating room Even more precise characterization of the possible lesion depth with respect to the surgical margin might be possible with future developments in the underlying detection software algorithms [24]. In addition, confirming PSMA-positive lymph nodes (S/B > 3), the DROP-INβ probe concept might also support the intraoperative identification of metastatic lymph nodes. to account the guidelines from the International Com- mission on Radiological Protection [23], this would mean that the surgeon would be allowed to perform 200 of such [68Ga]Ga-PSMA-11 guided procedures a year, while the scrub nurse would be limited to 62 procedures a year. Funding This work was supported by Sapienza University of Rome [grant nos. RM116154C8EF4FBC, RM11715C7D2B14C5]. Acknowledgements Potential limitations of the proposed [68Ga]Ga-PSMA- 11-guided surgery concept are the radiation dose for the surgical staff (currently limited to about 62 procedures a year) and the contamination of the prostate margins by tracer containing urine. The DROP-INβ probes ability to detect lesions using < 70 MBq doses helps limit the expos- ure of the surgical staff. It is worth highlighting in particular that injecting the radiotracer directly in the operating room allowed ex vivo examination after ~ 2.5 × t1/2 (3 h, t1/2 = 68 m). Hence future in vivo application, e.g., 1 h p.i., would allow an even lower activity to be used to achieve a similar detection sensitivity, namely of the order of 40 MBq. Re- garding the urine contamination of the samples, as stated previously, the accumulation of PSMA tracers in healthy organs and in particular urine may yield background signals that complicate intraoperative margin detection [20]. How- ever, the direct detection of beta particles performed with a detector substantially transparent to gamma rays, as sug- gested in this paper, should drastically reduce the impact of such a background; only the signal originating from a few millimeters around the detector should be detected (i.e., thus only a small urine layer must be considered [24, 31]). Nonetheless, acknowledgement of this effect by radiochem- ists [32, 33] and the reduced renal clearance of for example The authors would like to thank Sven van Leeuwen (IMI-Lab, Department of Radiology, LUMC, the Netherlands) and Michael Boonekamp (Department of Technical Services subsection Development, LUMC, the Netherlands) for their assistance with the illustrations and prototyping of the probe housing. This study was supported in part by an NWO-TTW-VICI grant (no. TTW 16141). Abbreviations RGS: Radioguided surgery; PSMA: Prostatic Specific Membrane Antigen; S/ B: Signal to background ratio; OR: Operating room Discussion In this study, the first steps are made towards integra- tion of beta radioguided surgery within the robot- assisted setting. Using the DROP-IN concept, the sur- geon has full control of probe placement, yielding auton- omy and great maneuverability during radioguidance [8–11]. Direct beta detection provides, thanks to its spe- cificity and sensitivity, a useful way to probe prostate margins and suspect lymph nodes. Compared to the previously reported use of a DROP- INγ probe in combination with the tracer [99Tc]Tc- PSMA-I&S (i.e., salvage procedures for lymphatic Page 7 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research Table 2 Probe evaluation in relation to pathology Pt # Probe evaluation Pathologic evaluation Injected activity [MBq] Total activity left at time of scanning [MBq] and injection-surgery time interval [min] S/B prostate tumor* S/B PET positive LNs Extra-capsular tumor spread Positive resection margins Shortest tumor-border distance [mm] Tumor in PET Positive LNs Tumor stage 1 68 11 (180) 107/14 = 7.6 N.A. Yes Yes 0 N.A. pT3aN0 R1 2 88 15 (170) 40/16 = 2.5 N.A. No No > 3 N.A. pT2N0 R0 3 76 13 (175) 108/15 = 7.2 50/8 = 6.3 (ExR), 34/10 = 3.4 (ObR) Yes No < 1 Yes (ExR), no (ObR) pT3bN1 R0 4 97 32 (110) 88/40 = 2.2 N.A. Yes No > 1.5 N.A. pT3bN0 R0 5 65 14 (150) 108/15 = 7.2 N.A. No No < 0.5 N.A. pT2N0 R0 6 62 18 (120) 247/45 = 5.5 N.A. Yes No < 0.5 N.A. pT3aN1 R0 7 23 7 (120) 35/7 = 5.0 20/3 = 6.7 (ObL), 9/3 = 3.0 (ExR) Yes Yes 0 Yes(ObL), yes (ExR) pT3bN1 R1 Pt # patient number, S/B signal to background, N.A. not applicable, ExR external iliac right, ObR obturator right, ObL obturator left *As measured on the surface of the resected specimen. PET negative lymph nodes that were excised are not added in the table for clarity sake, since no sign of elevated tracer uptake was found in none of them with the probe either Collamati et al. EJNMMI Research (2020) 10:92 Page 8 of 10 Page 8 of 10 metastases) [10], the use of a DROP-INβ probe in com- bination with [68Ga]Ga-PSMA-11 possesses some unique advantages. First of all, this approach supports the use of more widely available PET tracers. Conclusion In this study, we presented the integration of two recent developments in RGS: a high efficiency beta detector and a flexible DROP-IN probe housing compatible with robot-assisted surgery. The first prototype of DROP-INβ probe has been successfully validated on ex vivo samples of prostate tumors with [68Ga]Ga-PSMA-11, being able to detect all PET positive resected specimens, with a smallest detected dimension in this data sample of 7 mm. Probe maneuverability was found to be the same of the DROP-INγ concept, which has already demonstrated its efficacy in in vivo tests. This DROP-INβ probe could thus help exploit the growing amount of disease specific PET tracers and may help provide a new powerful tool to perform tumor margin evaluation and confirm meta- static spread. Discussion Secondly, the limited tissue penetration of β particles (only a few millimeters) allows for an accurate surface scanning of the primary tumor margins [12], thus highlighting pos- sible tumor localizations on the prostate surface. Indeed, in the current study, beta radiation was severely attenu- ated when > 1.5 mm of healthy tissue was located be- tween the surface of the prostate specimen and the pathological tumor margins. In this sense, β-tracing benefits from similar positive features as fluorescence imaging [25], i.e., no ‘shine-through’ of neighboring or deeper lying tracer uptake and a superior spatial reso- lution [12, 26]. These features are essential when the extra-capsular spread of PSMA-overexpressing tumor lesions is pursued in a prostate with (significant) de- fault PSMA expression [27]. Consequently, β-tracing could provide a superior means for margin assess- ment during, e.g., nerve sparing surgery [28, 29]. Al- ternative to investigated beta-radiation detection for tumor margin assessment on the prostate surface, fully matured ex vivo technologies are available (e.g., NeuroSAFE [30]) and alternative β-emission-based imaging technologies are being explored (e.g., Ceren- kov [22]). Future research, and in particular random- ized trials, will have to show which technology is superior, or if different technologies can work in synergy. [18F]F-PSMA tracers [34, 35] may in the future help to overcome these issues. In addition, the influence of renal clearance might also be overcome by using β-emitting iso- topes that have a longer half-life, allowing the tumor resec- tion to take place after all renal clearance of non-bound tracer is realized, e.g. using alternative PET isotopes such as 64Cu (t1/2 = 12.7 hours), or even theranostic isotopes such as 67Cu (t1/2 = 2.5 days), 90Y (t1/2 = 2.66 days), or 177Lu (t1/2 = 6.6 days) [36, 37]. Authors’ contributions F. C., M. F., C. M. T., and R. M. developed and tuned the Monte Carlo simulation and analyzed its output. M. D. S., R. F., R. M., E. S. C., and Silvio M. took care of the hardware development of the detector including its testing. M. N. van O., J. o. H., F. v. B., H. G. v. d. P., R. A. V. O., P. J. v. L., and F. W.B. v. L. organized and performed the medical part of the experimentation, from the nuclear medicine and surgical point of view. The authors read and approved the final manuscript. Availability of data and materials The Monte Carlo simulation datasets used during the current study are available from the corresponding author on reasonable request. All data gained on patient samples during this study are included in this published article. Ethics approval and consent to participate Ethics approval and consent to participate The study was approved by the local ethics committee (NL66218.031.18) and all patients provided a written informed consent. References Morganti S, Bertani E, Bocci V, Colandrea M, Collamati F, Cremonesi M, et al. Tumor-non-tumor discrimination by a β- detector for Radio Guided Surgery on ex-vivo neuroendocrine tumors samples. Phys Med. 2020;72:96–102. 4. Van Oosterom MN, Rietbergen DDD, Welling MM, Van Der Poel HG, Maurer T, Van Leeuwen FWB. Recent advances in nuclear and hybrid detection modalities for image-guided surgery. Vol. 16, Expert Review of Medical Devices. Taylor and Francis Ltd; 2019. p. 711–734. 4. Van Oosterom MN, Rietbergen DDD, Welling MM, Van Der Poel HG, Maurer T, Van Leeuwen FWB. Recent advances in nuclear and hybrid detection modalities for image-guided surgery. Vol. 16, Expert Review of Medical Devices. Taylor and Francis Ltd; 2019. p. 711–734. 25. van Oosterom MN, van der Poel HG, van Leeuwen FWB, Meershoek P, Welling MM, Pinto F, et al. Extending the hybrid surgical guidance concept with freehand fluorescence tomography. IEEE Trans Med Imaging [Internet]. 2020 [cited 2020 Jan 27];39(1):226–235. Available from: http://www.ncbi.nlm. nih.gov/pubmed/31247546. 5. KleinJan GH, van Werkhoven E, van den Berg NS, Karakullukcu MB, Zijlmans HJMAA, van der Hage JA, et al. The best of both worlds: a hybrid approach for optimal pre- and intraoperative identification of sentinel lymph nodes. Eur J Nucl Med Mol Imaging. 2018;45(11):1915–25. 5. KleinJan GH, van Werkhoven E, van den Berg NS, Karakullukcu MB, Zijlmans HJMAA, van der Hage JA, et al. The best of both worlds: a hybrid approach for optimal pre- and intraoperative identification of sentinel lymph nodes. Eur J Nucl Med Mol Imaging. 2018;45(11):1915–25. 26. Bluemel C, Rubello D, Colletti PM, de Bree R, Herrmann K. Sentinel lymph node biopsy in oral and oropharyngeal squamous cell carcinoma: current status and unresolved challenges. Eur J Nucl Med Mol Imaging. Springer Berlin. 2015;42:1469–80. 6. Bowles H, Sánchez N, Tapias A, Paredes P, Campos F, Bluemel C, et al. Radioguided surgery and the GOSTT concept: from pre-operative image and intraoperative navigation to image-assisted excision. Rev Española Med Nucl e Imagen Mol (English Ed). 2017;36(3):175–84. 27. Fendler WP, Calais J, Allen-Auerbach M, Bluemel C, Eberhardt N, Emmett L, et al. 68Ga-PSMA-11 PET/CT interobserver agreement for prostate cancer assessments: an international multicenter prospective study. J Nucl Med. 2017;58(10):1617–23. 7. Pouw B, De Wit-Van Der Veen LJ, Stokkel MPM, Loo CE, Vrancken Peeters MJTFD, Valdés Olmos RA. Heading toward radioactive seed localization in non-palpable breast cancer surgery? A meta-analysis. J Surg Oncol. John Wiley and Sons Inc. 2015;111:185–91. 28. References 1. Herrmann K, Nieweg OE, Povoski SP. Radioguided surgery: current applications and innovative directions in clinical practice; 2016. 503 p. 1. Herrmann K, Nieweg OE, Povoski SP. Radioguided surgery: current applications and innovative directions in clinical practice; 2016. 503 p. 1. Herrmann K, Nieweg OE, Povoski SP. Radioguided surgery: current applications and innovative directions in clinical practice; 2016. 503 p. 20. Van Leeuwen FWB, Van Der Poel HG. Surgical guidance in prostate cancer: “from molecule to man” translations. Clin Cancer Res. 2016;22(6):1304–6. 2. Meershoek P, Buckle T, van Oosterom MN, KleinJan GH, van der Poel HG, van Leeuwen F. Can fluorescence-guided surgery help identify all lesions in unknown locations or is the integrated use of a roadmap created by preoperative imaging mandatory? A blinded study in prostate cancer patients. J Nucl Med [Internet]. 2019 [cited 2020 Jan 27];jnumed.119.235234. Available from: http://jnm.snmjournals.org/lookup/doi/10.2967/jnumed.11 9.235234. 2. Meershoek P, Buckle T, van Oosterom MN, KleinJan GH, van der Poel HG, van Leeuwen F. Can fluorescence-guided surgery help identify all lesions in unknown locations or is the integrated use of a roadmap created by preoperative imaging mandatory? A blinded study in prostate cancer patients. J Nucl Med [Internet]. 2019 [cited 2020 Jan 27];jnumed.119.235234. Available from: http://jnm.snmjournals.org/lookup/doi/10.2967/jnumed.11 9.235234. 21. Wang M, Tulman DB, Sholl AB, Kimbrell HZ, Mandava SH, Elfer KN, et al. Gigapixel surface imaging of radical prostatectomy specimens for comprehensive detection of cancer-positive surgical margins using structured illumination microscopy. Sci Rep. 2016;3:6. 22. olde, Heuvel J, de Wit-van der Veen BJ, van der Poel HG, Bekers EM, Grootendorst MR, Vyas KN, et al. 68Ga-PSMA Cerenkov luminescence imaging in primary prostate cancer: first-in-man series. Eur J Nucl Med Mol Imaging. 2020. https://link.springer.com/article/10.1007/s00259-020-04783-1#citeas. 3. Horn T, Krönke M, Rauscher I, Haller B, Robu S, Wester HJ, et al. Single lesion on prostate-specific membrane antigen-ligand positron emission tomography and low prostate-specific antigen are prognostic factors for a favorable biochemical response to prostate-specific membrane antigen- targeted radioguided surgery in recurrent prostate cancer. Eur Urol. 2019; 76(4):517–23. 3. Horn T, Krönke M, Rauscher I, Haller B, Robu S, Wester HJ, et al. Single lesion on prostate-specific membrane antigen-ligand positron emission tomography and low prostate-specific antigen are prognostic factors for a favorable biochemical response to prostate-specific membrane antigen- targeted radioguided surgery in recurrent prostate cancer. Eur Urol. 2019; 76(4):517–23. 23. The 2007 recommendations of the International Commission on Radiological Protection. ICRP publication 103. Ann ICRP. 2007;. 24. Received: 23 June 2020 Accepted: 28 July 2020 Received: 23 June 2020 Accepted: 28 July 2020 Received: 23 June 2020 Accepted: 28 July 2020 19. Allison J, Amako K, Apostolakis J, Arce P, Asai M, Aso T, et al. Recent developments in GEANT4. Nucl Instruments Methods Phys Res Sect A Accel Spectrometers, Detect Assoc Equip. 2016;835:186–225. Consent for publication Not applicable Page 9 of 10 Page 9 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research (2020) 10:92 Competing interests radioguidance: first-in-human translation. Eur J Nucl Med Mol Imaging. 2019;46(1):49–53. F.C. and R.F are listed as inventors on an Italian patent application (RM2013A000053) entitled “Utilizzo di radiazione beta- per la identificazione intraoperatoria di residui tumorali e la corrispondente sonda di rivelazione” dealing with the implementation of an intraoperative beta- probe for radioguided surgery according to the results presented in this paper. The same authors are also inventors in the PCT patent application (PCT/IT2014/ 000025) entitled “Intraoperative detection of tumor residues using beta- radiation and corresponding probes” covering the method and the instruments described in this paper. 10. Van Leeuwen FWB, Van Oosterom MN, Meershoek P, Van Leeuwen PJ, Berliner C, Van Der Poel HG, et al. Minimal-invasive robot-assisted image- guided resection of prostate-specific membrane antigen-positive lymph nodes in recurrent prostate cancer. Clin Nucl Med. 2019;44(7):580–1. 11. van Oosterom MN, Simon H, Mengus L, Welling MM, van der Poel HG, van den Berg NS, et al. Revolutionizing (robot-assisted) laparoscopic gamma tracing using a drop-in gamma probe technology. Am J Nucl Med Mol Imaging [Internet]. 2016 [cited 2020 Jan 27];6(1):1–17. Available from: http:// www.ncbi.nlm.nih.gov/pubmed/27069762. FWB van Leeuwen is a consultant for Hamamatsu Photonics and is Chief Innovation Officer at ORSI Academy. FWB van Leeuwen is a consultant for Hamamatsu Photonics and is Chief Innovation Officer at ORSI Academy. 12. Camillocci ES, Baroni G, Bellini F, Bocci V, Collamati F, Cremonesi M, et al. A novel radioguided surgery technique exploiting β−decays. Sci Rep [Internet]. 2015;4(1):4401 Available from: http://www.nature.com/articles/ srep04401. y No other potential conflicts of interest relevant to this article exist. No other potential conflicts of interest relevant to this article exist. No other potential conflicts of interest relevant to this article exist Author details 1 1Istituto Nazionale di Fisica Nucleare, Sezione di Roma, Piazzale Aldo Moro 2, 00185 Rome, Italy. 2Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Albinusdreef 2, 2333ZA Leiden, The Netherlands. 3Department of Urology, The Netherlands Cancer Institute—Antoni van Leeuwenhoek, Amsterdam, The Netherlands. 4Dipartimento di Fisica, Università di Roma Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy. 5Dipartimento Di Scienze di Base Applicate per l’Ingegneria, Sapienza Università di Roma, Rome, Italy. 6Scuola di specializzazione in Fisica Medica, Università Cattolica del Sacro Cuore, Rome, Italy. 7Department of Nuclear Medicine, The Netherlands Cancer Institute—Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands. 8Scuola di specializzazione in Fisica Medica, Sapienza Università di Roma, Rome, Italy. 9Section Nuclear Medicine, Department of Radiology, Leiden University Medical Center, Leiden, The Netherlands. 10ORSI Academy, Melle, Belgium. 13. Solfaroli Camillocci E, Schiariti M, Bocci V, Carollo A, Chiodi G, Colandrea M, et al. First ex vivo validation of a radioguided surgery technique with β- radiation. Phys Med. 2016;32(9):1139–44. 14. Mancini-Terracciano C, Donnarumma R, Bencivenga G, Bocci V, Cartoni A, Collamati F, et al. Feasibility of beta-particle radioguided surgery for a variety of “nuclear medicine” radionuclides. Phys Med. 2017;43. 15. Angelone M, Battistoni G, Bellini F, Bocci V, Collamati F, De Lucia E, et al. Properties of para-terphenyl as a detector for α, β and γ radiation. IEEE Trans Nucl Sci. 2014;61(3):1483–7. 16. Collamati F, Bocci V, Castellucci P, De Simoni M, Fanti S, Faccini R, et al. Radioguided surgery with β radiation: a novel application with Ga68. Sci Rep. 2018;8(1):16171. 16. Collamati F, Bocci V, Castellucci P, De Simoni M, Fanti S, Faccini R, et al. Radioguided surgery with β radiation: a novel application with Ga68. Sci Rep. 2018;8(1):16171. 17. Collamati F, Moretti R, Alunni-Solestizi L, Bocci V, Cartoni A, Collarino A, et al. Characterisation of a β detector on positron emitters for medical applications. Phys Med. 2019;67:85–90. 17. Collamati F, Moretti R, Alunni-Solestizi L, Bocci V, Cartoni A, Collarino A, et al. Characterisation of a β detector on positron emitters for medical applications. Phys Med. 2019;67:85–90. 18. Bocci V, Chiodi G, Iacoangeli F, Nuccetelli M, Recchia L. The ArduSiPM a compact trasportable software/hardware data acquisition system for SiPM detector. In: 2014 IEEE Nuclear Science Symposium and Medical Imaging Conference (NSS/MIC). 2014. p. 1–5. References F- 18 labelled PSMA-1007: biodistribution, radiation dosimetry and histopathological validation of tumor lesions in prostate cancer patients. Eur J Nucl Med Mol Imaging. 2017;44(4):678–88. 34. Giesel FL, Hadaschik B, Cardinale J, Radtke J, Vinsensia M, Lehnert W, et al. F- 18 labelled PSMA-1007: biodistribution, radiation dosimetry and histopathological validation of tumor lesions in prostate cancer patients. Eur J Nucl Med Mol Imaging. 2017;44(4):678–88. 35. Collamati F, Bellini F, Bocci V, De Lucia E, Ferri V, Fioroni F, et al. Time evolution of DOTATOC uptake in neuroendocrine tumors in view of a possible application of radioguided surgery with b2 decay. J Nucl Med. 2015;56(10):1501–6. 35. Collamati F, Bellini F, Bocci V, De Lucia E, Ferri V, Fioroni F, et al. Time evolution of DOTATOC uptake in neuroendocrine tumors in view of a possible application of radioguided surgery with b2 decay. J Nucl Med. 2015;56(10):1501–6. 36. Zia NA, Cullinane C, Van Zuylekom JK, Waldeck K, McInnes LE, Buncic G, et al. A bivalent inhibitor of prostate specific membrane antigen radiolabeled with copper-64 with high tumor uptake and retention. Angew Chemie Int Ed [Internet]. 2019 [cited 2020 Jan 27];58(42):14991–4. Available from: http://doi.wiley.com/10.1002/anie.201908964. 36. Zia NA, Cullinane C, Van Zuylekom JK, Waldeck K, McInnes LE, Buncic G, et al. A bivalent inhibitor of prostate specific membrane antigen radiolabeled with copper-64 with high tumor uptake and retention. Angew Chemie Int Ed [Internet]. 2019 [cited 2020 Jan 27];58(42):14991–4. Available from: http://doi.wiley.com/10.1002/anie.201908964. 37. Baur B, Solbach C, Andreolli E, Winter G, Machulla HJ, Reske SN. Synthesis, radiolabelling and in vitro characterization of the gallium-68-, yttrium-90- and lutetium-177-labelled PSMA Ligand, CHX-A-DTPA-DUPA-Pep. Pharmaceuticals. 2014;7(5):517–29. 37. Baur B, Solbach C, Andreolli E, Winter G, Machulla HJ, Reske SN. Synthesis, radiolabelling and in vitro characterization of the gallium-68-, yttrium-90- and lutetium-177-labelled PSMA Ligand, CHX-A-DTPA-DUPA-Pep. Pharmaceuticals. 2014;7(5):517–29. References van Leeuwen FWB, Winter A, van Der Poel HG, Eiber M, Suardi N, Graefen M, et al. Technologies for image-guided surgery for managing lymphatic metastases in prostate cancer. Nat Rev Urol. Nature Publishing Group. 2019; 16:159–71. 8. Fuerst B, Sprung J, Pinto F, Frisch B, Wendler T, Simon H, et al. First robotic SPECT for minimally invasive sentinel lymph node mapping. IEEE Trans Med Imaging. 2016;35(3):830–8. 8. Fuerst B, Sprung J, Pinto F, Frisch B, Wendler T, Simon H, et al. First robotic SPECT for minimally invasive sentinel lymph node mapping. IEEE Trans Med Imaging. 2016;35(3):830–8. 29. Yossepowitch O, Briganti A, Eastham JA, Epstein J, Graefen M, Montironi R, et al. Positive surgical margins after radical prostatectomy: a systematic review and contemporary update. Eur Urol. 2014;65:303–13. 9. Meershoek P, van Oosterom MN, Simon H, Mengus L, Maurer T, van Leeuwen PJ, et al. Robot-assisted laparoscopic surgery using DROP-IN Page 10 of 10 Collamati et al. EJNMMI Research (2020) 10:92 Collamati et al. EJNMMI Research (2020) 10:92 30. Beyer B, Schlomm T, Tennstedt P, Boehm K, Adam M, Schiffmann J, et al. A feasible and time-efficient adaptation of NeuroSAFE for da Vinci robot- assisted radical prostatectomy. Eur Urol. 2014;66(1):138–44. 30. Beyer B, Schlomm T, Tennstedt P, Boehm K, Adam M, Schiffmann J, et al. A feasible and time-efficient adaptation of NeuroSAFE for da Vinci robot- assisted radical prostatectomy. Eur Urol. 2014;66(1):138–44. 31. Collamati F, Maccora D, Alfieri S, Bocci V, Cartoni A, Collarino A, et al. Radioguided surgery with β −radiation in pancreatic neuroendocrine tumors: a feasibility study. Sci Rep. 2020;10(1):1–10. 31. Collamati F, Maccora D, Alfieri S, Bocci V, Cartoni A, Collarino A, et al. Radioguided surgery with β −radiation in pancreatic neuroendocrine tumors: a feasibility study. Sci Rep. 2020;10(1):1–10. 32. Hensbergen AW, Buckle T, van Willigen DM, Schottelius M, Welling MM, van der Wijk FA, et al. Hybrid tracers based on cyanine backbones targeting prostate-specific membrane antigen – tuning pharmacokinetic properties and exploring dye–protein interaction. J Nucl Med. 2019. https://doi.org/10. 2967/jnumed.119.233064. 33. Hensbergen AW, van Willigen DM, van Beurden F, van Leeuwen PJ, Buckle T, Schottelius M, et al. Image-guided surgery: are we getting the most out of small-molecule prostate-specific-membrane-antigen-targeted tracers? Bioconjug Chem [Internet]. 2020 [cited 2020 Jan 27];acs.bioconjchem. 9b00758. Available from: https://pubs.acs.org/doi/abs/10.1021/acs. bioconjchem.9b00758. 34. Giesel FL, Hadaschik B, Cardinale J, Radtke J, Vinsensia M, Lehnert W, et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W3000638802	https://www.scielo.br/j/rsp/a/ztLYnPcNFcszFNDrBCFRchq/?lang=en&format=pdf	English	null	Prenatal care in the Brazilian public health services	Revista de saúde pública/Revista de Saúde Pública	2,020	cc-by	8,080	Correspondence: Maria do Carmo Leal Escola Nacional de Saúde Pública. Fundação Oswaldo Cruz (ENSP/Fiocruz). Rua Leopoldo Bulhões, 1480 - sala 809, Manguinhos. Rio de Janeiro, RJ, Brasil. CEP 21041-210 Email: ducaleal@gmail.com http://www.rsp.fsp.usp.br/ Original Article Original Article Rev Saude Publica. 2020;54:8 Prenatal care in the Brazilian public health services Maria do Carmo LealI , Ana Paula Esteves-PereiraI , Elaine Fernandes ViellasI , Rosa Maria Soares Madeira DominguesII , Silvana Granado Nogueira da GamaI Maria do Carmo LealI , Ana Paula Esteves-PereiraI , Elaine Fernandes ViellasI , Rosa Maria Soares Madeira DominguesII , Silvana Granado Nogueira da GamaI I Fundação Oswaldo Cruz. Escola Nacional de Saúde Pública (ENSP/Fiocruz). Departamento de Epidemiologia e Métodos Quantitativos em Saúde (DEMQS). Rio de Janeiro, RJ, Brasil Q Q II Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em HIV/Aids (INI/Fiocruz). Rio de Janeiro, RJ, Brasil II Fundação Oswaldo Cruz. Instituto Nacional de Infectologia Evandro Chagas. Laboratório de Pesquisa Clínica em HIV/Aids (INI/Fiocruz). Rio de Janeiro, RJ, Brasil INTRODUCTION Prenatal care is a set of simultaneously preventive, health promotion, diagnostic and curative actions targeting favorable pregnancy outcomes for women and their children1. Prenatal care is a set of simultaneously preventive, health promotion, diagnostic and curative actions targeting favorable pregnancy outcomes for women and their children1. The Brazilian recommendation for prenatal care in 2012 was of at least six prenatal care visits, including vaccination, routine diagnostic laboratory tests, and the use of supplements or medical treatment for complications2. All the procedures should be registered in the hand-held prenatal notes, aiming reference and counter-reference at the time of birth. The bond between the pregnant woman and the place of birth is also recommended to prevent pilgrimage: the search for hospital care during labor3. Data from the Brazilian Live Birth Information System (SINASC) show the evolution of prenatal care coverage in Brazil. In the year of 1995, more than 10% of Brazilian pregnant women did not have any prenatal care visit, and in 2015, only 2.2%. Less than half of the pregnant women used to attend seven or more prenatal care visits in 1995, increasing to 66.5% in 2015, showing an expansion of this coverage and the importance of the Brazilian Unified Health System (SUS), created in 1990, to the dissemination of this benefit4. In 2013, the Ministry of Health (MH), the National Council of State Health Secretaries (CONASS), and the National Council of Municipal Health Secretaries (CONASEMS) agreed upon some health care indicators to strengthen the Integrated Planning of SUS and the implementation of the Public Health Action Organizational Contract (COAP)5. The coverage of prenatal care is among these health care indicators, and we evaluated it here according to the five geographic macro regions, within SUS, using the data from the Birth in Brazil survey. We also analyzed the effects of inadequate prenatal care on the health of women and newborns. Prenatal care in the Brazilian public sector Leal MC et al. ABSTRACT OBJECTIVE: To verify regional inequalities regarding access and quality of prenatal and birth care in Brazilian public health services and associated perinatal outcomes. METHODS: Birth in Brazil was a national hospital-based survey conducted between 2011 and 2012, which included 19,117 women with public-funded births. Regional differences in socio-demographic and obstetric characteristics, as well as differences in access and quality of prenatal and birth care were tested by the χ2 test. The following outcomes were assessed: spontaneous preterm birth, provider-initiated preterm birth, low birth weight, intrauterine growth restriction, Apgar in the 5th min < 8, neonatal and maternal near miss. Multiple and non-conditional logistic regressions were used for the analysis of the associated perinatal outcomes, with the results expressed in adjusted odds ratio and 95% confidence interval. RESULTS: Regional inequalities regarding access and quality of prenatal and birth care among users of public services are still evident in Brazil. Pilgrimage for birth associated with all perinatal outcomes studied, except for intrauterine growth restriction. The odds ratios ranged between 1.48 (95%CI 1.23–1.78) for neonatal near miss and 1.62 (95%CI 1.27–2.06) for provider-initiated preterm birth. Among women with clinical or obstetric complications, pilgrimage for birth associated with provider-initiated preterm birth and with Apgar in the 5th min < 8, odds ratio of 1.98 (95%CI 1.49–2.65) and 2.19 (95%CI 1.31–3.68), respectively. Inadequacy of prenatal care associated with spontaneous preterm birth in both groups of women, with or without clinical or obstetric complications. CONCLUSION: Improvements in the quality of prenatal care, appropriate coordination and comprehensive care at the time of birth have a potential to reduce prematurity rates and, consequently, infant morbidity and mortality rates in the country. Received: Jan 17, 2019 Approved: Apr 22, 2019 How to cite: Leal MC, Esteves-Pereira AP, Viellas EF, Domingues RMSM, Gama, SGN. Prenatal care in the Brazilian public sector. Rev Saude Publica. 2020;54:8. DESCRIPTORS: Prenatal Care. Maternal-Child Health Services. Health Care Quality, Access, and Evaluation. Socioeconomic Factors. Health Status Disparities. Copyright: 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 that the original author and source are credited. 1 http://doi.org/10.11606/s1518-8787.2020054001458 1 http://doi.org/10.11606/s1518-8787.2020054001458 Prenatal care in the Brazilian public sector Leal MC et al. INTRODUCTION METHODS The first indicator considered the gestational trimester at the time of prenatal onset and the total number of appointments, adjusted for the gestational age at the birth. The onset of prenatal care was considered appropriate when performed until the 12th gestational week, according to the recommendation of the MH. The minimum calendar of the MH was used to calculate the adequacy of the number of appointments. The Ministry of Health recommends at least one appointment in the first gestational trimester, two appointments in the second and three appointments in the last trimester2. The number of appointments was considered adequate when the pregnant woman attended 100% of the minimum appointments planned for the gestational age at birth. The indicator was considered adequate when both early onset and number of prenatal appointments were adequate. The second indicator, named overall adequacy 1, was created by Domingues et al.8, and it considers early onset, minimum number of appointments, routine examinations performed and guidance on reference maternity for birth. Prenatal care is considered adequate when the onset occurs until the 12th gestational week; the number of appointments is adequate (≥ 100% of the appointments planned for gestational age in birth); at least one test result must be recorded: fasting blood sugar, AES, VDRL, HIV and ultrasound; and there is guidance on the maternity reference for birth. The third indicator, named overall adequacy 2, considered the items of overall adequacy 1 plus having birthed in the hospital for which the woman was referred to during prenatal care. The following outcomes were assessed: spontaneous preterm birth, provider-initiated preterm birth, low birth weight (LBW < 2,500g), intrauterine growth restriction (IUGR), Apgar in the 5th min < 8, neonatal near miss and maternal near miss. We classified as spontaneous preterm birth the ones with less than 37 gestational weeks in which the beginning of labor (L) was spontaneous or with premature rupture of the membranes. Provider-initiated preterm births were initiated either by induction or cesarean section before L. The women with rupture of membranes that gave birth by induced L or cesarean section before L were classified in the category spontaneous births. Labor was considered as induced if women with intact membranes received medical intervention to initiate uterine contraction before the onset of spontaneous labor. Surgeries that occurred without spontaneous or induced labor were considered cesarean sections before L. METHODS This study is part of Birth in Brazil study, a research conducted in public and private services between 2011 and 2012. Birth in Brazil is a nationwide hospital survey, with sampling in three stages of selection. In the first stage, hospitals with 500 or more deliveries per year were selected, stratified according to the macro regions of the country, by location (capital or non-capital) and by the type of hospital (public, private or mixed). In the second stage, the number of days necessary to perform 90 interviews with puerperal women in each hospital was defined. In the third and last stage, eligible puerperal women were selected. More information about the sample design is detailed by Vasconcellos et al.6 In total, Birth in Brazil included 23,894 puerperal women admitted for birth in the 266 selected hospitals and their newborns with any weight and gestational age, or stillbirths with weight ≥ 500g and/or gestational age ≥ 22 weeks of gestation. For the current analysis, we included only 19,117 women with public-funded births, representing 80% of the national sample. Information were obtained from face-to-face interviews with women after birth, from medical records and from hand-held prenatal notes. Detailed information on data collection is available in another publication7. Post-hoc calculations showed that—with a significance level of 5% and spontaneous preterm birth of 6% in the group of unexposed—the subgroup with the lowest sample size in the multiple regression (3,807 women with clinical or gestational complications) would have 90% power to detect an increased risk corresponding to an odds ratio (OR) ≥ 1.3. For the rarest outcomes, Apgar score in the 5th minute < 8 and maternal near miss, the smallest Prenatal care in the Brazilian public sector Leal MC et al. subgroup of women would have 90% power to detect increased risks corresponding to OR ≥ 2.0 and 2.5, respectively. Prenatal care and access to maternity hospitals indicators were: type of prenatal care unit; trimester of onset of prenatal care; number of prenatal care appointments; had hand-held prenatal notes; had hand-held prenatal notes upon admission for birth; results of routine tests recorded in hand-held prenatal notes [fasting blood sugar test, urine (abnormal sediment elements—ASE), (VDRL), HIV and ultrasound]; received guidance on reference maternity for birth; birthed in the referenced maternity; pilgrimage for birth; type of hospital and location of hospital. For the assessment of the prenatal care adequacy, three indicators were used. METHODS We used the 10th percentile of weight for gestational age at birth according to the Intergrowth criterion9 to classify the IUGR. Gestational age at birth was calculated by an algorithm based on estimates of early ultrasounds10. The variable neonatal near miss was built based on recommendations from Pileggi-Castro et al.11, using information from hospital records. The presence of any of the following features indicates the neonatal near miss. Pragmatic criteria: Apgar score in the 5th min < 7, birth weight < 1,750g and gestational age < 33 weeks. Management criteria: antibiotic use, continuous positive airway pressure (CPAP), exposure to phototherapy in the first 72 hours; use of vasoactive drug, anticonvulsant, surfactant, cardiac massage, presence of hypoglycemia, and orotracheal intubation. The neonatal near miss is defined as a morbid event that almost resulted in newborn death in the first 28 days of life12. Prenatal care in the Brazilian public sector Leal MC et al. Maternal near miss is defined by the World Health Organization (WHO) as “a woman who nearly died but survived a complication that occurred during pregnancy, childbirth or within 42 days after termination of pregnancy13.” WHO proposes a classification using 25 criteria based on the presence of cardiac, respiratory, renal, hepatic, neurological, coagulation-related and uterine dysfunction, which represent a set of clinical, laboratory and management identifiers14. These criteria defined by WHO were adopted for the identification of maternal near miss cases using information from hospital records. The identified cases were independently reviewed by two specialists, who aimed to identify possible inconsistencies in the extraction and/or typing of data from the medical records. The following covariates were assessed: age (12–19, 20–34, ≥ 35 years), skin color (white, black, brown), years of schooling (≤ 7, 8–10, 11–14, ≥ 15), economic class (D+E, C, A+B), marital status (lives with a partner, does not live with a partner), wage labor (yes, no), parity (0, 1–2, ≥ 3), and clinical or gestational complications (hypertensive disorders such as chronic hypertension, pre-eclampsia and HELLP syndrome; eclampsia; pre-existing diabetes; gestational diabetes; kidney, cardiac, heart or autoimmune diseases; placenta praevia, and placenta abruption). The classification of complications was validated by two obstetricians using information from medical records. In statistical analysis, macroregional differences in sociodemographic characteristics and clinical or gestational complications were tested by the χ2 test, with statistical significance (p < 0.05). METHODS The same procedure was used to assess inequalities, stratified by women with and without clinical or gestational complications. We tested whether pilgrimage for birth and inadequacy of prenatal care were associated with neonatal outcomes in the two subgroups of women for the whole country. We used multiple non-conditional logistic regressions adjusting them by the following variables: region, age, schooling and parity. The selection of adjustment variables was due to their association, with statistical significance (p < 0.05), both with exposure and with the outcomes studied (data not shown). For maternal near miss we also adjusted for type of birth. The results were expressed in adjusted OR and 95% confidence interval (95%CI). Because this is a complex sample, the statistical analysis was thorough reviewed, including a data weighting process calculated by the reverse of each woman probability of inclusion in the sample, with a procedure of calibration in each selection stratum to correct the effect of the sampling strategy. The Statistical Package for the Social Sciences (SPSS) software, version 21, was used in the statistical analysis. RESULTS Table 1 compares the characteristics of the 19,117 women with public-funded births according to the macroregions of the country. In the North and Northeast regions, the frequency of births among adolescents, women with low education and lower economic level was higher than in the Southeast. The variable “does not live with a partner” was more common in the Southeast region, whereas “wage labor” was more frequent in the South. The North region concentrated the highest proportion of women with three or more previous deliveries. Clinical and gestational complications were more frequent in the South and Southeast, except for chronic hypertension, which was more prevalent in the Northeast, and gestational hypertensive syndromes, more frequent in the Midwest. Prenatal care showed important regional variations. Despite the high coverage, the proportion of women without any prenatal care was 60% higher in the North than the national average. The Southeast, South and Midwest regions had the highest prevalence of women with early prenatal onset, and the Southeast had the highest coverage of women with at least six prenatal appointments (Table 2). Prenatal care in the Brazilian public sector Leal MC et al. Table 1. Sociodemographic characteristics and clinical and gestational complications in women with birth funded by the Brazilian Unified Health System according to macroregions of Brazil, 2011–2012. Table 1. Sociodemographic characteristics and clinical and gestational complications in women with birth funded by the Brazilian Unified Health System according to macroregions of Brazil, 2011–2012. RESULTS North (n = 2,143) Northeast (n = 5,683) Southeast (n = 7,838) South (n = 2,244) Midwest (n = 1,209) Brazil (n = 19,117) p-valuea % % % % % % Age in years 12–19 28.1 24.9 19.8 20.4 24.4 22.6 < 0.001 20–34 66.4 66.5 71.3 68.9 68.5 68.8 ≥ 35 5.6 8.6 8.9 10.7 7.1 8.5 Skin color White 12.1 17.2 33.3 59.3 25.5 28.7 < 0.001 Black 6.0 11.6 10.8 6.9 8.3 9.9 Brown 81.9 71.2 55.9 33.8 66.2 61.4 Years of schooling ≤ 7 37.5 43.3 24.7 30.6 24.0 32.3 < 0.001 8–10 30.9 24.7 30.6 32.2 35.5 29.4 11–14 29.0 29.8 41.7 33.2 37.2 35.5 ≥ 15 2.7 2.2 3.0 3.9 3.3 2.9 Economic class D+E 39.4 48.4 18.4 12.0 20.7 29.0 < 0.001 C 51.2 45.5 63.3 60.9 63.9 56.4 A+B 9.4 6.1 18.3 27.1 15.4 14.5 Marital status Does not live with a partner 19.3 18.2 24.5 14.9 19.2 20.6 < 0.001 Lives with a partner 81.8 75.5 85.1 80.8 79.4 Wage labor No 75.0 75.1 63.3 57.4 64.2 67.5 < 0.001 Yes 25.0 24.9 36.7 42.6 35.8 32.5 Previous births 0 40.1 47.6 44.7 42.7 43.9 44.8 < 0.001 1–2 41.4 39.7 44.6 44.8 44.7 42.8 ≥ 3 18.5 12.6 10.7 12.5 11.4 12.4 Clinical or gestational complications Pre-existing hypertension 1.5 2.8 3.1 2.8 1.6 2.7 < 0.001 Hypertensive syndromesb 8.2 10.5 11.6 11.5 11.7 10.9 < 0.001 Pre-existing diabetes 0.5 1.0 1.3 1.4 0.8 1.1 0.015 Gestational diabetes 7.6 5.5 9.5 11.1 5.5 8.0 < 0.001 Other chronic diseasesc 0.6 0.8 0.8 0.8 1.2 0.8 0.465 Placenta praevia 0.7 0.4 0.4 0.8 0.4 0.4 0.261 Placental abruption 1.1 1.2 1.7 1.7 1.2 1.5 0.046 a χ2 test. b Pre-eclampsia, HELLP syndrome, eclampsia. The coverage of hand-held prenatal notes was almost universal. However, not all women took it to the hospital for admission for birth. The country coverage of at least one VDRL test and one HIV during pregnancy was 88% and 79%, respectively, with the North and Northeast regions with the lowest prevalence. The coverage of fasting blood sugar and ASE tests was near to 85%, with the South region with the highest prevalence. Considering ultrasound exams, the North region had the largest deficiency, with coverage under 70% (Table 2). Approximately half of the women were bonded with the maternity hospital during prenatal care. RESULTS The South region stood out with more than 90% of women with births in the indicated Prenatal care in the Brazilian public sector Leal MC et al. Table 2. Prenatal care of women with birth funded by the Brazilian Unified Health System according to maternal risk and macroregions in Brazil 2011–2012 Table 2. Prenatal care of women with birth funded by the Brazilian Unified Health System according to maternal risk and macroregions in Brazil, 2011–2012. Table 2. Prenatal care of women with birth funded by the Brazilian Unified Health System according to maternal risk and macroregions in Brazil, 2011–2012. Women without complications Women with complications All women N n = 1,773 NE n = 4,674 SE n = 6,143 S n = 1,727 MW n = 993 Brazil n = 15,310 p-valuea N n = 370 NE n = 1,008 SE n = 1,696 S n = 518 MW n = 215 Brazil n = 3,807 p-valuea N n = 2,143 NE n = 5,682 SE n = 7,839 S n = 2,245 MW n = 1,208 Brazil n = 19,117 p-valuea % % % % % % % % % % % % % % % % % % Received prenatal care No 2.8 1.7 1.5 0.9 1.6 1.6 0.001 0.5 1.8 0.5 0.0 1.6 0.8 < 0.001 2.5 1.7 1.3 0.7 1.6 1.5 < 0.001 Yes 97.2 98.3 98.5 99.1 98.4 98.4 99.5 98.2 99.5 100.0 98.4 99.2 97.5 98.3 98.7 99.3 98.4 98.5 Type of basic unitb FHP/birthing center/BHU 94.1 88.2 92.1 91.6 92.2 91.1 < 0.001 89.8 82.9 84.5 83.1 85.2 84.5 0.039 93.4 87.3 90.4 89.6 91.0 89.7 < 0.001 Outpatient clinic 5.9 11.8 7.9 8.4 7.8 8.9 11.2 17.1 15.5 16.9 14.8 15.5 6.6 12.7 9.6 11.4 9.0 10.3 Onset of prenatal careb Up to12 weeks 45.2 52.0 59.3 57.1 65.2 55.6 < 0.001 49.7 57.7 63.7 59.7 65.7 60.3 < 0.001 46.0 52.2 60.1 57.6 65.0 56.5 < 0.001 Second trimester 49.8 43.9 36.8 37.5 31.4 40.2 46.2 38.7 32.0 37.0 32.9 35.9 49.2 43.0 35.8 37.5 31.4 39.3 Third trimester 5.0 4.1 3.9 5.4 3.4 4.2 4.1 3.6 4.3 3.3 1.4 3.8 4.8 4.8 4.1 4.9 3.6 4.2 No. RESULTS of prenatal appointmentsb Between 1 and 3 17.7 14.1 9.0 9.9 8.5 11.7 < 0.001 15.1 8.4 5.7 6.9 4.3 7.4 < 0.001 17.2 13.1 8.3 9.2 7.7 10.9 < 0.001 Between 4 and 5 28.1 27.0 14.0 17.3 21.2 20.4 24.6 23.0 16.0 15.4 15.9 18.6 27.5 26.3 14.4 16.9 20.3 20.0 6 or more 54.2 58.9 77.0 72.8 70.3 67.9 60.3 68.6 78.3 77.7 79.8 74.0 55.3 60.6 77.3 73.9 72.0 69.1 Have received hand-held prenatal notesb No 0.7 1.1 1.4 0.8 2.4 1.2 0.001 0.5 1.6 0.5 0.6 0.9 0.9 0.043 0.7 1.2 1.1 0.8 2.1 1.1 0.002 Yes 99.3 98.9 98.6 99.2 97.6 98.8 99.5 98.4 99.5 99.4 99.1 99.1 99.3 98.8 98.9 99.2 97.9 98.9 Took the hand-held prenatal notes on admission for birthb No 35.9 25.8 20.0 16.4 54.0 25.4 < 0.001 22.9 17.9 13.5 10.4 38.4 16.5 < 0.001 33.6 24.4 18.5 15.0 51.2 23.6 < 0.001 Yes 64.1 74.2 80.0 83.6 46.0 74.6 77.1 82.1 86.5 89.6 61.6 83.5 66.4 75.6 81.5 85.0 48.8 76.4 Blood sugar levelc No 23.3 23.7 17.7 10.4 18.8 19.2 < 0.001 12.4 16.0 12.0 6.5 11.5 12.2 < 0.001 21.1 22.2 16.4 9.5 17.1 17.7 < 0.001 Yes, once 49.3 54.4 45.7 40.0 52.7 48.2 48.6 49.1 40.4 33.5 43.1 42.5 49.2 53.4 44.4 38.4 50.5 47.0 Yes, twice 27.4 21.9 36.6 49.5 28.6 32.6 39.0 34.9 47.7 60.0 45.4 45.3 29.7 24.4 39.2 52.1 32.4 35.4 Urine (AES)c No 24.8 19.1 14.9 8.4 18.3 16.4 < 0.001 12.5 13.7 11.5 5.8 10.0 11.2 < 0.001 22.3 18.0 14.1 7.8 16.4 15.3 < 0.001 Yes 75.2 80.9 85.1 91.6 81.7 83.6 87.5 86.3 88.5 94.2 90.0 88.8 77.7 82.0 85.9 92.2 83.6 84.7 Ultrasoundc No 32.9 18.9 9.6 9.2 14.3 14.8 < 0.001 27.0 15.8 8.2 6.7 8.5 11.6 < 0.001 31.7 18.3 9.3 8.6 13.0 14.1 < 0.001 Yes 67.1 81.1 90.4 90.8 85.7 85.2 73.0 84.2 91.8 93.3 91.5 88.4 68.3 81.7 90.7 91.4 87.0 85.9 VDRL test results during the pregnancyc No 22.8 17.7 9.5 5.5 17.2 13.1 < 0.001 14.6 11.5 7.4 4.5 9.3 8.7 < 0.001 21.1 16.5 9.3 5.3 15.4 12.1 < 0.001 Yes, one 50.0 54.2 46.9 38.7 45.8 48.3 53.7 50.5 44.0 34.2 45.0 45.2 50.7 53.2 46.0 37.6 45.6 47.7 Yes, two 27.2 28.1 43.6 55.8 37.0 38.6 31.7 38.0 48.6 61.3 45.7 46.1 28.2 30.3 44.7 57.1 39.0 40.2 HIV test results during the pregnancyc No 31.6 35.5 14.3 8.1 19.6 21.9 < 0.001 28.8 28.4 11.5 6.9 15.4 16.8 < 0.001 31.0 34.1 13.7 7.8 18.6 20.7 < 0.001 Yes, one 50.5 51.3 53.4 49.8 47.9 51.8 52.8 51.5 55.4 48.0 48.4 52.8 51.0 51.4 53.8 49.4 48.1 52.1 Yes, two 17.9 13.2 32.3 42.1 32.5 26.3 18.4 20.1 33.1 45.1 36.2 30.4 18.0 14.5 32.5 42.8 33.3 27.2 Bond with maternity hospitalb No 52.1 50.4 40.1 41.2 43.5 45.0 < 0.001 50.7 40.7 36.0 34.9 34.4 38.4 < 0.001 51.8 48.7 39.2 39.7 41.8 43.6 < 0.001 Yes 47.9 49.6 59.9 58.8 56.5 55.0 49.3 59.3 64.0 65.1 65.6 61.6 48.2 51.3 60.8 60.3 58.2 56.4 Birthed in the bonded maternity hospitald No 14.2 23.3 19.7 8.7 16.0 18.5 < 0.001 14.5 25.0 17.9 10.4 19.0 18.4 < 0.001 14.2 23.7 19.3 9.0 16.6 18.5 < 0.001 Yes 85.8 76.7 80.3 91.3 84.0 81.5 85.5 75.0 82.1 89.6 81.0 81.6 85.8 76.3 80.7 91.0 83.4 81.5 Continue Table 2. RESULTS Prenatal care of women with birth funded by the Brazilian Unified Health System according to maternal risk and macroregions in Brazil, 2011–2012. Continuation Pilgrimage for birth No 80.0 67.2 78.5 89.9 76.4 76.4 < 0.001 78.2 65.0 81.0 87.0 75.8 77.0 < 0.001 79.7 66.9 79.1 89.2 76.3 76.5 < 0.001 Yes, one hospital 17.3 27.7 17.2 9.2 20.1 19.7 19.6 29.6 16.0 12.2 21.9 19.8 17.7 28.0 16.9 9.9 20.4 19.7 Yes, two hospitals 2.7 5.1 4.3 0.9 3.5 3.9 2.2 5.4 3.0 0.8 2.3 3.2 2.6 5.1 4.0 0.9 3.3 3.8 Type of hospital Public 71.8 58.6 42.8 28.9 56.5 50.3 < 0.001 83.0 67.1 48.1 35.6 63.2 55.7 < 0.001 73.5 60.1 43.9 30.4 57.5 51.3 < 0.001 Mixed 28.2 41.4 57.2 71.1 43.5 49.7 17.0 32.9 51.9 64.4 36.8 44.3 26.5 39.9 56.1 69.6 42.5 48.7 Location of hospital Capital 45.2 34.7 32.2 22.3 64.3 35.4 < 0.001 45.8 50.2 39.2 30.5 70.4 43.4 < 0.001 45.2 37.5 33.7 24.1 65.2 37.0 < 0.001 Non-capital 54.8 65.3 67.8 77.7 35.7 64.6 54.2 49.8 60.8 69.5 29.6 56.6 54.8 62.5 66.3 75.9 34.8 63.0 FHP: Family Health Program; BHU: basic health unit a χ2 test. b h h d d h l i l d d i hi l i Table 2. Prenatal care of women with birth funded by the Brazilian Unified Health System according to maternal risk and macroregions Table 2. Prenatal care of women with birth funded by the Brazilian Unified Health System according to maternal χ b The women who attended the prenatal care are included in this analysis. c The women who attended the prenatal care and presented the prenatal card in the labor admission are included in this analysis. d The women who attended the prenatal care, presented the prenatal card in the labor admission and received guidance on the reference maternity for birth are included in this analysis . c The women who attended the prenatal care and presented the prenatal card in the labor admission are included in this analysis. d The women who attended the prenatal care, presented the prenatal card in the labor admission and received guidance on the reference maternity for birth are included in this analysis . Table 3. RESULTS Adequacy of prenatal care and neonatal and maternal outcomes in women with birth funded by the Unified Health System according to maternal risk and macroregions of Brazil, 2011–2012. RESULTS Table 3. Adequacy of prenatal care and neonatal and maternal outcomes in women with birth funded by the Unified Health System according to maternal risk and macroregions of Brazil, 2011–2012. care and neonatal and maternal outcomes in women with birth funded by the Unified Health System according ions of Brazil, 2011–2012. p y q g week and had an adequate number (100%) of appointments for gestational age at birth, considering the schedule of six appointments. c The women who attended the prenatal care and took the hand-held prenatal notes for admission for birth are included in this analysis. Prenatal care was considered adequate when it started until the 12th gestational week and had an adequate number (100%) of appointments for gestational age at birth, considering the schedule of six appointments, there was at least one result of each recommended tests in the prenatal routine and women received guidance on the reference maternity for birth care. ity for birth care. enatal care, took the hand-held prenatal notes for admission for birth and received guidance on the reference maternity sis . g y d The women who attended the prenatal care, took the hand-held prenatal notes for admission for birth and received guidance on the reference maternity for birth are included in this analysis . Prenatal care was considered adequate when it started until the 12th gestational week and had an adequate number (100%) of appointments for d The women who attended the prenatal care, took the hand-held prenatal notes for admission for birth and received guidance on the reference maternity for birth are included in this analysis . Prenatal care was considered adequate when it started until the 12th gestational week and had an adequate number (100%) of appointments for for birth are included in this analysis . Prenatal care in the Brazilian public sector Leal MC et al. RESULTS Women without complications Women with complications All women N n = 1,773 NE n = 4,674 SE n = 6,143 S n = 1,727 MW n = 993 Brazil n = 15,310 p-valuea N n = 1,773 NE n = 4,674 SE n = 6,143 S n = 1,727 MW n = 993 Brazil n = 15,310 p-valuea N n = 1,773 NE n = 4,674 SE n = 6,143 S n = 1,727 MW n = 993 Brazil n = 15,310 p-valuea % % % % % % % % % % % % % % % % % % Adequacy of prenatal careb 47.3 52.6 67.1 65.8 63.4 60.1 < 0.001 55.5 64.5 72.0 69.5 72.6 68.2 < 0.001 48.7 54.7 68.2 66.7 65.0 61.7 < 0.001 Adequacy of overall prenatal care 1c 10.1 11.4 21.6 24.6 18.5 17.7 < 0.001 12.0 21.0 29.4 29.4 26.9 25.6 < 0.001 10.5 13.3 23.4 25.7 20.4 19.4 < 0.001 Adequacy of overall prenatal care 2d 9.6 8.8 17.3 22.5 15.1 14.5 < 0.001 11.7 16.2 24.0 26.1 24.8 21.2 < 0.001 10.0 10.2 18.9 23.4 17.3 16.0 < 0.001 Type of birth Vaginal 59.1 63.0 62.0 59.8 57.2 61.4 < 0.001 36.2 37.2 38.8 45.3 20.6 37.9 < 0.001 55.1 58.4 57.0 56.5 50.5 56.7 < 0.001 Intrapartum cesarean section 31.7 27.2 31.4 32.0 33.7 30.4 52.6 50.6 53.4 45.3 68.2 52.4 35.3 31.4 36.2 35.1 40.0 34.8 Antepartum cesarean section 9.3 9.8 6.6 8.2 9.1 8.2 11.1 12.2 7.8 9.4 11.2 9.7 9.6 10.2 6.8 8.5 9.5 8.5 Outcomes Spontaneous preterm birth 8.5 9.0 6.7 7.4 8.4 7.8 < 0.001 9.8 8.1 5.8 8.4 5.8 7.2 0.023 8.8 8.9 6.5 7.6 7.9 7.7 < 0.001 Provided-initiated preterm birth 3.5 1.9 2.1 2.5 1.4 2.2 < 0.001 11.9 11.8 11.9 10.5 12.1 11.7 0.949 4.9 3.7 4.2 4.4 3.4 4.1 0.096 Low birth weight (< 2,500g) 7.9 8.9 7.6 7.7 7.8 8.1 0.124 17.9 18.7 13.5 12.7 17.6 15.4 0.001 9.6 10.7 8.9 8.9 9.6 9.5 0.001 IUGR (< 10th percentile) 5.9 7.7 7.9 7.0 8.8 7.6 < 0.001 11.6 10.3 7.1 6.3 8.1 8.4 < 0.001 6.9 8.2 7.8 6.8 8.7 7.7 < 0.001 Apgar in the 5th minute < 8 1.6 3.4 1.9 1.3 1.2 2.2 < 0.001 3.2 3.0 2.5 1.8 2.9 2.6 0.615 1.9 3.3 2.0 1.4 1.5 2.3 < 0.001 Neonatal near miss 9.2 9.1 10.4 10.5 8.9 9.8 0.100 15.9 17.6 18.9 17.2 17.9 18.0 0.670 10.3 10.6 12.2 12.0 10.6 11.4 0.061 Maternal near miss 0.5 0.3 0.3 0.6 0.2 0.4 0.318 3.4 5.5 3.3 3.3 7.6 4.1 0.004 1.0 1.2 1.0 1.2 1.6 1.1 0.333 IUGR: intrauterine growth restriction (< 10th percentile in intergrowth curve) a χ2 test. Inadequate prenatal care (onset and number of appointments) Inadequate prenatal care (onset and number of appointments) Inadequate prenatal care (onset and number of appointments) OR: odds ratio; 95%CI: 95% confidence interval; LBW: low birth weight (< 2,500g); IUGR: intrauterine growth restriction (< 10th percentile in the Intergrowth curve). a Adj t d b i h li d it OR: odds ratio; 95%CI: 95% confidence interval; LBW: low birth weight (< 2,500g); IUGR: intrauterine growth restriction (< 10th percentile in the Intergrowth curve). a Adjusted by region, age, schooling and parity. Intergrowth curve). a Adjusted by region, age, schooling and parity. a Adjusted by region, age, schooling, parity and type of birth. a Adjusted by region, age, schooling and parity. a Adjusted by region, age, schooling, parity and type of birth. maternities. Pilgrimage affected more than 20% of women in the country, reaching more than 30% in the Northeast (Table 2). Women with clinical or gestational complications had better prenatal indicators than women without complications. For the former, a higher prevalence of prenatal care, early onset and six or more appointments were observed. The coverage of VDRL and HIV tests was also higher for these women. In the Northeast, the difference observed among women with or without complications was more prominent than in other regions (Table 2). For the minimal adequacy of prenatal care, which considered both early onset and the minimum number of appointments, the Southeast and South regions had the highest prevalence. For the overall adequacy 1, the prevalence decreased substantially in all regions. When considering the most restricted criterion, the overall adequacy 2, the prevalence in the country was only 16%, with the Northeast region with the worst result, 10% (Table 3). The Midwest region had the highest rate of cesarean section (49.5%), whereas the Northeast the lowest (41.6%). The Southeast had the lowest rate of antepartum cesarean section (6.8%), as observed in Table 3. The North, Northeast and Midwest regions had the worst neonatal results, mainly for spontaneous preterm birth, LBW and IUGR. We found more regional inequalities and worse outcomes among women with complications (Table 3). Pilgrimage was associated with all neonatal outcomes, except IUGR, even after adjustment for confounding variables. The odds ratios ranged from 1.48 (95%CI 1.23–1.78) for neonatal near miss to 1.62 (95%CI 1.27–2.06) for provider-initiated preterm birth. Inadequate prenatal care (onset and number of appointments) Among women with clinical or gestational complications, pilgrimage was strongly associated with provider-initiated preterm birth and Apgar in the 5th min < 8, with OR of 1.98 (95%CI 1.49–2.65) and 2.19 (95%CI 1.31–3.68), respectively. The prenatal inadequacy was associated with spontaneous preterm birth in both groups of women (Table 4). RESULTS Prenatal care was considered adequate when it started until the 12th gestational week and had an adequate number (100%) of appointments for gestational age at birth, considering the schedule of six appointments, there was at least one result of each recommended tests in the prenatal routine, i d id h f i f bi h d bi h d i h f i y Prenatal care was considered adequate when it started until the 12th gestational week and had an adequate number (100%) of appointments for gestational age at birth, considering the schedule of six appointments, there was at least one result of each recommended tests in the prenatal routine, women received guidance on the reference maternity for birth care, and birth occurred in the reference maternity. Prenatal care in the Brazilian public sector Leal MC et al. Table 4. Neonatal and maternal outcomes associated with journey and inadequate prenatal care in women with birth financed by the Brazilian Unified Health System according to maternal risk in Brazil. 2011–2012. Table 4. Neonatal and maternal outcomes associated with journey and inadequate prenatal care in women with birth financed by the Brazilian Unified Health System according to maternal risk in Brazil. 2011–2012. Spontaneous preterm birth Provided-initiated preterm birth LBW IUGR Apgar 5 min < 8 Neonatal near miss Maternal near miss ORa (95%CI) ORa (95%CI) ORa (95%CI) ORa (95%CI) ORa (95%CI) ORa (95%CI) ORb (95%IC) Pilgrimage for birth All women 1.61 (1.29–2.01) 1.62 (1.27–2.06) 1.56 (1.28–1.89) 1.19 (0.95–1.49) 1.56 (1.19–2.03) 1.48 (1.23–1.78) 1.38 (0.81–2.36) Women without complications 1.60 (1.25–2.04) 1.32 (0.92–1.91) 1.52 (1.23–1.87) 1.14 (0.86–1.51) 1.41 (1.03–1.94) 1.50 (1.21–1.85) 1.34 (0.70–2.56) Women with complications 1.67 (1.06–2.61) 1.98 (1.49–2.65) 1.69 (1.23–2.31) 1.37 (1.03–1.83) 2.19 (1.31–3.68) 1.45 (1.16–1.81) 1.39 (0.70–2.77) Inadequate prenatal care (onset and number of appointments) All women 1.51 (1.24–1.83) 1.06 (0.73–1.55) 1.25 (0.99–1.56) 1.11 (0.88–1.38) 0.96 (0.68–1.36) 1.14 (0.95–1.37) 0.96 (0.54–1.68) Women without complications 1.47 (1.18–1.82) 0.95 (0.62–1.46) 1.21 (0.94–1.54) 1.19 (0.95–1.49) 1.00 (0.69–1.43) 1.08 (0.88–1.34) 0.82 (0.29–2.36) Women with complications 1.73 (1.02–2.94) 1.14 (0.66–1.97) 1.32 (0.90–1.92) 1.14 (0.86–1.51) 0.81 (0.39–1.71) 1.28 (0.85–1.93) 0.97 (0.55–1.73) OR: odds ratio; 95%CI: 95% confidence interval; LBW: low birth weight (< 2,500g); IUGR: intrauterine growth restriction (< 10th percentile in the Intergrowth curve). a Adjusted by region, age, schooling and parity. a Adjusted by region age schooling parity and type of birth 8 http://doi.org/10.11606/s1518-8787.2020054001458 DISCUSSION Prenatal care coverage in Brazil is almost universal for women using SUS, considering at least one prenatal appointment. However, this panorama changes as other parameters are Prenatal care in the Brazilian public sector Leal MC et al. progressively included. By including recommendations such as a minimum set of exams and the bond with the maternity hospital for birth, prenatal adequacy is reduced to slightly over a quarter of women, being further reduced if we consider effective bonding with the maternity hospital for birth. Tomasi et al.15 analyzed prenatal care in SUS in the same period of this study, observing that 89% of the pregnant women had six or more appointments, but only 15% received adequate prenatal care. The differences in coverage percentages between the two studies derive from two main reasons. Firstly, Tomasi’s study included only women aged 18 years or older in basic health units (BHU) by occasion of their evaluation; whereas Birth in Brazil included a representative sample of women at the time of birth. Secondly, the criterion used for prenatal adequacy was different in both studies. Since 99% of the deliveries occurred in hospitals, our data are closer to the Brazilian prenatal coverage. Our results indicate that the continuity and quality of care provided by SUS is still deficient. In the state of São Paulo, Monteiro et al.16 verified that, despite a nearly universal access to health care services, there were still problems related to quality, which were experienced mainly by lower socioeconomic groups and users of SUS. The social and economic inequalities among the geographical regions of the country are evident in this study. In the sphere of reproductive health, women are younger in the most disadvantaged regions, with a higher proportion of teenage pregnancy and greater parity. Women from the South and Southeast regions had higher proportions of clinical complications, probably because they were older and had more access to clinical diagnosis. However, in the North region, the proportion of women with no prenatal care was the same for both groups of women, with and without obstetric complications. This poorer performance in the North region may be due to geographical difficulties, large distances and barriers in the access of centers for diagnosis and treatment, absence of qualified professionals, etc. The North region had the highest proportion of home births, that when performed by unqualified professionals, associate with higher infant mortality rates17. DISCUSSION This fact shows that primary care has been effective in identifying these problems and in attending at-risk pregnant women. However, the system has failed the process of integrality care, not giving continuity to access to maternity care. The failure of the system in the coordination and integrality of care at the time of birth, with many women in pilgrimage, associated with great damage to newborns. In obstetrics, the role of delays in care is known. In the 1990s, English authors proposed a theoretical model called “three delays” that classifies the delays in: phase I, the delay in deciding to seek care; phase II, the delay in reaching an appropriate care unit; and phase III, the delay in receiving adequate care at the reference institution25. When a woman search for more than one health service to give birth, we are certainly facing phase II and III. Pacagnella et al.26 have used these concepts and analyzed the role of delays in maternal morbidity in Brazil and found an association between this outcome and the delays in care. In the current analysis, pilgrimage associated with nearly all negative outcomes in the newborn, especially in the group of pregnant women with complications, possibly because they require more clinical interventions. A recent analysis with data from Birth in Brazil showed that in SUS 32% of women with a obstetric risk were treated in hospitals without intensive care units, while 29.5% of low-risk patients delivered in hospitals with this type of resource27. This fact shows that the system is not adequately articulated to provide high complexity attention when necessary, although the system offers unnecessary support to those who do not need it. The consequences of this disarticulation can be seen in this study and deserve attention from managers to avoid suffering, complications and abbreviation of lives. Prenatal care is a programmatic action typical of primary care, and the results of this study prove this fact and its relationship with obstetric results. Since 90% of the interviewees did their prenatal care in the basic health network, the qualification actions of the teams and work processes play a fundamental role in improving care for the baby and pregnant women. Fachini et al.28 highlight the importance of increasing the effectiveness of the Family Health Strategy, considering its mediating effect on health care. DISCUSSION Regarding prenatal care in Brazil, Viellas et al.18 found that the low coverage and late onset of prenatal care in women with low schooling from the North and Northeast regions were more related to barriers in the access to care than with the lack of knowledge of pregnancy or personal problems. In this study, pregnant women with inadequate prenatal care were more susceptible to give birth to preterm newborns spontaneously. A study with this same sample of women observed that spontaneous preterm birth was associated with poverty and inadequacy of prenatal care19, factors which contribute to the maintenance of the high infant mortality rates in the country20, since preterm birth is the greatest risk factor for morbidity and mortality in the first year of life21. Furthermore, our study identified the lack of bond between the levels of outpatient and hospital care, an important aspect, since knowing the maternity hospital for birth contributes to the well-being of women and to the progress of the labor22. The law of maternity bond has completed ten years, but it has not been properly implemented in the country. Notably, the Southern region has achieved effective bond for more than 90% of pregnant women, thus, this region evidences a better organization of the system, better care coordination, better continuity and hierarchization of actions in maternal and child health. The cesarean section rate in all regions was high — especially for women with obstetric complications —, 75% higher than in the low-risk group. The average rate of 30.4% in the low-risk group approaches the value of the U.S. overall rate and is above the rate of European countries, whose rate is between 20% and 25%23. The Southeast region, which had the lowest rate of intrapartum cesarean section, also had the lowest overall cesarean section rate in the public sector of SUS (data not shown). In European countries, intrapartum Prenatal care in the Brazilian public sector Leal MC et al. cesarean sections are higher than antepartum, differently from what occurs in Brazil, where antepartum or elective cesarean section predominates24. The findings from the Southeast region may be a consequence of the ongoing movement of change in the birth care model of their SUS hospitals; focused on the best scientific evidence, without disregarding the women’s movement for a less medicalized birth care. Women who had clinical or gestational complications received better prenatal care than low-risk ones. DISCUSSION In conclusion, regional inequalities, barriers to access and inadequate prenatal care persist, thus, they contribute to adverse outcomes for newborns. The improvement in the quality of prenatal care and the coordination and integrality of care at the time of birth have a potential impact on preterm birth rates and consequently on the reduction of infant morbidity and mortality rates in the country. 1. World Health Organization. WHO Recommendations on antenatal care for a positive pregnancy experience. Geneva: WHO; 2016. REFERENCES 2. Ministério da Saúde (BR), Secretaria de Atenção à Saúde, Departamento de Atenção Básica. Atenção ao pré-natal de baixo risco. Brasília, DF; 2012. (Série A. Normas e Manuais Técnicos) (Cadernos de Atenção Básica, 32). 3. Brasil. Lei Nº 11.634, de 27 de dezembro de 2007. Dispõe sobre o direito da gestante ao conhecimento e a vinculação à maternidade onde receberá assistência no âmbito do Sistema Único de Saúde. Brasília, DF; 2007 [citado 10 out 2017]. Available from: http://www.planalto. gov.br/ccivil_03/_Ato2007-2010/2007/Lei/L11634.htm 3. Brasil. Lei Nº 11.634, de 27 de dezembro de 2007. Dispõe sobre o direito da gestante ao conhecimento e a vinculação à maternidade onde receberá assistência no âmbito do Sistema Único de Saúde. Brasília, DF; 2007 [citado 10 out 2017]. Available from: http://www.planalto. gov.br/ccivil_03/_Ato2007-2010/2007/Lei/L11634.htm Prenatal care in the Brazilian public sector Leal MC et al. 4. Ministério da Saúde (BR), DATASUS. SINASC: nascimentos por residência da mãe por ano do nascimento segundo número de consultas de pré-natal. Brasília, DF; 2015 [citado 10 out 2017]. Available from: http://tabnet.datasus.gov.br/cgi/deftohtm.exe?sinasc/cnv/nvuf.def 5. Ministério da Saúde (BR), Secretaria de Gestão Estratégica e Participativa, Departamento de Articulação Interfederativa. Caderno de diretrizes, objetivos, metas e indicadores: 2013-2015. Brasília, DF; 2013. (Série Articulação Interfederativa; v.1). 6. Vasconcellos MT, Silva PL, Pereira AP, Schilithz AO, Souza Junior PR, Szwarcwald CL. Sampling design for the Birth in Brazil: national survey into labor and birth. Cad Saude Publica. 2014;30 Supl 1:S1-10. https://doi.org/10.1590/0102-311X00176013 7. Leal MC, Silva AA, Dias MA, Gama SG, Rattner D, Moreira ME, et al. Birth in Brazil: national survey into labour and birth. Reprod Health. 2012;9:15. https://doi.org/10.1186/1742-4755-9-15 8. Domingues RMSM, Viellas EF, Dias MAB, Torres JA, Theme-Filha MM, Gama SGN, et al. Adequação da assistência pré-natal segundo as características maternas no Brasil. Rev Panam Salud Publica. 2015;37(3):140-7. 9. Villar J, Ismail LC, Victora CG, Ohuma EO, Bertino E, Altman DG, et al. International standards for newborn weight, length, and head circumference by gestational age and sex: the Newborn Cross-Sectional Study of the INTERGROWTH-21st Project. Lancet. 2014;384(9946):857-68. https://doi.org/10.1016/S0140-6736(14)60932-6 10. Pereira AP, Leal MC, Gama SGN, Domingues RMSM, Schilithz AOC, Bastos MH. Determining gestational age based on information from the Birth in Brazil study. Cad Saude Publica. 2014;30 Supl 1:S1-12. https://doi.org/10.1590/0102-311X00160313 11. Pileggi-Castro C, Camelo Jr JS, Perdoná GC, Mussi-Pinhata MM, Cecatti JG, Mori R, et al. Development of criteria for identifying neonatal near-miss cases: analysis of two WHO multicountry cross-sectional studies. BJOG. 2014;121 Suppl 1:110-8. https://doi.org/10.1111/1471-0528.12637 12. Avenant T. REFERENCES Neonatal near miss: a measure of the quality of obstetric care. Best Pract Res Clin Obstet Gynaecol. 2009;23(3):369-74. https://doi.org/10.1016/j.bpobgyn.2008.12.005 13. Say L, Souza JP, Pattinson RC; WHO Working Group on Maternal Mortality and Morbidity Classifications. Maternal near miss: towards a standard tool for monitoring quality of maternal health care. Best Pract Res Clin Obstet Gynaecol. 2009;23(3):287-96. https://doi.org/10.1016/j.bpobgyn.2009.01.007 14. Souza JP, Cecatti JG, Haddad SM, Parpinelli MA, Costa ML, Katz L, et al. The WHO maternal near-miss approach and the maternal severity index model (MSI): tools for assessing the management of severe maternal morbidity. PloS One. 2012;7(8):e44129. https://doi.org/10.1371/journal.pone.0044129 15. Tomasi E, Fernandes PAA, Fischer T, Siqueira FCV, Silveira DS, Thumé E, et al. Qualidade da atenção pré-natal na rede básica de saúde do Brasil: indicadores e desigualdades sociais. Cad Saude Ppublica. 2017;33(3):e00195815. https://doi.org/10.1590/0102-311x00195815 16. Monteiro CN, Beenackers MA, Goldbaum M, Barros MBA, Gianini RJ, Cesar CLG, et al. Use, access, and equity in health care services in São Paulo, Brazil. Cad Saude Publica. 2017;33(4):e00078015. https://doi.org/10.1590/0102-311X00078015 17. Leal MC, Bittencourt SDA, Torres RMC, Niquini RP, Souza Jr PRB. Determinants of infant mortality in the Jequitinhonha Valley and in the North and Northeast regions of Brazil. Rev Saude Publica. 2017;51:12. https://doi.org/10.1590/S1518-8787.2017051006391 18. Viellas EF, Domingues RMSM, Dias MAB, Gama SGN, Theme Filha MM, Costa JV, et al. Prenatal care in Brazil. Cad Saude Publica. 2014;30 Supl 1:S1-15. https://doi.org/10.1590/0102-311X00126013 19. Leal MC, Esteves-Pereira AP, Nakamura-Pereira M, Torres JA, Theme-filha M, Domingues RMSM, et al. Prevalence and risk factors related to preterm birth in Brazil. Reprod Health. 2016;13 Suppl 3:127. https://doi.org/10.1186/s12978-016-0230-0 20. Lansky S, Friche AAL, Silva AAM, Campos D, Bittencourt SDA, Carvalho ML, et al. Birth in Brazil survey: neonatal mortality, pregnancy and childbirth quality of care. Cad Saude Publica. 2014;30 Supl 1:S1-15. https://doi.org/10.1590/0102-311X00133213 21. GBDCM 2015 Child Mortality Collaborators. Global, regional, national, and selected subnational levels of stillbirths, neonatal, infant, and under-5 mortality, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016;388(10053): 1725-74. https://doi.org/10.1016/S0140-6736(16)31575-6 11 http://doi.org/10.11606/s1518-8787.2020054001458 Prenatal care in the Brazilian public sector Leal MC et al. 22. Beaton J, Gupton A. Childbirth expectations: a qualitative analysis. Midwifery 1990;6(3):133-9. https://doi.org/10.1016/S0266-6138(05)80170-6 23. Betrán AP, Ye J, Moller AB, Zhang J, Gulmezoglu AM, Torloni MR. The Increasing Trend in Caesarean Section Rates: global, regional and national estimates: 1990-2014. PloS One. 2016;11(2):e0148343. https://doi.org/10.1371/journal.pone.0148343 24. Nakamura-Pereira M, Leal MC, Esteves-Pereira AP, Domingues RMSM, Torres JA, Dias MAB, et al. REFERENCES Use of Robson classification to assess cesarean section rate in Brazil: the role of source of payment for childbirth. Reprod Health. 2016;13 Suppl 3:128. https://doi.org/10.1186/s12978-016-0228-7 25. Thaddeus S, Maine D. Too far to walk: maternal mortality in context. Soc Sci Med. 1994;38(8):1091-110. https://doi.org/10.1016/0277-9536(94)90226-7 26. Pacagnella RC, Cecatti JG, Parpinelli MA, Sousa MH, Haddad SM, Costa ML, et al. Delays in receiving obstetric care and poor maternal outcomes: results from a national multicentre cross-sectional study. BMC Pregnancy Childbirth. 2014;14:159. https://doi.org/10.1186/1471-2393-14-159 27. Bittencourt SD, Domingues RMSM, Reis LG, Ramos MM, Leal MC. Adequacy of public maternal care services in Brazil. Reprod Health. 2016;13 Suppl 3:120. https://doi.org/10.1186/s12978-016-0229-6 28. Facchini LA, Tomasi E, Dilélio AS. Qualidade da Atenção Primária à Saúde no Brasil: avanços, desafios e perspectivas. Saude Debate. 2018;42 nº espec 1:208-23. https://doi.org/10.1590/0103-11042018s114 Funding: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) - processo nº 557366/2009-7. Departamento de Ciência e Tecnologia, Secretaria de Ciência, Tecnologia e Insumos Estratégicos, Ministério da Saúde - Processo 25000.096149/2010-97. Escola Nacional de Saúde Pública Sergio Arouca, Fundação Oswaldo Cruz (Projeto INOVA) - Processo 25388.000773/2009-66. Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (Faperj) - processo nº E-26/103.083/2011 Authors’ Contributions: Conception, planning, analysis and interpretation of data: MCL, APEP. Writing and critical revision of the manuscript: MCL, APEP, EFV, RMSMD, SGNG. Conflict of Interest: The authors declare no conflict of interest.
https://openalex.org/W1491519194	https://europepmc.org/articles/pmc3515902?pdf=render	English	null	Comparison of Two Methodologies for CD4+ T Lymphocytes Relative Counting on Immune Monitoring of Patients with Human Immunodeficiency Virus	The scientific world journal/TheScientificWorldjournal	2,012	cc-by	3,937	Danielle Cristyane Kalva Borato,1 Emerson Carraro,2 Sˆonia Regina Weber Ribas,3 Carlos Augusto Kalva-Filho,4 and Jos´e Carlos Rebuglio Vellosa1 1Pharmaceutical Sciences Post Graduate Program, State University of Ponta Grossa (UEPG), General Carlos Cavalcanti Avenu 4748 Uvaranas, 84030-900 Ponta Grossa, PR, Brazil 2Department of Pharmacy, State University in the Paran´a Midwestern (UNICENTRO), CEDETEG Campus, 85040-080 Guarapuava, PR, Brazil p 3County Health Department, Expert Assistance Service (EAS), City Hall, 84051-900 Ponta Grossa, PR, Brazil 4Department of Physiotherapy, State University Paulista (UNESP), Presidente Prudente Campus, 19060-900 Presidente Prudente, SP, Brazil p 3County Health Department, Expert Assistance Service (EAS), City Hall, 84051-900 Ponta Grossa, PR, Brazil 4Department of Physiotherapy, State University Paulista (UNESP), Presidente Prudente Campus, 19060-900 Presidente Prudente, SP, Brazil 3County Health Department, Expert Assistance Service (EAS), City Hall, 84051-900 Ponta Grossa, PR, Brazil 4Department of Physiotherapy, State University Paulista (UNESP), Presidente Prudente Campus, 19060-900 Presidente Prudente, SP, Brazil Correspondence should be addressed to Jos´e Carlos Rebuglio Vellosa, josevellosa@yahoo.com.b Correspondence should be addressed to Jos´e Carlos Rebuglio Vellosa, josevellosa@yahoo.com.br Received 1 September 2012; Accepted 8 November 2012 Academic Editors: A. Banerjee and E. L. Hooghe-Peters Copyright © 2012 Danielle Cristyane Kalva Borato et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Considering that counting the percentage of CD4 T lymphocytes can add prognostic information regarding patients infected with HIV, the aim of this study was to evaluate the percentage values of CD4+ T lymphocytes from 81 patients determined by ﬂow cytometry and estimated by ﬂow cytometry in conjunction with a hematology counter. Means were compared through the Student’s t-test. Pearson’s correlation was determined, and the agreement between results was tested by Bland-Altman. The level of signiﬁcance was P < 0.05. It was found a signiﬁcantly higher mean diﬀerence between the relative values of CD4+ T lymphocytes to the hematologic counter (P < 0.05), for all strata studied. Positive and signiﬁcant correlations (P < 0.01) were found between the strata CD4 < 200 cells/mL (r = 0.93), between 200 and 500 cells/mL (r = 0.65), and >500 cells/mL (r = 0.81). The limits of agreement were 1.0 ± 3.8% for the stratum of CD4 < 200 cells/mL, approximately 2.2 ± 13.5% for the stratum of CD4 between 200 and 500 cells/mL, and approximately 6.2 ± 20.4% for the stratum > 500 cells/mL. The Scientiﬁc World Journal Volume 2012, Article ID 906873, 5 pages doi:10.1100/2012/906873 The Scientiﬁc World Journal Volume 2012, Article ID 906873, 5 pages doi:10.1100/2012/906873 The cientiﬁcWorldJOURNAL Danielle Cristyane Kalva Borato,1 Emerson Carraro,2 Sˆonia Regina Weber Ribas,3 Carlos Augusto Kalva-Filho,4 and Jos´e Carlos Rebuglio Vellosa1 The diﬀerences in the percentages of CD4+ T lymphocytes obtained by diﬀerent methodologies could lead to conﬂict when used in clinical decisions related to the treatment and care of people infected with HIV. 1. Introduction The absolute count of lymphocytes may be inﬂuenced by biological factors that aﬀect the total count of leukocytes and lymphocytes, such as the use of drugs that suppress the bone marrow, acute infections (e.g., sepsis, malaria, and tuberculosis), and pregnancy, which can lead to hemodilu- tion [9]. Besides these biological factors, there could also be a variation due to methodological factors such as diﬀerences in the methods and equipment used [3, 10, 11]. 2.4. Percentage Values of CD4+ T Lymphocytes Obtained by Flow Cytometry. The immunophenotyping of each sample was carried out using the protocol for T-cell count of the Multitest/TruCount standard (monoclonal antibodies CD45+/CD3+/CD8+/CD4+) by FACSCalibur ﬂow cytome- ter (Becton Dickinson-Biosciences, San Jose, CA, USA) to obtain the relative count of CD4+ cells. 2.4. Percentage Values of CD4+ T Lymphocytes Obtained by Flow Cytometry. The immunophenotyping of each sample was carried out using the protocol for T-cell count of the Multitest/TruCount standard (monoclonal antibodies CD45+/CD3+/CD8+/CD4+) by FACSCalibur ﬂow cytome- ter (Becton Dickinson-Biosciences, San Jose, CA, USA) to obtain the relative count of CD4+ cells. Several studies have reported that variations in the percentage count of CD4+ T lymphocytes are more stable parameters than variations in the absolute count to assess the progression of the disease [12–15]. Moreover, the relative values of CD4+ T lymphocytes in the initiation of antiretroviral therapy were associated with the risk of disease progression independent of other clinical factors, including absolute counts of CD4+ T lymphocytes [16]. 2.5. Statistical Analysis. The Kolmogorov-Smirnov test was conducted to ensure normality, and the values showed normal distribution. The statistical procedures used involved a descriptive analysis (mean and standard deviation), corre- lation, and comparison between the two methodologies. The data were analyzed using Student’s t-test for comparison of means between paired values. To investigate the correlations between the variables, we used the Pearson correlation. In the analysis of diﬀerent methodologies, the correlation between the results was veriﬁed through the graphical representation of the Bland-Altman method. The level of signiﬁcance adopted was P < 0.05. The data were processed by MedCalc statistical program. The main concern regarding the use of counting the percentage of CD4+ T cells is how the variation of results could have an inﬂuence on decisions related to the clinical treatment and care of people infected with HIV. 1. Introduction infections) to AIDS (human immunodeﬁciency syndrome) and death due to conditions associated with infection [3]. The natural history of infection with the human immunode- ﬁciency virus (HIV) is characterized by a progressive decline of T helper (CD4+) lymphocytes [1]. This depletion occurs because the virus infects and kills CD4+ T lymphocytes, the main mechanism for programmed cell death apoptosis [2]. These cells act as regulators and ampliﬁers of the immune response and are associated with the immunopathogenesis of HIV infection. Thus, the decline of CD4+ T cells results in an impaired immune system and the progression of infec- tion (the main consequence of the onset of opportunistic The level of CD4+ T cells is considered to be one of the most important immunological parameters in HIV-infected individuals to evaluate their prognosis and state of immune deﬁciency, to determine the start of antiretroviral therapy, to monitor the eﬀectiveness of this treatment, to evaluate the need to start or discontinue prophylaxis for opportunist infections [4], and to establish the diagnosis of AIDS [5]. Thus, quantiﬁcation of CD4+ lymphocytes (immunoph- enotyping by ﬂow cytometry) is an indispensable procedure 2 The Scientiﬁc World Journal 2.3. Percentage Values of CD4+ T Lymphocytes Estimated by Hematologic Equipment Associated with Flow Cytometry. The samples were subjected to cell count using the Cell-Dyn hematology counter 3700 (Abbott, QC, Canada) and FAC- SCalibur ﬂow cytometer (Becton Dickinson-Biosciences, San Jose, CA, USA). First, we obtained the total absolute lymphocyte count using hematology equipment. Then, this absolute value of total lymphocyte was combined with the absolute values of CD4+ T lymphocytes obtained by ﬂow cytometry in order to calculate the relative value of CD4+ T lymphocytes. in the evaluation of patients with HIV [6]. Immunopheno- typing provides important information about the leukocytes of the immune system, distinguishing total lymphocytes (CD45+), T lymphocytes (CD3+), and subtypes of T lym- phocytes which comprise two subsets: helper T cells (T lym- phocytes CD3+/CD4+) and cytotoxic T cells (T lymphocytes CD3+/CD8+) [7]. Thus, the total lymphocyte count and percentage values of lymphocyte subsets may be determined by using ﬂow cytometry, using CD45+ monoclonal antibody, in association with CD3+, CD4+, and CD8+ antibodies [8]. 1. Introduction Therefore, the aim of this study was to determine the variation of relative counts for CD4+ T cells using two diﬀerent methodologies: (i) estimating the percentage values using a hematology counter and a ﬂow cytometer and (ii) determination of these values only using the ﬂow cytometer. 3. Results and Discussion In this study, the variability between relative counts for CD4+ T lymphocytes generated by ﬂow cytometry and those estimated by an alternative methodology was analyzed. The estimated method necessitates the combination of results of hematologic equipment (absolute count of total lymphocytes) and cytometry ﬂow (absolute count of CD4+ T lymphocytes). 2. Methods 2.1. Participants and Ethics. There were 81 selected indi- viduals with HIV. All participants were informed about the survey, and they freely signed and dated a consent form. The protocol was approved by the Ethics in Research Committee of the State University of Ponta Grossa (no. 0443710-21/2010) and was conducted in accordance with the Helsinki Declaration. Samples were grouped according to the absolute count of CD4+ T cells, resulting in the following stratiﬁcation for the 81 samples analyzed: 18 samples with CD4+ T-cell counts below 200 cells/mL (132 ± 47 cells/mL), 34 samples between 200 and 500 cells/mL (342 ± 74 cells/mL), and 29 samples with counts above 500 cells/mL (701 ± 156 cells/mL). 2.2. Laboratory Analysis. As in immunophenotyping, the determination of CD4+ T cells is the most important immunological parameter in HIV-infected individuals. The percentages of lymphocyte count obtained only by ﬂow cytometry and the combination of the two methods (ﬂow cytometry and hematology counter) were compared. The results of the percentage counts of CD4+ T cells obtained directly by ﬂow cytometry were 10.99 ± 3.99 for stratum CD4+ < 200 cells/mL, 22.89 ±6.47 for stratum 200– 500 cells/mL, and 29.84 ± 10.46 for stratum > 500 cells/mL. However, the estimated values obtained by the hematological counter were 11.86 ± 5.10, 25.08 ± 9.07, and 36.07 ± 16.78, respectively, for each of these strata. There were identiﬁed signiﬁcant diﬀerences between values for the relative counts from these two methodologies for every studied stratum (P < 0.05). Biological samples were collected by a vacuum system (Vacutainer) containing the anticoagulant EDTA-K3, and two 5 mL tubes of venous blood were collected for analysis, one by ﬂow cytometry (immunophenotyping) and one for analysis by traditional hematologic equipment (identiﬁca- tion by impedance and roughness). All tests were performed within 6 hours of collection. 2. Methods r = 0.65 10 15 20 25 30 35 40 45 50 10 15 20 25 30 35 40 CD4% ﬂow cytometry and hematology counter CD4% ﬂow cytometry (a) 10 15 20 25 30 35 40 45 Mean of CD4% hematology counter and ﬂow cytometry −15 −10 −5 5 0 10 15 20 Mean 2.2 −1.96 SD −11.4 +1.96 SD 15.7 (b) CD4% hematology counter, CD4% ﬂow (b) (a) Figure 2: Correlation of percentage values of CD4+, P < 0.05 (a) and limits of agreement between the values estimated by Bland-Altman analysis (b) obtained by the hematology counter and the ﬂow cytometer in the stratum of CD4 count between 200 and 500 cells/mL. The correlation between the percentages of CD4+ T lymphocytes obtained by the two methodologies for the three strata of CD4 cells studied is shown in Figures 1(a), 2(a), and 3(a), as well as the agreement represented by the Bland-Altman analysis shown in Figures 1(b), 2(b), and 3(b). values for the three strata studied, ranging from about 1% for the stratum CD4+ < 200 cells/mL up to 6% for the stratum > 500 cells/mL. A possible explanation for these diﬀerences is the form used for the determination of total lymphocytes by the two devices. The additional variability of the count is due to a greater inaccuracy in the way in which the hematologic equipment classiﬁes total lymphocytes [17]. The correlation between the percentages of CD4+ T lymphocytes obtained by the two methodologies for the three strata of CD4 cells studied is shown in Figures 1(a), 2(a), and 3(a), as well as the agreement represented by the Bland-Altman analysis shown in Figures 1(b), 2(b), and 3(b). Studying the Bland-Altman analysis, it can be seen that the diﬀerence between the two measures was 1.0% for the stratum of CD4+ < 200 cells/mL, and the limits of agreement were from −2.8% to 4.8%. In the CD4 strata between 200 and 500 cells/mL, lymphocyte counts above 500 cells/mL were observed as well as broader concordance limits between 2.2% (−11.4% to 15.7%) and 6.2% (−14.1% to 26.6%), respectively, compared to the extract of CD4+ < 200 cells/mL. Studying the Bland-Altman analysis, it can be seen that the diﬀerence between the two measures was 1.0% for the stratum of CD4+ < 200 cells/mL, and the limits of agreement were from −2.8% to 4.8%. 2. Methods 3 The Scientiﬁc World Journal 3 r = 0.93 0 5 10 15 20 25 2 4 6 8 10 12 14 16 18 20 CD4% ﬂow cytometry and hematology counter CD4% ﬂow cytometry (a) −6 −4 −2 0 2 4 6 Mean of CD4% hematology counter and ﬂow cytometry CD4% hematology counter, CD4% ﬂow cytometry Mean 1 −1.96 SD −2.8 +1.96 SD 4.8 0 5 10 15 20 25 (b) Figure 1: Correlation of percentage values of CD4+, P < 0.05 (a) and limits of agreement between the values estimated by Bland-Altman analysis (b) obtained by the hematology counter and the ﬂow cytometer in the stratum of CD4 count <200cells/mL. −6 −4 −2 0 2 4 6 Mean of CD4% hematology counter and ﬂow cytometry CD4% hematology counter, CD4% ﬂow cytometry Mean 1 −1.96 SD −2.8 +1.96 SD 4.8 0 5 10 15 20 25 (b) r = 0.93 0 5 10 15 20 25 2 4 6 8 10 12 14 16 18 20 CD4% ﬂow cytometry and hematology counter CD4% ﬂow cytometry (a) CD4% hematology counter, CD4% ﬂow (b) (a) Figure 1: Correlation of percentage values of CD4+, P < 0.05 (a) and limits of agreement between the values estimated by Bland-Altman analysis (b) obtained by the hematology counter and the ﬂow cytometer in the stratum of CD4 count <200cells/mL. r = 0.65 10 15 20 25 30 35 40 45 50 10 15 20 25 30 35 40 CD4% ﬂow cytometry and hematology counter CD4% ﬂow cytometry (a) 10 15 20 25 30 35 40 45 Mean of CD4% hematology counter and ﬂow cytometry −15 −10 −5 5 0 10 15 20 CD4% hematology counter, CD4% ﬂow cytometry Mean 2.2 −1.96 SD −11.4 +1.96 SD 15.7 (b) Figure 2: Correlation of percentage values of CD4+, P < 0.05 (a) and limits of agreement between the values estimated by Bland-Altman analysis (b) obtained by the hematology counter and the ﬂow cytometer in the stratum of CD4 count between 200 and 500 cells/mL. Acknowledgments The authors are grateful to Departamento de An´alises Cl´ınicas e Toxicol´ogicas (UEPG) and Ponta Grossa Expert Assistance Service to allow this study to be realized. The main importance of using percentage values of CD4+ T lymphocytes is in the absolute count changes in response to stimuli that are independent of HIV infection, and the percentages are less subject to this variability [13]. Considering the percentage values of CD4+ T lymphocytes for evaluation of HIV-infected individuals, the stratum CD4+ < 200 cells/mL counting could be underestimated by up to 4.8% or overestimated up to 2.8%, while for the strata 200 < CD4+ < 500 cells/mL and CD4+ cells > 500 cells/mL, the count could be underestimated by up to 15.7% and 26.6% or overestimated by up to 11.4% and 14.1%, respectively, as presented in Figures 1, 2, and 3. 2. Methods In the CD4 strata between 200 and 500 cells/mL, lymphocyte counts above 500 cells/mL were observed as well as broader concordance limits between 2.2% (−11.4% to 15.7%) and 6.2% (−14.1% to 26.6%), respectively, compared to the extract of CD4+ < 200 cells/mL. The results corroborate information reported earlier showing that the lymphocyte count obtained from hema- tologic analyzers is prone to errors [17–20], and, on the other hand, the use of a gate on CD45+ cells labeled with an associated dispersion parameter light using the ﬂow cytometer provides better precision and accuracy in the quantiﬁcation of lymphocytes in relation to the parameters of cell volume and conductance of hematologic counters [6]. It was noted that the estimate of the count of CD4+ T cells from the hematology counter was higher in relative The Scientiﬁc World Journal 4 r = 0.81 10 20 30 40 50 60 70 80 10 15 20 25 30 35 40 45 50 55 60 CD4% ﬂow cytometry and hematology counter CD4% ﬂow cytometry (a) 10 20 30 40 50 60 70 −20 −10 0 10 20 30 40 Mean of CD4% hematology counter and ﬂow cytometry CD4% hematology counter, ﬂow cytometry Mean 6.2 −1.96 SD −14.1 +1.96 SD 26.6 (b) Figure 3: Correlation of percentage values of CD4+, P < 0.05 (a) and limits of agreement between the values estimated by Bland-Altman analysis (b) obtained by the hematology counter and the ﬂow cytometer in the stratum of CD4 count >500 cells/mL. r = 0.81 10 20 30 40 50 60 70 80 10 15 20 25 30 35 40 45 50 55 60 CD4% ﬂow cytometry and hematology counter CD4% ﬂow cytometry (a) 10 20 30 40 50 60 70 −20 −10 0 10 20 30 40 Mean of CD4% hematology counter and ﬂow cytometry CD4% hematology counter, ﬂow cytometry Mean 6.2 −1.96 SD −14.1 +1.96 SD 26.6 (b) (b) Figure 3: Correlation of percentage values of CD4+, P < 0.05 (a) and limits of agreement between the values estimated by Bland-Altman analysis (b) obtained by the hematology counter and the ﬂow cytometer in the stratum of CD4 count >500 cells/mL. 4. Conclusions Comparing the percentage of CD4+ T cells in the stratum of CD4+ < 200 cells/mL by the hematology counter and ﬂow cytometry showed that the measures have a strong correlation (r = 0.93). However, they do not show a good agreement, since the Bland-Altman plot (Figure 2(a)) shows that the diﬀerence between the two methods was 1.0% of CD4+ T lymphocytes, and the limits of agreement were ±3.8%. In this study, the analysis of agreement between the hema- tology meter and the ﬂow cytometer showed relatively large limits for the analyzed strata, indicating high variability. So, although there was a good correlation between the percentage values of CD4+ T lymphocytes estimated by the two methods association, the correlation between individual measurements indicated relatively large limits for all strata of CD4+ cells studied. From a clinical standpoint, the diﬀerences given by the limits of agreement of the percentage values of CD4+ T lymphocytes could cause a conﬂict in decisions regarding treatment and care of people infected with HIV. Therefore, the interpretation of the percentage count of CD4+ T lymphocytes for immune monitoring of patients with human immunodeﬁciency virus should carefully take into account variations that may occur due to the methodology used. To the stratum of CD4+ between 200 and 500 cells/mL, it is noted that the measures are moderately correlated (r = 0.65). Despite the correlation, Figure 2(b) (Bland-Altman) demonstrates broader limits of agreement of approximately 2.2 ± 13.5%. This is similar to the lymphocyte count above 500 cells/mL with limits of agreement approximately 6.2 ± 20.4%, as shown in Figure 3(b) (Bland-Altman), although this stratum showed a strong correlation (r = 0.81). Similarly, MacLennan et al. [20], assessing the use of ﬂow cytometry to provide only the absolute count of CD4+ T cells (associated with total lymphocyte count in hematology analyzers to obtain the percentage of CD4+ T lymphocytes), obtained bias of 0.92% and limits of agreement between 5.83% and 7.66% through FacsCount and Multitest/Tubs Trucount method and in FACSCalibur ﬂow cytometer, for the absolute count of CD4+ T cells below 200 cells/mL. References [1] T. H. Mogensen, J. Melchjorsen, C. S. Larsen, and S. R. Paludan, “Innate immune recognition and activation during HIV infection,” Retrovirology, vol. 7, article 54, 2010. [2] B. Moll, E. E. Emeson, and C. B. Small, “Inverted ratio of inducer to suppressor T lymphocyte subsets in drug abusers with opportunistic infections,” Clinical Immunology and Immunopathology, vol. 25, no. 3, pp. 417–423, 1982. [3] J. B. Alimonti, T. B. Ball, and K. R. Fowke, “Mechanisms of CD4+ T lymphocyte cell death in human immunodeﬁciency virus infection and AIDS,” Journal of General Virology, vol. 84, no. 7, pp. 1649–1661, 2003. [3] J. B. Alimonti, T. B. Ball, and K. R. Fowke, “Mechanisms of CD4+ T lymphocyte cell death in human immunodeﬁciency virus infection and AIDS,” Journal of General Virology, vol. 84, no. 7, pp. 1649–1661, 2003. The Scientiﬁc World Journal 5 [4] X. Li, C. Breukers, A. Ymeti, B. Lunter, L. W. M. M. Terstappen, and J. Greve, “CD4 and CD8 enumeration for HIV monitoring in resource-constrained settings,” Cytometry B, vol. 76, no. 2, pp. 118–126, 2009. [19] D. Glencross, L. E. Scott, I. V. Jani, D. Barnett, and G. Janossy, “CD45-assisted PanLeucogating for accurate, cost-eﬀective dual-platform CD4+ T-cell enumeration,” Clinical Cytometry, vol. 50, no. 2, pp. 69–77, 2002. [5] Centers for Disease Control and Prevention, “1997 revised guidelines for performing CD4+ T-cell determinations in persons infected with human immunodeﬁciency virus (HIV),” Morbidity and Mortality Weekly Report, vol. 46, no. RR-2, pp. 1–29, 1997. [20] C. A. MacLennan, M. K. P. Liu, S. A. White et al., “Diagnostic accuracy and clinical utility of a simpliﬁed low cost method of counting CD4 cells with ﬂow cytometry in Malawi: diagnostic accuracy study,” British Medical Journal, vol. 335, no. 7612, pp. 190–194, 2007. [6] M. R. G. O’Gorman and J. K. A. Nicholson, “Adoption of single-platform technologies for enumeration of absolute T lymphocyte subsets in peripheral blood,” Clinical and Diagnostic Laboratory Immunology, vol. 7, no. 3, pp. 333–335, 2000. [7] C. A. Janeway, T. Paul, and M. Walport, Immunobiology, Garland Publishing, New York, NY, USA, 6th edition, 2005. [8] G. Janossy, I. V. Jani, N. J. Bradley, A. Bikoue, T. Pitﬁeld, and D. K. Glencross, “Aﬀordable CD4+-T-cell counting by ﬂow cytometry: CD45 gating for volumetric analysis,” Clinical and Diagnostic Laboratory Immunology, vol. 9, no. 5, pp. 1085– 1094, 2002. [9] J. Hoﬀman, J. Van Griensven, R. Colebunders, and M. References McKellar, “Role of the CD4 count in HIV management,” HIV Therapy, vol. 4, no. 1, pp. 27–39, 2010. [10] H. Bussmann, C. W. Wester, K. V. Masupu et al., “Low CD4+ T lymphocyte values in human immunodeﬁciency virus-negative adults in Botswana,” Clinical and Diagnostic Laboratory Immunology, vol. 11, no. 5, pp. 930–935, 2004. [11] J. P. Aboulker, B. Autran, K. Beldjord, F. Touraine, and P. Debre, “Consistency of routine measurements of CD4+, CD8+ peripheral blood lymphocytes,” Journal of Immunological Methods, vol. 154, no. 2, pp. 155–161, 1992. [12] D. M. Moore, R. S. Hogg, B. Yip, K. Craib, E. Wood, and J. S. G. Montaner, “CD4 percentage is an independent predictor of survival in patients starting antiretroviral therapy with absolute CD4 cell counts between 200 and 350 cells/microL,” HIV Medicine, vol. 7, no. 6, pp. 383–388, 2006. [13] Y. Pirzada, S. Khuder, and H. Donabedian, “Predicting AIDS- related events using CD4 percentage or CD4 absolute counts,” AIDS Research and Therapy, vol. 3, no. 1, article 20, 2006. [14] M. Guiguet, E. Kendjo, G. Carcelain et al., “CD4+ T-cell percentage is an independent predictor of clinical progression in AIDS-free antiretroviral-naive patients with CD4+ T-cell counts >200 cells/mm3,” Antiviral Therapy, vol. 14, no. 3, pp. 451–457, 2009. [15] T. Hulgan, S. Raﬀanti, A. Kheshti et al., “CD4 lympho- cyte percentage predicts disease progression in HIV-infected patients initiating highly active antiretroviral therapy with CD4 lymphocyte counts >350 Lymphocytes/mm3,” Journal of Infectious Diseases, vol. 192, no. 6, pp. 950–957, 2005. [16] T. Hulgan, B. E. Shepherd, S. P. Raﬀanti et al., “Absolute count and percentage of CD4+ lymphocytes are independent predictors of disease progression in HIV-infected persons initiating highly active antiretroviral therapy,” Journal of Infectious Diseases, vol. 195, no. 3, pp. 425–431, 2007. [17] E. Simson and W. Groner, “Variability in absolute lymphocyte counts obtained by automated cell counters,” Communications in Clinical Cytometry, vol. 22, no. 1, pp. 26–34, 1995. [18] B. Brando, D. Barnett, G. Janossy et al., “Cytoﬂuorometric methods for assessing absolute numbers of cell subsets in blood,” Cytometry, vol. 42, no. 6, pp. 327–346, 2000.
https://openalex.org/W2086131760	https://europepmc.org/articles/pmc2527517?pdf=render	English	null	Toll-Like Receptor-2 Mediates Diet and/or Pathogen Associated Atherosclerosis: Proteomic Findings	PloS one	2,008	cc-by	10,357	Toll-Like Receptor-2 Mediates Diet and/or Pathogen Associated Atherosclerosis: Proteomic Findings Abstract doi:10.1371/journal.pone.0003204 Editor: William Giannobile, University of Michigan, United States of America Editor: William Giannobile, University of Michigan, United States of America Received June 29, 2008; Accepted August 13, 2008; Published September 12, 2008 Received June 29, 2008; Accepted August 13, 2008; Published September 12, 2008 Copyright: 2008 Madan 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: 2008 Madan et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: NHLBI grant R01 HL076801 supported the study. Competing Interests: The authors have declared that no competing interests exist. * E mail: samar@bu edu Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: samar@bu.edu Monika Madan, Salomon Amar* tment of Periodontology and Oral Biology, School of Dental Medicine, Boston University, Boston, Massachusetts, United States of Am PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 Abstract Background: Accumulating evidence implicates a fundamental link between the immune system and atherosclerosis. Toll- like receptors are principal sensors of the innate immune system. Here we report an assessment of the role of the TLR2 pathway in atherosclerosis associated with a high-fat diet and/or bacteria in ApoE+/2 mice. Methods and Results: To explore the role of TLR2 in inflammation- and infection-associated atherosclerosis, 10 week-old ApoE+/2-TLR2+/+, ApoE+/2-TLR2+/2 and ApoE+/2-TLR22/2 mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 ml live Porphyromonas gingivalis (P.g) (107 CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. ApoE+/2-TLR2+/+ mice showed a significant increase in atheromatous lesions in proximal aorta and aortic tree compared to ApoE+/2-TLR2+/2 and ApoE+/2-TLR22/2 mice for all diet conditions. They also displayed profound changes in plaque composition, as evidenced by increased macrophage infiltration and apoptosis, increased lipid content, and decreased smooth muscle cell mass, all reflecting an unstable plaque phenotype. SAA levels from ApoE+/2-TLR2+/+ mice were significantly higher than from ApoE+/ 2-TLR2+/2 and ApoE+/2-TLR22/2 mice. Serum cytokine analysis revealed increased levels of pro-inflammatory cytokines in ApoE+/2-TLR2+/+ mice compared to ApoE+/2-TLR2+/2 and TLR22/2 mice, irrespective of diet or bacterial challenge. ApoE+/2- TLR2+/+ mice injected weekly for 24 weeks with FSL-1 (a TLR2 agonist) also demonstrated significant increases in atherosclerotic lesions, SAA and serum cytokine levels compared to ApoE+/2-TLR22/2 mice under same treatment condition. Finally, mass-spectrometry (MALDI-TOF-MS) of aortic samples analyzed by 2-dimentional gel electrophoresis differential display, identified 6 proteins upregulated greater than 2-fold in ApoE+/2-TLR2+/+ mice fed the high fat diet and inoculated with P.g compared to any other group. Conclusion: Genetic deficiency of TLR2 reduces diet- and/or pathogen-associated atherosclerosis in ApoE+/2 mice, along with differences in plaque composition suggesting greater structural stability while TLR-2 ligand-specific activation triggers atherosclerosis. The present data offers new insights into the pathophysiological pathways involved in atherosclerosis and paves the way for new pharmacological interventions aimed at reducing atherosclerosis. Citation: Madan M, Amar S (2008) Toll-Like Receptor-2 Mediates Diet and/or Pathogen Associated Atherosclerosis: Proteomic Findings. PLoS ONE 3(9): e3204. Introduction In mice fed with chow diet and injected with P.g the proximal aorta occupied by lesion was 16.163.2% in ApoE+/2-TLR2+/+, 7.662.3 in ApoE+/2-TLR2+/2 and 1.560.5 in ApoE+/-TLR22/2 Histomorphometric analysis revealed significantly smaller lesions in the proximal aortas of ApoE+/2-TLR2+/2 mice compared to ApoE+/2-TLR2+/+ mice and ApoE+/-TLR22/2 mice. In mice fed with high fat diet and injected with P.g the proximal aorta occupied by lesion was 46.266.6% in ApoE+/2-TLR2+/+, 18.662.4 in ApoE+/2-TLR2+/2 and 3.0460.8 in ApoE+/-TLR22/2 mice. In mice fed with high fat diet and injected with saline the proximal aorta occupied by lesion was 22.762.9% in ApoE+/2-TLR2+/+, 11.762.5 in ApoE+/2-TLR2+/2 and 2.260.6 in ApoE+/-TLR22/2 mice. In mice fed with chow diet and injected with P.g the proximal aorta occupied by lesion was 16.163.2% in ApoE+/2-TLR2+/+, 7.662.3 in ApoE+/2-TLR2+/2 and 1.560.5 in ApoE+/-TLR22/2 mice. (Fig. 3) Similar results were obtained at 14 weeks post inoculation also (data not shown). However, we did not observe any lesions in the chow-fed, vehicle-treated mice (CS), irrespective of their TLR2 genotype. Histomorphometric analysis of the proximal aortas revealed that 37.564.6% of the aortic lumen was occupied by lesion in ApoE+/2-TLR2+/+ mice injected with FSL-1 and maintained on a high fat diet, compared with only 10.461.3% of ApoE+/2-TLR2+/+ mice kept on a chow diet (Fig 4A). No lesions were observed in ApoE+/2-TLR22/2 mice after 24 weeks of FSL-1 injections and a standard chow diet (data not shown). Furthermore, there was no statistically significant difference in the percentage of aortic lumen occupied by lesions in mice injected with P. g when compared to FSL-1 treatment, irrespective of diet (Fig 4B). Two important observations suggest TLR2 as a novel target to consider for therapeutic intervention in atherosclerosis. One is that TLR2 mediates responses to lipoproteins derived from multiple pathogens. Its unique ability to heterodimerize with TLR1 or TLR6 thus results in a relatively broad range of ligand specificity [19,20,21] which may contribute to atherogenesis in the context of exposure to a variety of pathogens. The potential importance of infectious agents of oral/periodon- tal origin, such as Porphyromonas gingivalis (P.g) in the development of atherosclerosis has recently been described [22,23,24,25,26,27]. To our knowledge, the direct role of TLR2 in the bacteria- enhanced atherogenic process had never been addressed and warranted further investigation. Imunohistochemical Analysis of Proximal Aorta Imunohistochemical Analysis of Proximal Aorta After establishing the involvement of TLR2 in diet/bacteria induced atherosclerosis, we performed a detailed examination of plaque compositions by immunofluorescence staining. Five sections of the proximal aorta per animal (n = 8) each separated by 80 mm, were selected and staining specific for macrophages, smooth muscle cells, and apoptotic cells using MOMA-2, a-SMA and TUNEL were performed, respectively. Significant differences in the plaque composition were observed between atherosclerotic lesions of all ApoE+/2-TLR2+/+ mice when compared to those from ApoE+/2-TLR2+/2 and ApoE+/2-TLR22/2 mice, irrespec- tive of the treatment. The atherosclerotic lesions in ApoE+/2- TLR2+/+ mice exhibited a greater percentage of infiltrating macrophages than smooth muscle cell accumulation. In contrast, in ApoE+/2-TLR2+/2 and ApoE+/2-TLR22/2 mice, macro- phage content was either less than or equal to the smooth muscle cell accumulation in plaques (Figs. 5A–5G). Furthermore, the marked increase in the inflammatory component of the lesions in ApoE+/2-TLR2+/+mice was associated with a substantial increase in the occurrence of apoptosis within their plaques. In ApoE+/- TLR2+/+ mice we observed: 6.3%, 5.2% and 4.9% inflammatory component in the HP, HS and CP groups, respectively (Figs. 5I–L) Introduction [6,7,8]. Macrophages and endothelial cells (EC) express receptors that recognize a broad range of molecular patterns foreign to the mammalian organism but commonly found on pathogens. These molecules include lipopolysaccharides and lipoproteins from Gram- negative bacteria, peptidoglycan and lipoteichoic acids from Gram- positive bacteria, lipoproteins from mycoplasma, and zymosan from yeast [9]. These pattern-recognition receptors include various scavenger receptors (ScRs) and Toll-like receptors (TLRs). Atherosclerosis is a multifactorial chronic inflammatory disease characterized by the accumulation of cells of both the innate and acquired immune systems within the intima of the arterial wall [1,2]. In atherosclerosis, the normal homeostatic functions of the endothelium are altered, promoting an inflammatory response that results in increased expression of adhesion molecules. This in turn leads to recruitment of leukocytes, including monocytes, which penetrate into the intima, predisposing the vessel wall to lipid accretion [1,3,4]. Inflammatory mediators enhance uptake of modified lipoprotein particles and formation of lipid-laden macro- phages. The adaptive immune response in atherosclerosis is mediated by T cells that enter the intima and secrete cytokines, which subsequently amplify the inflammatory response and promote the migration and proliferation of intimal smooth muscle cells. [2,5]. TLRs are members of a large superfamily containing the interleukin-1 receptors (IL-1R) that share significant homology in their cytoplasmic domain, which is known as the Toll/IL-1R (TIR) domain [10]. Ligation of most TLRs transmits transmem- brane signals that activate the NF-kB and mitogen-activated protein kinase (MAPK) pathways [8,11,12,13]. Both in vitro and in vivo knockout mouse studies have implicated TLRs in neointima formation and intimal hyperplasia involving modulation of inflammatory responses to exogenous and endog- enous stimuli [14]. Although TLRs mediate protection against The innate immune system involves several different cell types, most importantly those of the mononuclear phagocyte lineage September 2008 \| Volume 3 \| Issue 9 \| e3204 1 September 2008 \| Volume 3 \| Issue 9 \| e3204 TLR-2 Mediated Atherosclerosis with FSL-1 for 24 weeks,11.260.6% of the aorta was covered by lesion. In contrast, in mice maintained on standard chow, FSL-1 treatment for 24 weeks resulted in significantly smaller atheroscle- rotic lesions that occupied only 1.94%60.39% of the aorta (Fig. 2A). No statistically significant differences could be observed in the extent of aortic lesions in ApoE+/2-TLR2+/+ mice injected with P.g or FSL-1, irrespective of the diet (Fig. 2B). Introduction Therefore, in the work reported here, we examined the effect of genetic deletion of TLR2 on the progression of atherosclerosis driven by a high fat diet and/or P. g infection in the ApoE+/2 murine model. Stimulation by the specific agonist FSL-1 was used to further establish the role of TLR2 in modulating the progression of diet and/or bacteria enhanced atherosclerosis in mice with normal expression of TLR2. En face and Histomorphometric Analysis of Atheroma Lesions Quantitative en face analysis revealed statistically significant smaller lesions in ApoE+/2-TLR2+/2 mice compared to ApoE+/2-TLR2+/+ mice after 24 week treatments (p,0.05): High fat diet inoculated with P. g (HP) demonstrated 12.561.7%, High fat diet injected with vehicle (HS) 5.261.8% and Chow diet inoculated with P. g (CP) demonstrated 2.660.8% of the aorta occupied by lesion. Interestingly, we did not observe any lesions on the aortic surface in ApoE+/2-TLR22/2 mice, irrespective of the diet or inoculum (Figs 1A–J). We also did not observe any lesions in any of the mice that were maintained on a chow diet and injected with vehicle (CS), regardless of their genetic backgrounds. We previously observed similar results from en face analysis performed after 14 weeks of these inoculation treatments of mice these same three genotypes (data not shown). Results Levels of Glucose, Total Serum Cholesterol, LDL, and HDL Mice were monitored for metabolic status by measuring blood glucose and lipids. No significant differences in body weight were observed in mice in connection to genotype or treatment, (e.g. P. g, FSL-1). Total serum cholesterol, LDL, HDL or glucose levels also revealed no significant differences among ApoE+/2-TLR2+/+, ApoE+/2-TLR2+/2 and ApoE+/2-TLR22/2 mice that received similar treatments and were maintained on a similar diet. However, we observed a tendency of increased cholesterol, LDL and decreased HDL level in ApoE+/2-TLR2+/+ mice injected with P. g as compared to vehicle injected group. Furthermore, the lipid and glucose profiles did not reveal any differences between mice of the same genotype that were injected with P. g or FSL-1 (supplemental data: Table S1 and Table S2). PLoS ONE \| www.plosone.org Introduction infection, various studies have demonstrated increased expression of TLR1, 2, and 4 in human atherosclerotic lesion, mechanistically linking TLRs, inflammation and atherosclerosis [6,15,16] with downstream signaling of TLR directly regulating inflammatory genes. In vitro stimulation of TLRs in human fibroblasts with a synthetic fibroblast stimulating lipopeptide (FSL-1; Pam2CGDPK- HPKSF) leads to activation of NF-kB and the production of inflammatory cytokines in a MyD88-dependent manner [17,18]. g , p ( g ) Histomorphometric analysis revealed significantly smaller lesions in the proximal aortas of ApoE+/2-TLR2+/2 mice compared to ApoE+/2-TLR2+/+ mice and ApoE+/-TLR22/2 mice. In mice fed with high fat diet and injected with P.g the proximal aorta occupied by lesion was 46.266.6% in ApoE+/2-TLR2+/+, 18.662.4 in ApoE+/2-TLR2+/2 and 3.0460.8 in ApoE+/-TLR22/2 mice. In mice fed with high fat diet and injected with saline the proximal aorta occupied by lesion was 22.762.9% in ApoE+/2-TLR2+/+, 11.762.5 in ApoE+/2-TLR2+/2 and 2.260.6 in ApoE+/-TLR22/2 mice. In mice fed with chow diet and injected with P.g the proximal aorta occupied by lesion was 16.163.2% in ApoE+/2-TLR2+/+, 7.662.3 in ApoE+/2-TLR2+/2 and 1.560.5 in ApoE+/-TLR22/2 mice. (Fig. 3) Similar results were obtained at 14 weeks post inoculation also (data not shown). However, we did not observe any lesions in the chow-fed, vehicle-treated mice (CS), irrespective of their TLR2 genotype. Histomorphometric analysis of the proximal aortas revealed that 37.564.6% of the aortic lumen was occupied by lesion in ApoE+/2-TLR2+/+ mice injected with FSL-1 and maintained on a high fat diet, compared with only 10.461.3% of ApoE+/2-TLR2+/+ mice kept on a chow diet (Fig 4A). No lesions were observed in ApoE+/2-TLR22/2 mice after 24 weeks of FSL-1 injections and a standard chow diet (data not shown). Furthermore, there was no statistically significant difference in the percentage of aortic lumen occupied by lesions in mice injected with P. g when compared to FSL-1 treatment, irrespective of diet (Fig 4B). Histomorphometric analysis revealed significantly smaller lesions in the proximal aortas of ApoE+/2-TLR2+/2 mice compared to ApoE+/2-TLR2+/+ mice and ApoE+/-TLR22/2 mice. In mice fed with high fat diet and injected with P.g the proximal aorta occupied by lesion was 46.266.6% in ApoE+/2-TLR2+/+, 18.662.4 in ApoE+/2-TLR2+/2 and 3.0460.8 in ApoE+/-TLR22/2 mice. In mice fed with high fat diet and injected with saline the proximal aorta occupied by lesion was 22.762.9% in ApoE+/2-TLR2+/+, 11.762.5 in ApoE+/2-TLR2+/2 and 2.260.6 in ApoE+/-TLR22/2 mice. Serum Cytokine Levels To further correlate the serum cytokine levels within advanced stage atherosclerotic lesions, serum samples were analyzed for 32 cytokines. Statistical significance was evaluated by ANOVA followed by the post-hoc Scheffe test. A level of p,0.05 was considered significant. Significantly altered cytokine levels were observed as a consequence of the high fat diet and of inoculation with P. g. These are presented in supplement sheet Fig-3. Compared to ApoE+/2-TLR2+/2 and ApoE+/2-TLR22/2 mice, ApoE+/2-TLR2+/+ mice displayed profoundly higher levels of most proinflammatory cytokines and chemokines, including IL-1a, IL-1b, IL-6, IL-12p40, IL-12p70, TNF-a, MCP-1, VEGF, M- CSF, and GM-CSF in high fat diet and/or bacterial-challenged animals. No significant differences were observed in the cytokine levels between all 3 genetic background mice fed the chow diet Two-dimensional Protein Maps of Aortic Tissues from ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 Mice Serum Amyloid A Level Also, there was no significant difference in serum SAA levels between ApoE+/2-TLR2+/+ mice injected with FSL-1 when compared to the P. g injected group, irrespective of the diet (Fig 6B). and saline-inoculated. The most significantly elevated cytokine levels were seen in ApoE+/2-TLR2+/+ mice fed the high fat diet and also inoculated with P.g (HP) (Fig. 7). / and saline-inoculated. The most significantly elevated cytokine levels were seen in ApoE+/2-TLR2+/+ mice fed the high fat diet and also inoculated with P.g (HP) (Fig. 7). Cytokine profiling in serum samples obtained from ApoE+/2- TLR2+/+ mice treated for 24 weeks with FSL-1 or wild type P.g. were compared. The expression of cytokines was increased with both stimuli in ApoE+/2-TLR2+/+ stimulated with FSL-1 or wild- type P.g. irrespective of the diet (Fig. 3 and 4 Supplemental Data S1). FSL-1 did not stimulate cytokine expression in ApoE+/2- TLR22/2 mice (Fig. S3 and S4 Supplemental Data S1). Cytokine profiling in serum samples obtained from ApoE+/2- TLR2+/+ mice treated for 24 weeks with FSL-1 or wild type P.g. were compared. The expression of cytokines was increased with both stimuli in ApoE+/2-TLR2+/+ stimulated with FSL-1 or wild- type P.g. irrespective of the diet (Fig. 3 and 4 Supplemental Data S1). FSL-1 did not stimulate cytokine expression in ApoE+/2- TLR22/2 mice (Fig. S3 and S4 Supplemental Data S1). Serum Amyloid A Level At the conclusion of the 24 week treatment period, SAA levels were highest in mice on high fat diets and also inoculated with P.g. High fat mice inoculated only with saline had the next highest SAA levels, followed by mice on a normal diet but challenged with weekly inoculations of P.g. Mice from all genetic backgrounds maintained on standard chow and inoculated with vehicle had the lowest SAA levels, regardless of genotype (Fig 6A). The serum Similarly, FSL-1 treatment for 24 weeks failed to induce any atherosclerotic changes in the aortas of ApoE+/2-TLR22/2 mice maintained on standard lab chow (data not shown). However, in ApoE+/2-TLR2+/+ mice maintained on a high fat diet and treated PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 2 TLR-2 Mediated Atherosclerosis Figure 1. P. g and/or high fat diet increases aortic atherosclerotic lesions in ApoE+/2-TLR2+/+ mice when compared to ApoE+/2- TLR2+/2, and ApoE+/2-TLR22/2 mice. En face analysis: (1A–1I): Representative en face view of aortic surface lesions in ApoE+/2-TLR2+/+, ApoE+/2- TLR2+/2, and ApoE+/2-TLR22/2 mice after 24 weeks of treatments. (1J): Calculated percentages of aortic surface area covered by lesions after 24 weeks of treatments (bacterial challenge or vehicle control) among mice of three genotypes maintained on standard chow or high fat diets. Values represent means6SD; p,0.05 for ApoE+/2-TLR2+/+ mice compared to ApoE+/2-TLR2+/2 mice and p,0.05 for ApoE+/2-TLR2+/+ mice compared to ApoE+/2-TLR22/2 mice in the same treatment condition and maintained on the same diet. Abbreviations are as defined in the text. doi:10.1371/journal.pone.0003204.g001 Figure 1. P. g and/or high fat diet increases aortic atherosclerotic lesions in ApoE+/2-TLR2+/+ mice when compared to ApoE+/2- TLR2+/2, and ApoE+/2-TLR22/2 mice. En face analysis: (1A–1I): Representative en face view of aortic surface lesions in ApoE+/2-TLR2+/+, ApoE+/2- TLR2+/2, and ApoE+/2-TLR22/2 mice after 24 weeks of treatments. (1J): Calculated percentages of aortic surface area covered by lesions after 24 weeks of treatments (bacterial challenge or vehicle control) among mice of three genotypes maintained on standard chow or high fat diets. Values represent means6SD; p,0.05 for ApoE+/2-TLR2+/+ mice compared to ApoE+/2-TLR2+/2 mice and *p,0.05 for ApoE+/2-TLR2+/+ mice compared to ApoE+/2-TLR22/2 mice in the same treatment condition and maintained on the same diet. Abbreviations are as defined in the text. doi:10.1371/journal.pone.0003204.g001 SAA levels were undetectable in ApoE+/2-TLR22/2 mice maintained on a chow diet and injected with FSL-1 (data not shown). Two-dimensional Protein Maps of Aortic Tissues from ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 Mice Two-dimensional Protein Maps of Aortic Tissues from ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 Mice Protein extracts from aortic tissues of ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice maintained on a high fat diet and/or inoculated with P. g were separated using 2-DGE. An example of the overall 2-DGE patterns of aortic protein extracts of ApoE+/2- TLR2+/+ mice fed a high fat diet and inoculated with P. g is shown in Fig 8. A total of 34 different protein spots were detected in response to P.g challenge: 21 protein spots increased least by 2-fold (Fig 8 red); 3 protein spot decreased at least by 2-fold (Fig 8 green) and 10 unmatched protein spots (Fig 8 black). Out of the 21 Protein extracts from aortic tissues of ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice maintained on a high fat diet and/or inoculated with P. g were separated using 2-DGE. An example of the overall 2-DGE patterns of aortic protein extracts of ApoE+/2- TLR2+/+ mice fed a high fat diet and inoculated with P. g is shown in Fig 8. A total of 34 different protein spots were detected in response to P.g challenge: 21 protein spots increased least by 2-fold (Fig 8 red); 3 protein spot decreased at least by 2-fold (Fig 8 green) and 10 unmatched protein spots (Fig 8 black). Out of the 21 PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 3 TLR-2 Mediated Atherosclerosis Figure 2. TLR2 activation through FSL-1 demonstrated no significant difference in aortic lesions when compared to P. g in ApoE+/2- TLR2+/+ and ApoE+/2-TLR22/2 mice. (2A) Percentage of aortic surface area covered by lesions in chow-fed groups for mice from two genetic backgrounds (ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2) injected with P. g or FSL-1 for 24 weeks. Values represent means6SD; p,0.05 between ApoE+/2-TLR2+/+ mice and ApoE+/2-TLR22/2 mice injected with P. g; *p,0.05 between ApoE+/2-TLR2+/+ mice and ApoE+/2-TLR22/2 mice injected with FSL-1. No lesions were detected in ApoE+/2-TLR22/2 mice irrespective of the treatment. (2B) Percentage of aortic surface area covered by lesions in ApoE+/2-TLR2+/+ mice maintained on either diet and injected weekly with P. g or FSL-1 for 24 weeks. Values represent means6SD. doi:10.1371/journal.pone.0003204.g002 Figure 2 TLR2 activation through FSL-1 demonstrated no significant difference in Figure 2. TLR2 activation through FSL-1 demonstrated no significant difference in aortic lesions when compared to P. g in ApoE+/2- TLR2+/+ and ApoE+/2-TLR22/2 mice. Two-dimensional Protein Maps of Aortic Tissues from ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 Mice (2A) Percentage of aortic surface area covered by lesions in chow-fed groups for mice from two genetic backgrounds (ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2) injected with P. g or FSL-1 for 24 weeks. Values represent means6SD; p,0.05 between ApoE+/2-TLR2+/+ mice and ApoE+/2-TLR22/2 mice injected with P. g; *p,0.05 between ApoE+/2-TLR2+/+ mice and ApoE+/2-TLR22/2 mice injected with FSL-1. No lesions were detected in ApoE+/2-TLR22/2 mice irrespective of the treatment. (2B) Percentage of aortic surface area covered by lesions in ApoE+/2-TLR2+/+ mice maintained on either diet and injected weekly with P. g or FSL-1 for 24 weeks. Values represent means6SD. doi:10.1371/journal.pone.0003204.g002 protein spots with at least 2 fold increase 6 proteins: Vesl-2 protein, Sod-2 protein, fumarate hydratase, myosin light chain polypeptide 3, aconitase, and gelsolin were identified (Table 1). Out of the 10 unmatched spots a protein: Hb was identified (table 1) only in ApoE+/2-TLR2+/+ mice maintained on the high fat diet and inoculated with P. g. Magnified gel regions corresponding to Hb from mice of both genotypes maintained on high fat diets after challenge with P.g or vehicle are compared in Figs 8A–D. previous studies showing that periodontal pathogens can influence the systemic lipoprotein profile [33]. Our data support previous studies showing that both endoge- nous (diet) and exogenous (Pam3CSK4) TLR2 ligands play important roles in the modulation of atherosclerosis [14,28]. Indeed, mice deficient in TLR4, TLR2 and MyD88 all have reduced atherosclerosis which establishes that TLR-dependent pathways contribute to disease development. Although it is likely that total ‘‘infectious burden’’ contributes to atherosclerosis progression, endogenous ligands may also initiate and modulate Toll-like receptor signaling pathways [29,30,31]. Discussion The unstable plaque phenotype is characterized by increased vulnerability to rupture and thrombosis. Histologically, an unstable plaque is identified by its thin fibrous cap, low smooth muscle cell count, high macrophage content, increased apoptosis and large lipid core [32,33,34,35,36]. The loss of the smooth muscle cells in particular is thought to be detrimental for plaque stability since most of the interstitial collagen fibers, which are important for the tensile strength of the fibrous cap, are produced by these cells [37]. Our detailed immunohistochemical analysis of atherosclerotic lesions for smooth muscle cells, macrophages, and apoptotic regions found that all the signs of plaque instability were consistently observed in ApoE+/2-TLR2+/+ mice, while ApoE+/2- TLR2+/2 and ApoE+/2-TLR22/2 mice showed lesions more characteristic of stable plaque, with less macrophage content, less apoptosis, smaller lipid cores, and higher smooth muscle cell mass (Fig. 5). A significant increase of apoptosis in the lesions of ApoE+/ In the present study we demonstrate that TLR2 plays an important role in the pathogenesis of bacteria-enhanced diet- dependent atherosclerosis in the ApoE+/2 murine model, establishing a key link between atherosclerosis and immune defense against foreign pathogens and/or endogenous inflamma- tory ligands. Both en face and histomorphometric data revealed that a greater percentage of the aorta and aortic lumen was occupied by the atherosclerotic lesions in ApoE+/2-TLR2+/+ mice as compared to either ApoE+/2-TLR2+/2 or ApoE+/2-TLR22/2 mice. ApoE+/2-TLR2+/+ mice fed a high fat diet and inoculated with P. g (HP) exhibited larger lesions compared to mice fed a high fat diet and inoculated only with saline vehicle (HS) or mice fed the standard lab chow diet and inoculated with P. g. (CP). Increased cholesterol, LDL and decreased HDL level seen in ApoE+/2- TLR2+/+ mice injected with P. g in current work, corroborates well PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 4 TLR-2 Mediated Atherosclerosis Figure 3. P.g and/or high fat diet increases atherosclerotic lesions in proximal aorta of ApoE+/2-TLR2+/+ mice when compared to ApoE+/2-TLR2+/2, and ApoE+/2-TLR22/2 mice. Microscopic cross-sections (10 mm) of the proximal aortic root were stained with Sudan IV and counterstained with hematoxylin to reveal lipid deposition, which was quantified by digital morphometry. (3A–D): results from mice maintained on a standard chow diet and inoculated weekly with P. g (CP). (3E–H): results from mice maintained on a high fat diet and inoculated weekly with vehicle (HS) (normal saline). Discussion (3I–L) results from mice maintained on a high fat diet and inoculated weekly with P. g (HP). (3D, H, l): data are presented graphically as percentage of total lumen of the proximal aorta occupied by lesions after 24 weeks of injections. Values represent means6SD; p,0.05 between ApoE+/2-TLR2+/+ mice and ApoE+/2-TLR2+/2 mice; *p,0.05 between ApoE+/2-TLR2+/+ mice and ApoE+/2-TLR22/2 mice in the same condition and maintained on the same diet. Abbreviations are as defined in the text. Photomicrographs shown are representative images obtained at the end of the 24 week treatment period. Original magnifications 206. Scale bar represents 0.5 mm. doi:10.1371/journal.pone.0003204.g003 Figure 3. P.g and/or high fat diet increases atherosclerotic lesions in proximal aorta of ApoE+/2-TLR2+/+ mice when compared to ApoE+/2-TLR2+/2, and ApoE+/2-TLR22/2 mice. Microscopic cross-sections (10 mm) of the proximal aortic root were stained with Sudan IV and counterstained with hematoxylin to reveal lipid deposition, which was quantified by digital morphometry. (3A–D): results from mice maintained on a standard chow diet and inoculated weekly with P. g (CP). (3E–H): results from mice maintained on a high fat diet and inoculated weekly with vehicle (HS) (normal saline). (3I–L) results from mice maintained on a high fat diet and inoculated weekly with P. g (HP). (3D, H, l): data are presented graphically as percentage of total lumen of the proximal aorta occupied by lesions after 24 weeks of injections. Values represent means6SD; p,0.05 between ApoE+/2-TLR2+/+ mice and ApoE+/2-TLR2+/2 mice; *p,0.05 between ApoE+/2-TLR2+/+ mice and ApoE+/2-TLR22/2 mice in the same condition and maintained on the same diet. Abbreviations are as defined in the text. Photomicrographs shown are representative images obtained at the end of the 24 week treatment period. Original magnifications 206. Scale bar represents 0.5 mm. doi:10.1371/journal.pone.0003204.g003 2-TLR2+/+ mice, along with upregulation of proinflammatory cytokines which regulate the release of the matrix-degrading proteinases and may favor the unstable plaque phenotype [38]. and the involvement of TLR2 in atherosclerosis. Our data show that TLR2 elicits differential expression of inflammatory cytokines and co-stimulatory molecules upon challenge with atherogenic stimuli (P.g. and/or high fat diet). Maximum induction of a host of proinflammatory cytokines (IL-1a, IL-1b, IL-6, IL-18, IFN-c, IL- 12p40, IL-12p70, TNF-a, MCP-1, VEGF, M-CSF, and GM-CSF) was observed in ApoE+/2-TLR2+/+ mice maintained on a high fat diet and challenged with P. g. Discussion Most of these cytokines and chemokines are proinflammatory factors, favoring cell migration, proliferation [40,41] and chemo-attraction of inflammatory cells, such as monocytes/macrophages and T cells [42,43,44]. These results further implicate an aspect of antigen-specific adaptive immunity mostly characteristic of a Th1 response, including cytokines IL-2, IL-18, IFNc and TNF-a [9]. The differential cytokine induction also implies that P. g and/or a high fat diet can activate different receptors to mediate intracellular signaling. It is known that the formation of heterodimers between TLR2 and other TLRs (TLR1 or TLR6) dictates the specificity of ligand recognition, thereby diversifying the possible outcomes of TLR2 and the involvement of TLR2 in atherosclerosis. Our data show that TLR2 elicits differential expression of inflammatory cytokines and co-stimulatory molecules upon challenge with atherogenic stimuli (P.g. and/or high fat diet). Maximum induction of a host of proinflammatory cytokines (IL-1a, IL-1b, IL-6, IL-18, IFN-c, IL- 12p40, IL-12p70, TNF-a, MCP-1, VEGF, M-CSF, and GM-CSF) was observed in ApoE+/2-TLR2+/+ mice maintained on a high fat diet and challenged with P. g. Most of these cytokines and chemokines are proinflammatory factors, favoring cell migration, proliferation [40,41] and chemo-attraction of inflammatory cells, such as monocytes/macrophages and T cells [42,43,44]. These results further implicate an aspect of antigen-specific adaptive immunity mostly characteristic of a Th1 response, including cytokines IL-2, IL-18, IFNc and TNF-a [9]. The differential cytokine induction also implies that P. g and/or a high fat diet can activate different receptors to mediate intracellular signaling. It is known that the formation of heterodimers between TLR2 and other TLRs (TLR1 or TLR6) dictates the specificity of ligand recognition, thereby diversifying the possible outcomes of TLR2 SAA, the mouse counterpart of human C-reactive protein, is an acute phase reactant known as a marker for systemic inflamma- tion. It has been demonstrated that CRP is produced in the liver in response to IL-6, IL-1b, and TNF-a [39]. A strong association has also been shown between circulating levels of SAA and the extent of atherosclerosis in the aorta [23,26,27]. Our data demonstrated significantly higher serum levels of SAA, IL-6, IL-1b, and TNF-a in ApoE+/2-TLR2+/+ mice compared with ApoE+/2-TLR22/2 mice, irrespective of the diet or bacterial challenge treatment. Furthermore, SAA levels significantly correlated with the extent of aortic lesions examined after 24 weeks of challenge. Interestingly, significantly lower levels of SAA in ApoE+/2-TLR22/2 mice suggest that TLR2 deficiency may also lead to a lower overall systemic inflammatory status. PLoS ONE \| www.plosone.org Discussion In this context, it is noteworthy that the ApoE+/2- TLR22/2 genotype conferred atheroprotective effects, which may result in part from reduced systemic inflammation as shown by reduced expression of proinflammatory cytokines and chemokines in all treatment groups. showed that both FSL-1 stimulation and P.g challenge resulted in a relatively similar expression of proinflammatory cytokines. Our expression proteomic approach extends the growing body of literature linking TLR2 and atherogenesis by identifying proteins involved in P. g- and/or diet-induced atherosclerosis in ApoE+/2 mice. Using 2D gel electrophoresis (2-DGE) in combination with mass spectrometry (MS), we found that in ApoE+/2-TLR2+/+ mice, P. g stimulation in combination with a high fat diet up-regulated the expression of a set of proteins (Hb, Vesl-2 protein, Sod-2 protein, fumarate hydratase, myosin light chain polypeptide 3, aconitase and gelsolin) compared to high fat diet alone. Some of these proteins were found of interest in improving our understanding of the mechanisms linking athero- genesis to infection, inflammation, and immune response. Our expression proteomic approach extends the growing body of literature linking TLR2 and atherogenesis by identifying proteins involved in P. g- and/or diet-induced atherosclerosis in ApoE+/2 mice. Using 2D gel electrophoresis (2-DGE) in combination with mass spectrometry (MS), we found that in ApoE+/2-TLR2+/+ mice, P. g stimulation in combination with a high fat diet up-regulated the expression of a set of proteins (Hb, Vesl-2 protein, Sod-2 protein, fumarate hydratase, myosin light chain polypeptide 3, aconitase and gelsolin) compared to high fat diet alone. Some of these proteins were found of interest in improving our understanding of the mechanisms linking athero- genesis to infection, inflammation, and immune response. To further establish the role of TLR2 in modulating the progression of atherosclerosis, we stimulated mice with the TLR2 agonist known as FSL-1. En face and histomorphometric analysis revealed that systemic exposure to FSL-1 dramatically increased lesion severity in a manner similar to P. g. In contrast, the absence of TLR2 resulted in complete prevention of lesions in mice on the chow diet and injected with FSL-1. Furthermore, the percentage of the lesions observed both in aorta and aortic sinus were comparable to the lesions observed in ApoE+/2-TLR2+/+ mice injected with P. g irrespective of the diet after 24 weeks. FSL-1 stimulation also altered the systemic inflammatory status as monitored by increased serum SAA levels in the ApoE+/2- TLR2+/+ mice when compared to the ApoE+/2-TLR22/2 mice. Discussion We performed cytokine profiling in order to further investigate the systemic inflammatory status associated with TLR2 deficiency PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 5 TLR-2 Mediated Atherosclerosis Figure 4. TLR2 activation through FSL-1 demonstrated no significant difference in proximal aortic lesions when compared to P. g in ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice. Microscopic cross-sections (10 mm) of the proximal aortic root were stained with Sudan IV and counterstained with hematoxylin to reveal lipid deposition, which was quantified by digital morphometry for samples from ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice. (4A): percentage of total lumen of the proximal aorta occupied by lesions after 24 weeks of treatment in ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice maintained on a standard chow diet and injected weekly with P. g or FSL-1. Values represent means6SD; p,0.05 for differences between mice injected with P. g; *p,0.05 for differences between mice injected with FSL-1. No lesions were detected in ApoE+/2-TLR22/ 2 mice irrespective of the treatment. (4B): percentage of total lumen of the proximal aorta occupied by lesions in ApoE+/2-TLR2+/+ mice maintained on a chow diet or a high fat diet after 24 weeks of injections with P. g or FSL-1. Values represent means6SD. doi:10.1371/journal.pone.0003204.g004 Figure 4. TLR2 activation through FSL-1 demonstrated no significant difference in proximal aortic lesions when compared to P. g in ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice. Microscopic cross-sections (10 mm) of the proximal aortic root were stained with Sudan IV and counterstained with hematoxylin to reveal lipid deposition, which was quantified by digital morphometry for samples from ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice. (4A): percentage of total lumen of the proximal aorta occupied by lesions after 24 weeks of treatment in ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice maintained on a standard chow diet and injected weekly with P. g or FSL-1. Values represent means6SD; p,0.05 for differences between mice injected with P. g; *p,0.05 for differences between mice injected with FSL-1. No lesions were detected in ApoE+/2-TLR22/ 2 mice irrespective of the treatment. (4B): percentage of total lumen of the proximal aorta occupied by lesions in ApoE+/2-TLR2+/+ mice maintained on a chow diet or a high fat diet after 24 weeks of injections with P. g or FSL-1. Values represent means6SD. doi:10.1371/journal.pone.0003204.g004 showed that both FSL-1 stimulation and P.g challenge resulted in a relatively similar expression of proinflammatory cytokines. activation [20]. PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 Figure 5. P. g and/or high fat diet results in unstable plaque in ApoE+/2-TLR2+/+ mice when compared to ApoE+/2-TLR2+/2, and ApoE+/2-TLR22/2 mice. Representative photomicrographs of atherosclerotic plaques from the aortic sinus of ApoE+/2-TLR2+/+, ApoE+/2-TLR2+/2 and ApoE+/-TLR22/2 mice maintained on a high fat diet and inoculated weekly with P. g (HP) for 24 weeks. Stains identify sections of macrophage infiltration (MOMA-2 red staining) (5a, 5b&5c); smooth muscle cells (a-SMA red staining) (5E, 5F&5G); TUNEL positive cells (green spots coinciding with nuclear stain DAPI) (5I, 5J&5K). Quantitative computer-assisted image analysis (as described in Materials and Methods) was used to quantify the percentage of macrophage-positive areas (5D), smooth muscle cell area (5H) and TUNEL/DAPI positive cells (5I) in proximal aortic lesions in ApoE+/2- TLR2+/+, ApoE+/2-TLR2+/2 and ApoE+/-TLR22/2 mice of all the groups at the conclusion of the 24 week treatment period. Data represent means6SD; p,0.05 for ApoE+/2-TLR2+/+ mice compared to ApoE+/2-TLR2+/2 mice, and *p,0.05 for ApoE+/2-TLR2+/+mice compared to ApoE+/2-TLR22/2 mice in the same treatment condition and maintained on the same diet. Abbreviations are as defined in text. Original magnifications 1006 for macrophages and smooth muscle and 2006 for TUNEL/DAPI staining. Scale bar represents 0.5 mm. doi:10.1371/journal.pone.0003204.g005 injected with P. g may lead to an increase in intracellular calcium, contributing to the increased atherosclerosis in this group of mice. phenotype. Its presumed mechanism of vascular injury would include both oxidative heme toxicity caused by its ineffective clearance and also the subsequent consumption of nitric oxide, an important mediator of vascular homeostasis. injected with P. g may lead to an increase in intracellular calcium, contributing to the increased atherosclerosis in this group of mice. Our observation of increased SOD-2 along with reduced smooth muscle cell content in ApoE+/2-TLR2+/+ mice fed a high fat diet and injected with P. g agrees well with a recent report in which SOD-2 deficient smooth muscle cells can exhibit a hypertrophic and hyperplastic phenotype. Thus we may speculate that P.g challenge upregulates mitochondrial SOD-2 and affects downstream pathways involving MAP kinases. Thus, increased SOD-2 may play a crucial role in determining plaque phenotype as it directly affects smooth muscle cell phenotype. Our observation of increased SOD-2 along with reduced smooth muscle cell content in ApoE+/2-TLR2+/+ mice fed a high fat diet and injected with P. g agrees well with a recent report in which SOD-2 deficient smooth muscle cells can exhibit a hypertrophic and hyperplastic phenotype. September 2008 \| Volume 3 \| Issue 9 \| e3204 PLoS ONE \| www.plosone.org Discussion It is noteworthy that the levels of serum SAA in ApoE+/2-TLR2+/+ mice stimulated with FSL-1 were comparable to the levels obtained when mice were challenged with P. g irrespective of the diet, thus confirming the role of TLR2 in upregulation of systemic inflammation produced by P. g challenge. Cytokine profiling Hemoglobin (Hb) is known to enhance the biological function of bacterial endotoxins [48] and therefore increased Hb can contribute to aheightened systemic response. Furthermore in- creased Hb content in the blood leads to increased viscosity, with detrimental effects on blood flow. Moreover, intraplaque hemor- rhage seen in advanced lesions also causes the deposition of Hb. In our study, the detection of Hb only in ApoE+/2-TLR2+/+ mice fed a high fat diet and injected with P. g indicates that elevated Hb levels may be useful as a biomarker for an unstable plaque September 2008 \| Volume 3 \| Issue 9 \| e3204 PLoS ONE \| www.plosone.org 6 TLR-2 Mediated Atherosclerosis Figure 5. P. g and/or high fat diet results in unstable plaque in ApoE+/2-TLR2+/+ mice when compared to ApoE+/2-TLR2+/2, and ApoE+/2-TLR22/2 mice. Representative photomicrographs of atherosclerotic plaques from the aortic sinus of ApoE+/2-TLR2+/+, ApoE+/2-TLR2+/2 and ApoE+/-TLR22/2 mice maintained on a high fat diet and inoculated weekly with P. g (HP) for 24 weeks. Stains identify sections of macrophage infiltration (MOMA-2 red staining) (5a, 5b&5c); smooth muscle cells (a-SMA red staining) (5E, 5F&5G); TUNEL positive cells (green spots coinciding with nuclear stain DAPI) (5I, 5J&5K). Quantitative computer-assisted image analysis (as described in Materials and Methods) was used to quantify the percentage of macrophage-positive areas (5D), smooth muscle cell area (5H) and TUNEL/DAPI positive cells (5I) in proximal aortic lesions in ApoE+/2- TLR2+/+, ApoE+/2-TLR2+/2 and ApoE+/-TLR22/2 mice of all the groups at the conclusion of the 24 week treatment period. Data represent means6SD; p,0.05 for ApoE+/2-TLR2+/+ mice compared to ApoE+/2-TLR2+/2 mice, and *p,0.05 for ApoE+/2-TLR2+/+mice compared to ApoE+/2-TLR22/2 mice in the same treatment condition and maintained on the same diet. Abbreviations are as defined in text. Original magnifications 1006 for macrophages and smooth muscle and 2006 for TUNEL/DAPI staining. Scale bar represents 0.5 mm. doi:10.1371/journal.pone.0003204.g005 Thus we may speculate that P.g challenge upregulates mitochondrial SOD-2 and affects downstream pathways involving MAP kinases. Thus, increased SOD-2 may play a crucial role in determining plaque phenotype as it directly affects smooth muscle cell phenotype. Vesl-2 (Homer 2) is a post-synaptic adaptor protein that has been shown to be present in multiple tissues such as brain and heart. It may be linked to atherogenesis through its associations with glutamate receptor complexes and also the actin cytoskeleton. These glutamate receptors are coupled with G-proteins and activate phospholipase C, ultimately activating the IP3 receptor (IP3R) to release intracellular calcium, which can alter the function of ECs. Endothelial dysfunction typically results in platelet aggregation at the damaged site. Elevated intracellular calcium also leads to increased uptake of macromolecules in plasma such as fibrinogen and LDL, eventually forming atherosclerotic plaque. Thus it may be speculated that increased vesl-2 protein in ApoE+/2-TLR2+/+ mice fed a high fat diet and Gelsolin is an actin-binding protein that is a key regulator of actin filament assembly and disassembly. It is regulated by Ca2+- and polyphosphoinositide 4, 5-bisphosphate (PIP2) and plays an important role in actin remodeling by regulating actin filament severing and capping. It has also been shown to play a role in apoptosis [45] and in sepsis-induced cell injury. Increased gelsolin PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 September 2008 \| Volume 3 \| Issue 9 \| e3204 7 i i h l b h i f ili h h [46] b bl i i h d f h i i Figure 6. TLR2 activation through FSL-1 demonstrated no significant difference in SAA levels when compared to P. g in ApoE+/2- TLR2+/+ and ApoE+/2-TLR22/2 mice. (6A) SAA levels in serum samples obtained after 24 weeks of inoculations, as determined by ELISA. Data represent means6SD; p,0.05 between ApoE+/2-TLR2+/+mice and ApoE+/2-TLR2+/2 mice; *p,0.05 between ApoE+/2-TLR2+/+mice and ApoE+/2- TLR22/2 mice maintained under the same conditions and on the same diet. Abbreviations are as defined in the text. (6B) SAA levels in serum samples obtained at the end of the study, determined by ELISA. Data represent means6SD in ApoE+/2-TLR2+/+ mice maintained on either standard chow or a high fat diet, and injected weekly with P. g or FSL-1. doi:10.1371/journal.pone.0003204.g006 TLR-2 Mediated Atherosclerosis TLR-2 Mediated Atherosclerosis Figure 6. TLR2 activation through FSL-1 demonstrated no significant difference in SAA levels when compared to P. g in ApoE+/2- TLR2+/+ and ApoE+/2-TLR22/2 mice. (6A) SAA levels in serum samples obtained after 24 weeks of inoculations, as determined by ELISA. Data represent means6SD; p,0.05 between ApoE+/2-TLR2+/+mice and ApoE+/2-TLR2+/2 mice; *p,0.05 between ApoE+/2-TLR2+/+mice and ApoE+/2- TLR22/2 mice maintained under the same conditions and on the same diet. Abbreviations are as defined in the text. (6B) SAA levels in serum samples obtained at the end of the study, determined by ELISA. Data represent means6SD in ApoE+/2-TLR2+/+ mice maintained on either standard chow or a high fat diet, and injected weekly with P. g or FSL-1. doi:10.1371/journal.pone.0003204.g006 activity has also been shown in failing human hearts [46] probably in reaction to the cell injury. Thus, increased level of gelsolin seen in ApoE+/2-TLR2+/+ mice fed a high fat diet and injected with P. g may be linked to the increased apoptosis and atherosclerosis observed in this group. not compromising host defenses or atheroprotective immune functions. Therefore manipulation of TLR2 pathways has great therapeutic potential. TLRs inhibitors or their associated signaling molecules hold great promise in the prevention of atherosclerosis. PLoS ONE \| www.plosone.org Materials and Methods Serum cytokine levels (pg/ml) in mice maintained on a high fat diet and injected with saline weekly for 24 weeks (Fig 7C&7D). Serum cytokine levels (pg/ml) in mice maintained on a high fat diet and inoculated weekly for 24 weeks with P. g (Fig 7E&7F). doi:10.1371/journal.pone.0003204.g007 Figure 7. P. g and/or high fat diet results in increased proinflamatory cytokines in ApoE+/2-TLR2+/+ mice when compared to ApoE+/ 2-TLR2+/2, and ApoE+/2-TLR22/2 mice. Serum cytokine levels (pg/ml) in mice maintained on a standard lab chow diet and inoculated weekly for 24 weeks with P. g. (Fig 7A&7B). Serum cytokine levels (pg/ml) in mice maintained on a high fat diet and injected with saline weekly for 24 weeks (Fig 7C&7D). Serum cytokine levels (pg/ml) in mice maintained on a high fat diet and inoculated weekly for 24 weeks with P. g (Fig 7E&7F). doi:10.1371/journal.pone.0003204.g007 Figure 7. P. g and/or high fat diet results in increased proinflamatory cytokines in ApoE+/2-TLR2+/+ mice when compared to ApoE+/ 2-TLR2+/2, and ApoE+/2-TLR22/2 mice. Serum cytokine levels (pg/ml) in mice maintained on a standard lab chow diet and inoculated weekly for 24 weeks with P. g. (Fig 7A&7B). Serum cytokine levels (pg/ml) in mice maintained on a high fat diet and injected with saline weekly for 24 weeks (Fig 7C&7D). Serum cytokine levels (pg/ml) in mice maintained on a high fat diet and inoculated weekly for 24 weeks with P. g (Fig 7E&7F). doi:10.1371/journal.pone.0003204.g007 Figure 8. P.g and/or high fat diet demonstrated changes in the aortic protein in ApoE+/2-TLR2+/+ mice when compared to ApoE+/2- TLR22/2 mice. Two-dimensional electrophoresis gel image of the proteins extracted from aortas (n = 5) from ApoE+/2-TLR2+/+ mice fed a high fat diet and injected weekly with P. g. Enlarged spots representing Hb were observed in aortas proteins from ApoE+/2-TLR2+/+ mice maintained on a high fat diet and injected with P. g (8B). Corresponding gel regions from aorta proteins from ApoE+/2-TLR2+/+ mice maintained on a high fat diet alone (8A) or ApoE+/2-TLR22/2 mice fed a high fat diet alone (8C) or also injected with P.g. (8D) did not exhibit the spots. Gels were stained by SYPRO RUBY stain. The spot numbers correspond to proteins listed in Table 1. doi:10.1371/journal.pone.0003204.g008 j p g Figure 8. P.g and/or high fat diet demonstrated changes in the aortic protein in ApoE+/2-TLR2+/+ mice when compared to ApoE+/2- TLR22/2 mice. Materials and Methods Aconitase and fumarate hydratase are Krebs cycle enzymes. These enzymes area lso known to play important roles in the response to oxidant stress, which can inactivate aconitase and other Krebs cycle enzymes [47]. We observed increased aconitase and fumarate hydratase levels in ApoE+/2-TLR2+/+ mice fed a high fat diet and injected with P. g. These proteins probably represent an adaptive response to increased oxidative stress but they are also important predisposing factors in the progression of the atherosclerotic process. Please refer to the supplemental data and Fig. S1 and S2 for details. Briefly, all animal protocols were approved by the Boston University Medical Campus Institutional Animal Care and Use Committee. To investigate the role of TLR2 in inflammation- and/or infection-associated atherosclerosis, 10 week-old ApoE+/2- TLR2+/+, ApoE+/2-TLR2+/2 and ApoE+/2-TLR22/2 mice were fed either a high fat diet or a regular chow diet. All mice were inoculated intravenously, once per week for 24 consecutive weeks, with 50 ml live P. g (107 CFU) or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. Histomorphometric analysis of the aortic lesions and the proximal aorta using Sudan red stain were performed. Immunofluroscent staining for macrophage, smooth muscle cell and apoptosis were performed on the proximal aortic sections. Metabolic profile, Taken together, our results confirm the important role for TLR2 signaling in diet and/or bacteria enhanced atherosclerosis in an ApoE+/2 mouse model, providing a link between innate immunity, inflammation and atherosclerosis. Due to TLR2 central role in the disease process, it represent a target of immunomod- ulatory therapy with the goal of tipping the balance from excessive chronic inflammation towards resolution of inflammation, while September 2008 \| Volume 3 \| Issue 9 \| e3204 8 TLR-2 Mediated Atherosclerosis PLoS ONE \| www.plosone.org 9 September 2008 \| Volume 3 \| Issue 9 \| e3204 PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 September 2008 \| Volume 3 \| Issue 9 \| e3204 PLoS ONE \| www.plosone.org 9 TLR-2 Mediated Atherosclerosis Figure 7. P. g and/or high fat diet results in increased proinflamatory cytokines in ApoE+/2-TLR2+/+ mice when compared to ApoE+/ 2-TLR2+/2, and ApoE+/2-TLR22/2 mice. Serum cytokine levels (pg/ml) in mice maintained on a standard lab chow diet and inoculated weekly for 24 weeks with P. g. (Fig 7A&7B). Materials and Methods Two-dimensional electrophoresis gel image of the proteins extracted from aortas (n = 5) from ApoE+/2-TLR2+/+ mice fed a high fat diet and injected weekly with P. g. Enlarged spots representing Hb were observed in aortas proteins from ApoE+/2-TLR2+/+ mice maintained on a high fat diet and injected with P. g (8B). Corresponding gel regions from aorta proteins from ApoE+/2-TLR2+/+ mice maintained on a high fat diet alone (8A) or ApoE+/2-TLR22/2 mice fed a high fat diet alone (8C) or also injected with P.g. (8D) did not exhibit the spots. Gels were stained by SYPRO RUBY stain. The spot numbers correspond to proteins listed in Table 1. doi:10.1371/journal.pone.0003204.g008 Table 1. 1 Protein identified by MALDI TOF. Table 1. 1 Protein identified by MALDI TOF. Spot number Protein Expectation Accession no. ApoE+/2 TLR2+/+ HFD+P.g 111,108,109 Gelsolin 5.30E-05 8606238 .2fold increase 104,95,80 Aconitase 1.20E-07 18079339 .2 fold increase 599,597,596,595 Hemoglobin 8.30E-06 31982300 Unmatched 689 SOD-2 6.05E-06 17390379 .2 fold increase 682 Vesl-2 2.00E-04 3766297 .2 fold increase 404 Fumarate hydratase 1.30E-05 33859554 .2 fold increase 417 Myosin, light polypeptide 3 5.70E-04 33563264 .2 fold increase Identification of proteins differentially expressed in ApoE+/2-TLR2+/+ mice fed a high fat diet and inoculated with P. g as compared to other groups. doi:10.1371/journal.pone.0003204.t001 differentially expressed in ApoE+/2-TLR2+/+ mice fed a high fat diet and inoculated with P. g as compared to other groups. 003204.t001 with 5 mg FSL-1in 50 ml saline or vehicle (normal saline). Animals were euthanized 24 weeks after the first inoculation. To compare and further establish an absolute effect of TLR2 in bacteria- enhanced atherosclerotic lesions, a third set of experiments involved only ApoE+/2-TLR22/2 mice fed only the standard serum amyloid A and serum cytokine levels were also performed for all the three genotypes. For the TLR2 agonist study a second set of four week old male ApoE+/2-TLR2+/+ fed either a HFD or a regular chow diet for 6 weeks (n = 10) were used. All mice were inoculated intravenously, once per week for 24 consecutive weeks, September 2008 \| Volume 3 \| Issue 9 \| e3204 September 2008 \| Volume 3 \| Issue 9 \| e3204 10 TLR-2 Mediated Atherosclerosis chow diet. Four week old ApoE+/2-TLR22/2 mice maintained on a regular chow diet for 6 weeks (n = 10), were inoculated with 50 ml saline vehicle or 5 mg FSL-1 in 50 ml saline. Supporting Information Figure S4 TLR2 activation through FSL-1 demonstrated similar expression of increased proinflamatory cytokines as compared high fat fed and injected with P. g in ApoE+/2-TLR2+/+ mice. Serum cytokine levels (pg/ml) in ApoE+/2-TLR2+/+ mice fed a high fat diet and injected weekly with P. g or FSL-1. Data represent mean+SD. Found at: doi:10.1371/journal.pone.0003204.s005 (0.77 MB TIF) Data S1 Supplemental material document Found at: doi:10.1371/journal.pone.0003204.s001 (0.08 MB DOC) Data S1 Supplemental material document Found at: doi:10.1371/journal.pone.0003204.s001 (0.08 MB DOC) Figure S1 Animal grouping and experimental time schedule for P.gingivalis expeiments. Four week old male ApoE+/2-TLR2+/ +, ApoE+/2-TLR2+/2 and ApoE+/2-TLR22/2 mice were fed either a HFD or a regular chow diet for 6 weeks (n = 8), then inoculated once per week for 24 weeks with 50 ml of either vehicle (normal saline) or 107 CFU) P. g while maintained on the chosen diet. Thus, there were 4 groups for each genotype of mice: Group 1 was fed a standard chow diet and inoculated weekly with 50 ml saline vehicle (CS); Group 2 was fed a standard chow diet and inoculated with 50 ml (107 CFU) P. g. (CP); Group 3 was fed a high fat diet and inoculated with 50 ml saline vehicle (HS); Group 4 was fed a high fat diet and inoculated with 50 ml (107 CFU) P. g (HP). In summary, mice (n = 8) in each group received 24 tail vein injections of either vehicle or P. g once weekly. d d i /j l ( B ) Found at: doi:10.1371/journal.pone.0003204.s005 (0.77 MB TIF) Table S1 Metabolic profiles of ApoE+/2-TLR2+/+, ApoE+/ 2-TLR2+/2 and ApoE+/2-TLR22/2 mice maintained on a standard lab chow diet or a high fat diet, and injected weekly with either saline or with P. g, at 24 weeks. Significance between ApoE+/2-TLR2+/+, ApoE+/2-TLR2+/2 and ApoE+/2- TLR22/2 for respective groups. Abbreviations are as defined in the text. Found at: doi:10.1371/journal.pone.0003204.s006 (0.07 MB DOC) Table S2 Metabolic profile of ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice fed with either a standard lab chow diet or a high fat diet, and injected weekly with either P. g or FSL- 1; measurements were obtained after 24 weeks of treatments. Found at: doi:10.1371/journal.pone.0003204.s007 (0.04 MB DOC) Table S2 Metabolic profile of ApoE+/2-TLR2+/+ and ApoE+/2-TLR22/2 mice fed with either a standard lab chow diet or a high fat diet, and injected weekly with either P. Supporting Information g or FSL- 1; measurements were obtained after 24 weeks of treatments. j g y Found at: doi:10.1371/journal.pone.0003204.s002 (0.83 MB TIF) Figure S2 Animal grouping and experimental time schedule for FSL-1expeiments. Effects of FSL-1 were tested in two sets of experiments. In the first, four week old male ApoE+/2-TLR2+/+ were fed either a HFD or a regular chow diet for 6 weeks (n = 10) then inoculated once per week for 24 weeks with 50 ml of either vehicle (normal saline) or 5 mg FSL-1 while maintained on the chosen diet. The resulting 4 groups were: Group 1a was fed a standard chow diet and inoculated with 50 ml saline vehicle (CS); Group 2a was fed a standard chow diet and inoculated weekly Found at: doi:10.1371/journal.pone.0003204.s007 (0.04 MB DOC) Statistical Analysis y All histomorphometric measurements were made by an examiner blinded to the identity of the samples. All quantitative measurements were confirmed by random analysis of one fourth of the specimens by the same examiner (R.0.92) and by another independent examiner (a pathologist) to ensure consistency. The intra-examiner and inter-examiner variation were each ,10%. All histomorphometric and serum assay data were analyzed by ANOVA followed by the post-hoc Scheffe test. A level of p,0.05 was considered significant. Found at: doi:10.1371/journal.pone.0003204.s003 (0.53 MB TIF) Figure S3 TLR2 activation through FSL-1 demonstrated similar expression of increased proinflamatory cytokines as compared chow fed and injected with P. g in ApoE+/2-TLR2+/+ mice. Serum cytokine levels (pg/ml) in ApoE+/2-TLR2+/+ mice fed a standard chow diet and injected weekly with P. g or FSL-1. Data represent mean+SD. Figure S3 TLR2 activation through FSL-1 demonstrated similar expression of increased proinflamatory cytokines as compared chow fed and injected with P. g in ApoE+/2-TLR2+/+ mice. Serum cytokine levels (pg/ml) in ApoE+/2-TLR2+/+ mice fed a standard chow diet and injected weekly with P. g or FSL-1. Data represent mean+SD. Found at: doi:10.1371/journal.pone.0003204.s004 (0.78 MB TIF) Materials and Methods All groups were analyzed for atherosclerotic lesions, metabolic profile, serum amyloid A and serum cytokine levels after 24 weeks of inoculations. with 50 ml (5 mg) FSL-1; Group 3a was fed a high fat diet and inoculated weekly with 50 ml saline vehicle (HS); Group 4a was fed a high fat diet and inoculated with 50 ml (5 mg) FSL-1. All groups were tested after 24 weeks of their diet and inoculation regimens. For the second set of experiments, four week old ApoE+/2- TLR22/2 mice maintained on only a regular chow diet for 6 weeks (n = 10), then were divided into 2 groups: Group 1b was inoculated weekly with 50 ml vehicle saline (CS); Group 2b was inoculated weekly with 50 ml (5 mg) FSL-1. All groups were tested after 24 weeks of inoculations. Author Contributions Conceived and designed the experiments: SA. Performed the experiments: MM SA. Analyzed the data: MM SA. Contributed reagents/materials/ analysis tools: SA. Wrote the paper: MM SA. Conceived and designed the experiments: SA. Performed the experiments: MM SA. Analyzed the data: MM SA. Contributed reagents/materials/ analysis tools: SA. Wrote the paper: MM SA. 14. Schoneveld, Oude N, van M, Laman, de K, et al. (2005) Toll-like receptor 2 stimulation induces intimal hyperplasia and atherosclerotic lesion development. Cardiovascular Research 66: 162–169. 13. Gordon S (2002) Pattern Recognition Receptors: Doubling Up for the Innate Immune Response. Cell 111: 927–930. 15. Xu XH, Shah PK, Faure E, Equils O, Thomas L, et al. (2001) Toll-Like Receptor-4 Is Expressed by Macrophages in Murine and Human Lipid-Rich 8. Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20: 197–216. 7. Medhitov RHC (2000) Advances in Immunology. N Engl J Med. TLR-2 Mediated Atherosclerosis Embo J 19: 3325–3336. 22. Gibson FC III, Hong C, Chou H-H, Yumoto H, Chen J, et al. (2004) Innate Immune Recognition of Invasive Bacteria Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice. Circulation 109: 2801–2806. 39. Rifai N, Ridker PM (2001) High-Sensitivity C-Reactive Protein: A Novel and Promising Marker of Coronary Heart Disease. Clin Chem 47: 403–411. 23. Li L, Messas E, Batista EL Jr, Levine RA, Amar S (2002) Porphyromonas gingivalis Infection Accelerates the Progression of Atherosclerosis in a Heterozygous Apolipoprotein E-Deficient Murine Model. Circulation 105: 861–867. 40. Barillari G, Albonici L, Incerpi S, Bogetto L, Pistritto G, et al. (2001) Inflammatory cytokines stimulate vascular smooth muscle cells locomotion and growth by enhancing [alpha]5[beta]1 integrin expression and function. Atherosclerosis 154: 377–385. 24. Hajishengallis G, Martin M, Schifferle RE, Genco RJ (2002) Counteracting interactions between lipopolysaccharide molecules with differential activation of toll-like receptors. Infect Immun 70: 6658–6664. 41. Yue TL, Wang X, Sung CP, Olson B, McKenna PJ, et al. (1994) Interleukin-8. A mitogen and chemoattractant for vascular smooth muscle cells. Circ Res 75: 1–7. 25. Hajishengallis G SA, Russell MW, Genco RJ (2002) Interactions of oral pathogens with toll-like receptors: possible role in atherosclerosis. Ann Period- ontol 7: 72–78. 42. Reape TJ, Groot PHE (1999) Chemokines and atherosclerosis. Atherosclerosis 147: 213–225. 26. Chi H, Messas E, Levine RA, Graves DT, Amar S (2004) Interleukin-1 receptor signaling mediates atherosclerosis associated with bacterial exposure and/or a high-fat diet in a murine apolipoprotein E heterozygote model: pharmacother- apeutic implications. Circulation 110: 1678–1685. 43. Shah PK (2000) Circulating Markers of Inflammation for Vascular Risk Prediction: Are they Ready for Prime Time. Circulation 101: 1758–1759. 44. Shin WS, Szuba A, Rockson SG (2002) The role of chemokines in human cardiovascular pathology: enhanced biological insights. Atherosclerosis 160: 91–102. 27. Madan M, Bishayi B, Hoge M, Messas E, Amar S (2007) Doxycycline affects diet- and bacteria-associated atherosclerosis in an ApoE heterozygote murine model: Cytokine profiling implications. Atherosclerosis 190: 62–72. 45. Sun HQ, Yamamoto M, Mejillano M, Yin HL (1999) Gelsolin, a Multifunc- tional Actin Regulatory Protein. J Biol Chem 274: 33179–33182. 28. Mullick AE, Tobias PS, Curtiss LK (2005) Modulation of atherosclerosis in mice by Toll-like receptor 2. J Clin Invest 115: 3149–3156. 46. Yang J, Moravec CS, Sussman MA, DiPaola NR, Fu D, et al. (2000) Decreased SLIM1 Expression and Increased Gelsolin Expression in Failing Human Hearts Measured by High-Density Oligonucleotide Arrays. Circulation 102: 3046–3052. 29. TLR-2 Mediated Atherosclerosis Atherosclerotic Plaques and Upregulated by Oxidized LDL. Circulation 104: 3103–3108. 31. O’Neill LA, Greene C (1998) Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants. J Leukoc Biol 63: 650–657. 16. Edfeldt K, Swedenborg J, Hansson GK, Yan Z-q (2002) Expression of Toll-Like Receptors in Human Atherosclerotic Lesions: A Possible Pathway for Plaque Activation. Circulation 105: 1158–1161. J 32. Chiu B (1999) Multiple infections in carotid atherosclerotic plaques. Am Heart J 138: S534–536. 17. Nakamura J, Shibata K, Hasebe A, Into T, Watanabe T, et al. (2002) Signaling pathways induced by lipoproteins derived from Mycoplasma salivarium and a synthetic lipopeptide (FSL-1) in normal human gingival fibroblasts. Microbiol Immunol 46: 151–158. 33. Madan M, Bishayi B, Hoge M, Amar S (2008) Atheroprotective role of interleukin-6 in diet- and/or pathogen-associated atherosclerosis using an ApoE heterozygote murine model. Atherosclerosis 197: 504–14. heterozygote murine model. Atherosclerosis 197: 504–14. 34. Mallat Z, Corbaz A, Scoazec A, Graber P, Alouani S, et al. (2001) Interleukin- 18/Interleukin-18 Binding Protein Signaling Modulates Atherosclerotic Lesion Development and Stability. Circ Res 89: 41e–45. 18. Okusawa T, Fujita M, Nakamura J-i, Into T, Yasuda M, et al. (2004) Relationship between Structures and Biological Activities of Mycoplasmal Diacylated Lipopeptides and Their Recognition by Toll-Like Receptors 2 and 6. Infect Immun 72: 1657–1665. 35. Virmani R, Kolodgie FD, Burke AP, Farb A, Schwartz SM (2000) Lessons From Sudden Coronary Death: A Comprehensive Morphological Classification Scheme for Atherosclerotic Lesions. Arterioscler Thromb Vasc Biol 20: 1262–1275. 19. Takeuchi O, Sato S, Horiuchi T, Hoshino K, Takeda K, et al. (2002) Cutting Edge: Role of Toll-Like Receptor 1 in Mediating Immune Response to Microbial Lipoproteins. J Immunol 169: 10–14. 36. Shiomi M, Ito T, Hirouchi Y, Enomoto M (2001) Stability of atheromatous plaque affected by lesional composition: study of WHHL rabbits treated with statins. Ann N Y Acad Sci 947: 419–423. 20. Ozinsky A, Smith KD, Hume D, Underhill DM (2000) Co-operative induction of pro-inflammatory signaling by Toll-like receptors. J Endotoxin Res 6: 393–396. 37. Kockx MM, Herman AG (2000) Apoptosis in atherosclerosis: beneficial or detrimental? Cardiovasc Res 45: 736–746. 21. Takeuchi O, Kawai T, Muhlradt PF, Morr M, Radolf JD, et al. (2001) Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol 13: 933–940. 38. Aliprantis AO, Yang RB, Weiss DS, Godowski P, Zychlinsky A (2000) The apoptotic signaling pathway activated by Toll-like receptor-2. References 10. Takeda K, Akira S (2004) Microbial recognition by Toll-like receptors. J Dermatol Sci 34: 73–82. 1. Ross R (1999) Atherosclerosis–an inflammatory disease. N Engl J Med 340: 115–126. 11. Muzio M, Natoli G, Saccani S, Levrero M, Mantovani A (1998) The Human Toll Signaling Pathway: Divergence of Nuclear Factor kappa B and JNK/SAPK Activation Upstream of Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6). J Exp Med 187: 2097–2101. 2. Libby P, Ridker PM, Maseri A (2002) Inflammation and atherosclerosis. Circulation 105: 1135–1143. 3. Prescott SM, McIntyre TM, Zimmerman GA, Stafforini DM (2002) Sol Sherry lecture in thrombosis: molecular events in acute inflammation. Arterioscler Thromb Vasc Biol 22: 727–733. 12. Faure E, Equils O, Sieling PA, Thomas L, Zhang FX, et al. (2000) Bacterial Lipopolysaccharide Activates NF-kappa B through Toll-like Receptor 4 (TLR-4) in Cultured Human Dermal Endothelial Cells. DIFFERENTIAL EXPRES- SION OF TLR-4 AND TLR-2 IN ENDOTHELIAL CELLS. J Biol Chem 275: 11058–11063. 4. Ross R (1999) Atherosclerosis is an inflammatory disease. Am Heart J 138: S419–420. 5. Libby P (2002) Inflammation in atherosclerosis. Nature 420: 868–874. 6. Medzhitov R (2001) Toll-like receptors and innate immunity. Nat Rev Immunol 1: 135–145. 7. Medhitov RHC (2000) Advances in Immunology. N Engl J Med. 8. Janeway CA Jr, Medzhitov R (2002) Innate immune recognition. Annu Rev Immunol 20: 197–216. 9. Hansson GK, Libby P, Schonbeck U, Yan Z-Q (2002) Innate and Adaptive Immunity in the Pathogenesis of Atherosclerosis. Circ Res 91: 281–291. PLoS ONE \| www.plosone.org 11 September 2008 \| Volume 3 \| Issue 9 \| e3204 TLR-2 Mediated Atherosclerosis Bjorkbacka H, Kunjathoor VV, Moore KJ, Koehn S, Ordija CM, et al. (2004) Reduced atherosclerosis in MyD88-null mice links elevated serum cholesterol levels to activation of innate immunity signaling pathways. Nat Med 10: 416–421. 47. Hausladen A, Fridovich I (1996) Measuring nitric oxide and superoxide: rate constants for aconitase reactivity. Methods Enzymol 269: 37–41. 30. Michelsen KS, Wong MH, Shah PK, Zhang W, Yano J, et al. (2004) Lack of Toll-like receptor 4 or myeloid differentiation factor 88 reduces atherosclerosis and alters plaque phenotype in mice deficient in apolipoprotein E. PNAS 101: 10679–10684. 48. Howe J, Garidel P, Roessle M, Richter W, Alexander C, et al. (2008) Structural investigations into the interaction of hemoglobin and part structures with bacterial endotoxins. Innate Immun 14: 39–49. PLoS ONE \| www.plosone.org September 2008 \| Volume 3 \| Issue 9 \| e3204 12
https://openalex.org/W2105060187	https://aab.copernicus.org/articles/48/94/2005/aab-48-94-2005.pdf	English	null	Some egg and hatching traits of local ducks, Turkish Pekins and Muscovy ducks in Isparta/Turkey (short communication)	Archives animal breeding/Archiv für Tierzucht	2,005	cc-by	4,664	Abstract The purpose of the research was to study some egg and hatching traits of Boz, Yeşilbaş, Kara ducks (as the local names) in comparison with Turkish Pekin and Muscovy ducks in intensive conditions in Isparta province of Turkey. Average egg weights of Boz, Yeşilbaş, Kara, Turkish Pekin, Black Muscovy, Black-white Muscovy, White Muscovy ducks were 68.6, 71.5, 71.0, 71.5, 73.3, 76.3, 75.5g; average percentages of fertility were 70.2, 73.0, 54.2, 78.5, 63.4, 33.5, 64.5 %, respectively. p y Black-white Muscovy ducks had the highest egg weight. Minor differences were found in the values of egg shape index and values of fertility. The percentage of hatchability of fertile eggs and embryo mortality did not differ among genotypes. The weight of day-old bird there were significant differences between the genotypes. A high weight reached Black-White Muscovy ducks with 46.2 g and the lowest weight were found in Boz genotype with 41.7 g. The results were discussed. Key Words: local duck, Pekin, Muscovy, egg, hatching, trait Titel der Arbeit: Einige Ei- und Bruteigenschaften von einheimischen Entenrassen, Türkischen Peking- und Muscovy-Enten in Isparta/Türkei (Kurzmitteilung) Zweck dieser Arbeit war, einige Ei- und Bruteigenschaften von Boz, Yeşilbaş, Kara (lokale Namen) Enten im Vergleich zu Türkisch Peking- und Muscovy-Enten in der Isparta Provinz der Türkei zu untersuchen. Die durchschnittlichen Eigewichte der Rassen Boz, Yeşilbaş, Kara, Türkisch Peking, Schwarz Muscovy, Schwarz-Weiss Muscovy und Weiss Muscovy waren 68,6, 71,5, 71,0, 71,5, 73,3, 76,3, 75,5 g; die durchschnittliche Befruchtungsrate bei diesen Rassen lag bei 70,2, 73,0, 54,2, 78,5, 63,4, 33,5, 64,5 %. Schwarz- Weiss Muscovy Enten hatten die höchsten Eigewichte. Geringe Unterschiede wurden bei dem Merkmal Eiformindex gefunden. Für die Schlupfrate der befruchteten Eier und die Embryomortalität fanden sich keine Unterschiede zwischen den Genotypen (P<0,05). Die größten Eintagskückengewichte erreichten die Black- White Muskovy mit 46,2 g, die niedrigsten die Tiere der Boz Population mit 41,2 g. Die Ergebnisse werden diskutiert. Schlüsselwörter: Lokale Entenrassen, Peking-Enten, Muscovy-Enten, Eier, Brut Zusammenfassung Titel der Arbeit: Einige Ei- und Bruteigenschaften von einheimischen Entenrassen, Türkischen Peking- und Muscovy-Enten in Isparta/Türkei (Kurzmitteilung) Arch. Tierz., Dummerstorf 48 (2005) 1, 94-100 Arch. Tierz., Dummerstorf 48 (2005) 1, 94-100 Arch. Tierz., Dummerstorf 48 (2005) 1, 94-100 Süleyman Demirel University, Faculty of Agriculture, Department of Animal Breeding, Isparta, Turkey ERGUL ISGUZAR Introduction In some regions of Turkey the local duck genotypes have been extensively raised for many years in small family farms. But, there is no information available on characteristics and performances of local duck breeds in Turkey (SELCUK and AKYURT, 1986). We have no statistical data on duck breeding potential in Turkey, it is only known that there are very few small-scale duck farms raising Pekin ducks (TESTIK, 1995). In Turkey, on this subject, the first development was in 1984 and with Pekin ducks which the number reached 46.000 ducklings (TURKOGLU, 1993). Different duck genotypes are brought by various lines from abroad to Turkey. On the other hand, the 95 Arch. Tierz. 48 (2005) 1 local duck genotypes, Turkey’s animal protein sources, are bred by the rural areas population under the extensive conditions as primitive and innocuous (ISGUZAR and TESTIK, 1999). ISGUZAR et al. (2002) reported about growth, carcass traits and meat quality of local ducks and could prove that these breeds could be important for crossbreed programs. p g The purpose of this research was to bring to light of the local duck’s some egg and hatching traits, to compare with different duck genotypes. Material and method Research materials consisted of breeder eggs of local duck genotypes (Kara, Yeşilbaş, Boz) which are widespread around Şarkikaraağaç/Isparta province, Turkish Pekin and Turkish Muscovy duck genotypes (black, black-white and white varieties). Turkish Muscovy duck genotypes (black, black white and white varieties). The local duck genotypes were determined by ISGUZAR and TESTIK (1999) of Boz, Yeşilbaş, Kara (as the local names) in Isparta region of Turkey. And then, these genotypes (three types of local duck, three types of Muscovy duck and one type Turkish Pekin duck) were bought from the duck farms in Isparta region. The genotypes were 1/3 ratio of male/female, and in hen house there were hay litter on the ground and the long plastic feeder and the water canal of 25x20 cm for only drinking water under intensive conditions. The genotypes had no artificial nesting-box and they had built the himself natural nesting into the hay litter on the ground. The breeders were only fed with a commercial layer breeding feed (pellets of 16% protein and 2850kcal/kgME) since appropriate hatching duration and storage conditions were also provided by the laying period. The breeder ducks had have ad libitum feed and water with no grazing, no access to water-pool and no meadow, ect. They were kept under intensive conditions in the hen house. Breeder ducks started to lay and each genotype group’s eggs consisted of 30-126, and identified with the different numbers according to each genotype groups and weighted by electronic weight tool before each hatching time. All egg’s length and width were measured by scale. The egg shape indexes were calculated by formula that were the egg’s width / the egg’s length x 100. Thus, egg shapes were determined. The breeder duck’s eggs were placed into pre-incubator at ones in the end of two weeks as three different hatching in the spring laying period. The hatching of fertile eggs and the mortality of embryo were determined by the finishing data of hatching and at 14 days of fertility. g g y y Statistical analyses were done with MINITAB for WINDOWS (version 10.5). MstatC was used for the test of significance between groups. The proportional values related with hatching which are fertility %, hatching of fertile egg % and embryo mortality % were transformed to angular values for variance analysis. Results Results Table 2 Average egg shape indexes in different hatching (Durchschnittlicher Eiformindex bei unterschiedlichen Brutzeiten) H A T C H I N G DUCK (1) (2) (3) GENERAL weights in different hatching (g) (Durchschnittliche Eigewichte in unterschiedlichen Brutzeiten (g)) Table 2 Average egg shape indexes in different hatching (Durchschnittlicher Eiformindex bei unterschiedlichen Brutzeiten) H A T C H I N G DUCK GENOTYPES n (1) x S X ± n (2) x S X ± n (3) x S X ± n GENERAL x S X ± Boz 95 0.77 ± 0.01a 105 0.74 ± 0.00b 71 0.75 ± 0.00a 271 0.75 ± 0.00ab Yeşilbaş 126 0.78 ± 0.01a 115 0.73 ± 0.00c 79 0.72 ± 0.00b 320 0.75 ± 0.00b Kara 76 0.74 ± 0.00bc 87 0.75 ± 0.00ab 52 0.75 ± 0.00a 215 0.75 ± 0.00b Turkish Pekin 63 0.76 ± 0.01ab 41 0.75 ± 0.00ab 25 0.75 ± 0.01a 129 0.75 ± 0.01ab Black Muscovy 31 0.76 ± 0.01ab 45 0.75 ± 0.00ab 25 0.75 ± 0.01a 101 0.75 ± 0.00ab Black-white Muscovy 44 0.76 ± 0.01ab 46 0.76 ± 0.00a 32 0.76 ± 0.00a 122 0.76 ± 0.00a White Muscovy 30 0.72 ± 0.00c 39 0.76 ± 0.00a 24 0.76 ± 0.01a 93 0.75 ± 0.01b a,b = Different letters in the same column show significant difference (P<0.05). able 2 verage egg shape indexes in different hatching (Durchschnittlicher Eiformindex bei unterschiedlichen t it ) Table 2 Average egg shape indexes in different hatching (Durchschnittlicher Eiformindex bei unterschiedlich Brutzeiten) The Tables 3 to 6 shows the results of hatching traits. The values of fertility in different hatching times at the same laying period (Table 3) show high differences between the genotypes from 33.5 % to 78.5 %. The best values reached Turkish Pekin (78.5 %), and the two local populations Boz (70.2 %) and Yesilbas (73.0 %). White Muscovy reached only 33.5 %. Results Table 1 and 2 show the means of egg weights and egg shape index of genotypes according to hatching times in the same laying period, respectively. The average egg weights of local ducks, Turkish Pekin and Muscovy ducks were 68.6 to 71.5 g respectively 71.0, the egg weight of the other races lay about these (Table 1). General average egg shape indexes was similar at all races 0.75. Only Black-White Muscovy reached 0.76 (Table 2). 96 ISGUZAR: Some egg and hatching traits of local ducks, Turkish Pekins and Muscovy ducks in Isparta/Turkey Table 1 Average egg weights in different hatching (g) (Durchschnittliche Eigewichte in unterschiedlichen Brutzeiten (g)) H A T C H I N G DUCK GENOTYPES n (1) x S X ± n (2) x S X ± n (3) x S X ± n GENERAL x S X ± Boz 95 70.3 ± 0.5d 105 67.6 ± 0.5c 71 67.8 ± 0.5c 271 68.6 ± 0.3d Yeşilbaş 126 76.3 ± 0.6a 115 69.2 ± 0.6bc 79 69.1 ± 0.8c 320 71.5 ± 2.4c Kara 76 72.2 ± 0.7bcd 87 70.3 ± 0.7b 52 70.7 ± 1.0c 215 71.0 ± 0.6c Turkish Pekin 63 73.1 ± 0.6bc 41 70.7 ± 0.9b 25 70.8 ± 1.2c 129 71.5 ± 0.8c Black Muscovy 31 71.1 ± 1.0cd 45 74.9 ± 0.6a 25 74.0 ± 0.7b 101 73.3 ± 1.2b Black-white Muscovy 44 74.1 ± 1.3ab 46 77.2 ± 0.9a 32 77.5 ± 1.0a 122 76.3 ± 1.1a White Muscovy 30 71.3 ± 1.2cd 39 77.2 ± 1.3a 24 78.0 ± 1.8a 93 75.5 ± 2.1a a,b = Different letters in the same column show significant difference (P<0.05). Results Table 2 Average egg shape indexes in different hatching (Durchschnittlicher Eiformindex bei unterschiedlichen Brutzeiten) H A T C H I N G DUCK GENOTYPES n (1) x S X ± n (2) x S X ± n (3) x S X ± n GENERAL x S X ± Boz 95 0.77 ± 0.01a 105 0.74 ± 0.00b 71 0.75 ± 0.00a 271 0.75 ± 0.00ab Yeşilbaş 126 0.78 ± 0.01a 115 0.73 ± 0.00c 79 0.72 ± 0.00b 320 0.75 ± 0.00b Kara 76 0.74 ± 0.00bc 87 0.75 ± 0.00ab 52 0.75 ± 0.00a 215 0.75 ± 0.00b Turkish Pekin 63 0.76 ± 0.01ab 41 0.75 ± 0.00ab 25 0.75 ± 0.01a 129 0.75 ± 0.01ab Black Muscovy 31 0.76 ± 0.01ab 45 0.75 ± 0.00ab 25 0.75 ± 0.01a 101 0.75 ± 0.00ab Black-white Muscovy 44 0.76 ± 0.01ab 46 0.76 ± 0.00a 32 0.76 ± 0.00a 122 0.76 ± 0.00a White Muscovy 30 0.72 ± 0.00c 39 0.76 ± 0.00a 24 0.76 ± 0.01a 93 0.75 ± 0.01b a,b = Different letters in the same column show significant difference (P<0.05). Table 1 Average egg weights in different hatching (g) (Durchschnittliche Eigewichte in unterschiedlichen Brutzeiten (g)) H A T C H I N G DUCK GENOTYPES n (1) x S X ± n (2) x S X ± n (3) x S X ± n GENERAL x S X ± Boz 95 70.3 ± 0.5d 105 67.6 ± 0.5c 71 67.8 ± 0.5c 271 68.6 ± 0.3d Yeşilbaş 126 76.3 ± 0.6a 115 69.2 ± 0.6bc 79 69.1 ± 0.8c 320 71.5 ± 2.4c Kara 76 72.2 ± 0.7bcd 87 70.3 ± 0.7b 52 70.7 ± 1.0c 215 71.0 ± 0.6c Turkish Pekin 63 73.1 ± 0.6bc 41 70.7 ± 0.9b 25 70.8 ± 1.2c 129 71.5 ± 0.8c Black Muscovy 31 71.1 ± 1.0cd 45 74.9 ± 0.6a 25 74.0 ± 0.7b 101 73.3 ± 1.2b Black-white Muscovy 44 74.1 ± 1.3ab 46 77.2 ± 0.9a 32 77.5 ± 1.0a 122 76.3 ± 1.1a White Muscovy 30 71.3 ± 1.2cd 39 77.2 ± 1.3a 24 78.0 ± 1.8a 93 75.5 ± 2.1a a,b = Different letters in the same column show significant difference (P<0.05). Table 4 Average values of the hatchability of fertile eggs in different hatching (%) (Schlupfrate befruchteter Eier bei unterschiedlichen Brutzeiten (%)) Table 5 Average values of the embryo mortality in different hatching (%) (Durchschnittliche Embryonenmortalität bei unterschiedlichen Brutzeiten (%)) Average values of the embryo mortality in different hatching (%) (Durchschnittliche Embryonenmortalität bei unterschiedlichen Brutzeiten (%)) Average values of the embryo mortality in different hatching (%) (Durchschnittliche Embryonenmortalität bei unterschiedlichen Brutzeiten (%)) H A T C H I N G DUCK GENOTYPES n (1) x S X ± n (2) x S X ± n (3) x S X ± n GENERAL x S X ± Boz 95 53.3 ± 14.2a 105 63.4 ± 10.8a 71 63.6 ± 15.5a 271 60.1 ± 7.3a Yeşilbaş 126 63.6 ± 7.1a 115 74.9 ± 5.3a 79 66.4 ± 11.4a 320 68.3 ± 4.6a Kara 76 41.7 ± 25.0a 87 58.7 ± 12.8a 52 53.7 ± 21.7a 215 51.4 ±10.9a Pekin 63 51.6 ± 20.5a 41 67.9 ± 7.4a 25 60.8 ± 9.8a 129 60.1 ± 7.5a Black Muscovy 31 58.3 ± 10.8a 45 57.9 ± 10.0a 25 60.4 ± 6.3a 101 58.9 ± 4.8a Black-white Muscovy 44 57.3 ± 20.9a 46 44.2 ± 15.1a 32 68.5 ± 11.1a 122 56.6 ± 9.0a White Muscovy 30 66.7 ± 23.6a 39 52.7 ± 12.1a 24 55.4 ± 12.8a 93 58.3 ± 9.1a a,b = Different letters in the same column show significant difference (P<0.05). Table 4 values of the hatchability of fertile eggs in different hatching (%) (Schlupfrate befruchteter Eier bei edlichen Brutzeiten (%)) Table 4 Average values of the hatchability of fertile eggs in different hatching (%) (Schlupfrate befruchteter Eier bei unterschiedlichen Brutzeiten (%)) H A T C H I N G DUCK GENOTYPES n (1) x S X ± n (2) x S X ± n (3) x S X ± n GENERAL x S X ± Boz 95 46.8 ± 14.2a 105 36.6 ± 10.8a 71 36.4 ± 15.5a 271 39.9 ± 7.3a Yeşilbaş 126 36.4 ± 7.1a 115 25.1 ± 5.3a 79 33.6 ± 11.4a 320 31.7 ± 4.6a Kara 76 58.3 ± 25.0a 87 41.3 ± 12.8a 52 46.2 ± 21.7a 215 48.6 ±10.9a Turkish Pekin 63 48.4 ± 20.5a 41 32.1 ± 7.4a 25 39.2 ± 9.8a 129 39.9 ± 7.5a Black Muscovy 31 41.7 ± 10.8a 45 42.2 ± 10.0a 25 39.6 ± 6.3a 101 41.1 ± 4.8a Black-white Muscovy 44 42.7 ± 20.9a 46 30.8 ± 10.8a 32 31.5 ± 11.1a 122 35.0 ± 8.0a White Muscovy 30 33.3 ± 23.6a 39 47.3 ± 12.1a 24 44.6 ± 12.8a 93 41.7 ± 9.1a a,b = Different letters in the same column show significant difference (P<0.05). Table 4 Table 6 Average weights of day-old birds in different hatching (g) (Durchschnittliches Gewicht der Eintagsküken bei unterschiedlichen Brutzeiten (g)) Average weights of day-old birds in different hatching (g) (Durchschnittliches Gewicht der Eintagsküken bei unterschiedlichen Brutzeiten (g)) Average weights of day-old birds in different hatching (g) (Durchschnittliches Gewicht der Eintagsküken bei unterschiedlichen Brutzeiten (g)) H A T C H I N G DUCK GENOTYPES n (1) x S X ± n (2) x S X ± n (3) x S X ± n GENERAL x S X ± Boz 95 40.6 ± 1.0b 105 42.1 ± 0.4c 71 42.3 ± 0.2bc 271 41.7 ± 0.4c Yeşilbaş 126 42.5 ± 0.2b 115 42.9 ± 1.3bc 79 42.5 ± 1.1bc 320 42.6 ± 0.5c Kara 76 43.6 ± 0.6ab 87 44.0 ± 0.1abc 52 43.9 ± 0.7ab 215 43.9 ± 0.3bc Pekin 63 44.3 ± 0.6ab 41 46.6 ± 1.2ab 25 46.4 ± 0.2a 129 45.8 ± 0.5ab Black Muscovy 31 40.3 ± 0.9b 45 42.8 ± 0.9bc 25 42.2 ± 1.0c 101 41.8 ± 0.6c Black-white Muscovy 44 47.1 ± 3.3a 46 47.7 ± 1.3a 32 43.7 ± 1.1ab 122 46.2 ± 1.2a White Muscovy 30 41.0 ± 0.4b 39 42.7 ± 2.0c 24 42.3 ± 1.9bc 93 42.0 ± 0.9c a,b = Different letters in the same column show significant difference (P<0.05). Results Table 3 Average values of fertility in different hatching (%) (Durchschnittliche Befruchtungsrate bei unterschiedlichen Brutzeiten (%)) H A T C H I N G DUCK GENOTYPES n (1) x S X ± n (2) x S X ± n (3) x S X ± n GENERAL x S X ± Boz 95 64.4 ± 13.4ab 105 76.1 ± 5.8a 71 70.0 ± 9.7ab 271 70.2 ± 5.5a Yeşilbaş 126 66.7 ± 2.8ab 115 79.1 ± 6.2a 79 73.4 ± 4.4ab 320 73.0 ± 2.9a Kara 76 67.9 ± 18.8a 87 50.5 ± 4.6ab 52 44.2 ± 3.7ab 215 54.2 ± 6.7b Turkish Pekin 63 82.8 ± 15.2a 41 75.9 ± 5.8a 25 76.8 ± 9.4a 129 78.5 ± 5.7a Black Muscovy 31 65.2 ± 13.6ab 45 64.8 ± 12.4a 25 60.1 ± 14.2ab 101 63.4 ± 7.0ab Black-white Muscovy 44 27.3 ± 16.2b 46 35.6 ± 15.3b 32 37.5 ± 18.4b 122 33.5 ± 8.8c White Muscovy 30 67.0 ± 10.5ab 39 64.2 ± 8.5ab 24 62.5 ± 8.0ab 93 64.5 ± 4.8ab a,b = Different letters in the same column show significant difference (P<0.05). Table 3 Average values of fertility in different hatching (%) (Durchschnittliche Befruchtungsrate bei unterschiedlichen Brutzeiten (%)) 3 age values of fertility in different hatching (%) (Durchschnittliche Befruchtungsrate bei unterschiedlichen eiten (%)) The values of hatchability of fertile eggs and of the embryo mortality (Table 4 and Table 5) show that there were not significant differences (P<0.05) among genotypes, 97 Arch. Tierz. 48 (2005) 1 but, there were significant differences for the day-old bird weight among the genotypes. The low and high weights reached Boz and Black-White Muscovy respectively. (Table 6) but, there were significant differences for the day-old bird weight among the genotypes. The low and high weights reached Boz and Black-White Muscovy respectively. (Table 6) Discussion Conservation of genetically different duck races, existing in small populations and threatened with extinction is required for economic, scientific and histological reasons 98 ISGUZAR: Some egg and hatching traits of local ducks, Turkish Pekins and Muscovy ducks in Isparta/Turkey (KSIAZKIEWICZ, 2003). Such flocks are being used in the duck to create new breeding or synthetic groups as well as to search for heterosis effects in commercial crossbreeds (ISGUZAR et al., 2002). (KSIAZKIEWICZ, 2003). Such flocks are being used in the duck to create new breeding or synthetic groups as well as to search for heterosis effects in commercial crossbreeds (ISGUZAR et al., 2002). The purpose of this study was to compare some reproduction traits of local ducks of different genotypes. In this way , these values of egg traits of local ducks were similar to the results of KARACA et al. (1996) who found Turkish Pekin with 69.9 g egg weight or TESTIK and SARICA (1991) with 71.5 to 74.2 g. Generally the studies related to egg weight of Pekins ducks in literature show a high variability between 61.5 to 84.4g. ISGUZAR and TESTIK (1999) found in local ducks with extensive conditions lower egg weight’s with 54.8 to 64.2 g, other authors similar results. For example Khaki Campell and Desi Ducks with 37.8 to 43.9 g (GAJENDRAN et al., 1990), Shaoxin ducks and Campbel ducks with 51.7 to 64.6 g (GE et al., 2000). Some authors reported about high egg weight’s in Muscovy populations for example with 70.6 g (NIKOLOVA and GERZILOW, 2000) respectively 84.5 g (HARUN et al., 2001). ROMBOLI et al. (1987) found in Black, White and Sepia varieties Muscovy 81.2, and 84.3 and 84.8 g respectively. In this study the results of Boz, Yesilbas and Kara were similar to Turkish Pekin’s, but expected lower to Muscovy ducks. The results had shown that there were significant differences among genotypes for egg weight, similarities between local and Pekin ducks and significant differences from those to Muscovy ducks for egg weight. A compare with literature results is difficult because many influences works. y No differences were found in the values of egg shape index in the different hatching in the same laying period. The results of fertility show that there were significant differences among genotypes. The best values reached Turkish Pekin and the two local races Boz and Yesilbas. Despite the high egg weight Black-White Muskovy obtained the lowest values. References FATTOUH, M. H.; TAG EL DIN, T. H.; GAD, H. A. M.; KHALIFAH, M. M.: Effect of force moulting method on some post moulting traits in ducks 2- Fertility and hatchability. 2nd World Waterfowl Conference, 7- 9 October, Alexandria/Egypt (2003) GAJENDRAN, K.; KOTHANDARAMAN, P.; SHERIFF, FR.: Performance of Khaki Campbell and desi ducks under intensive system of rearing in maritime monsoon type of climate. Cheiron 19 (1992) 5, 213-219; Rec. 386 of 408-CAB Abst., 1990 Performance of Khaki Campbell and desi ducks under intensive system of rearing in maritime monsoon type of climate. Cheiron 19 (1992) 5, 213-219; Rec. 386 of 408-CAB Abst., 1990 GE, S.F.; ZHAO, R.Q.; CHEN, J.; LU, L.Z.; SHEN, J.D.: Comparative study on the laying performances of Shaoxin Duck and Khaki-Campbell Duck. C Chinese Journal of Animal Science 36 (2000) 5, 22-23 Comparative study on the laying performances of Shaoxin Duck and Khaki-Campbell Duck. C Chinese Journal of Animal Science 36 (2000) 5, 22-23 ( ) HANH, D.T.; HAI, L.TH.; HUNG, D.S.; TINH, N.H.: Improving the productivity of local muskovy duck and initial production experiment of Mulard in Vietnam. 10th European Symposium on waterfowl (1995) proceeding, 31 , ; , ; , ; , Improving the productivity of local muskovy duck and initial production experiment of Mulard in Vietnam. 10th European Symposium on waterfowl (1995) proceeding, 31 p y p p g HARUN, M.A.S.; VEENEKLAAS, R.J.; VISSER, G.H.; KAMPEN, M VAN.; VAN KAMPEN, M.: Artificial incubation of Muscovy duck eggs: why some eggs hatch and others do not. Poultry Science 80 ((2001) 2, 219-224 ISGUZAR E TESTIK A HARUN, M.A.S.; VEENEKLAAS, R.J.; VISSER, G.H.; KAMPEN, M VAN.; VAN KAMPEN, M.: Artificial incubation of Muscovy duck eggs: why some eggs hatch and others do not. Poultry Science 80 ((2001) 2, 219-224 ISGUZAR, E.; TESTIK, A.: An investigation on local genotypes of waterfowl in Isparta province of Turkey. 12th European Symp. on waterfowl (1999) proceeding 51 56 ((2001) 2, 219-224 ISGUZAR, E.; TESTIK, A.: An investigation on local genotypes of waterfowl in Isparta province of Turkey. 12th European Symp. on waterfowl, (1999), proceeding, 51-56 ISGUZAR, E.; KOCAK, C.; PINGEL, H.: Growth, carcass traits and meat quality of different local ducks and Turkish Pekins. Arch. Tierz., Dummerstorf 45 (2002) 4, 413-418 ISGUZAR, E.; KOCAK, C.; PINGEL, H.: Growth, carcass traits and meat quality of different local ducks and Turkish Pekins. Arch. Discussion The literature described very various values higher and lower than in this investigation. These values were higher to the native ducks having 25.0 to 55.0 % (SELCUK and AKYRUT, 1986) and to local Moscovy ducks having 17.8 to 36.8 % (HANH et al., 1995). FATTOUH et al. (2003) reported about Muscovy ducks with fertility values of 87.8 %. The values of the hatchability of fertile eggs were similar in the literature (SELCUK and AKYURT, 1986). Other authors reported about higher values (KARAMAN and TESTIK, 1995; FATTOUH et al., 2003). Hatchability fertile eggs just like the values of embryo mortality there were not significant difference between local ducks, Turkish Pekin and Muscovy ducks. The percentage of the fertility, the hatchability of fertile eggs and the embryo mortality were relatively low (Tables 3 to 5). This is due to the fact that the breeder ducks were completely kept indoors without having access to outdoors and only fed with a commercial layer breeding feed since appropriate hatching duration and storage conditions were also provided. The breeder ducks had have no grazing, no meadow, no access to water-pool, no running ect., and they have the fresh drinking water, the clean hay litter and appropriate management in the breeder laying house under the intensive conditions. The weight of day-old birds were similar to Pekin ducks with 45.0.to 59.0 g and 45.0 g respectively (KNIZETOVA et al., 1992; SHAHIN et al., 2000) but lower than results of KARAMAN and TESTIK with 47.8 to 55.8 g. Between genotypes in this investigation there were significant differences between genotypes. High weight 99 Arch. Tierz. 48 (2005) 1 reached Black-White Muscovy ducks and the lowest were found in Boz genotype. There is certainly a positive correlation to the egg weight (KARAMAN and TESTIK, 1995). Egg and hatching traits are strongly influenced by environment therefore are compares with the literature heavily. reached Black-White Muscovy ducks and the lowest were found in Boz genotype. There is certainly a positive correlation to the egg weight (KARAMAN and TESTIK, 1995). Egg and hatching traits are strongly influenced by environment therefore are compares with the literature heavily. The aim of this study was to compare some reproductive traits of different Turkish local and other duck populations. The proved performances make possible a better judgment of this genotypes, hers conservation, further development or to create new commercial crossbreds. References Tierz., Dummerstorf 45 (2002) 4, 413-418 A ACA O O A KARACA, O.; OKUT, H.; ALTIN, T.: The estimation on some parameters of Pekin ducks’s egg productions. I. National Congrees of Zootecnical, Antalya/Turkey (1996) C , O ; O U , ; , The estimation on some parameters of Pekin ducks’s egg productions. I. National Congrees of Zootecnical, Antalya/Turkey (1996) KARAMAN M ; TESTIK A : KARAMAN, M.; TESTIK, A.: The study of effects on hatching and growth performances of Pekin ducks’s different weight eggs. International congrees of poultry husbandry, Istambul (1995) proceeding, 485-492, KNIZETOVA, H.; HYANEK, J.; CERVENY, J.: Egg size and post-hatch growth of Pekin duck. Arch. Geflügelkunde 56 (1992) 3, 128-131 KSIAZKIEWICZ, J. M.: Comparison of reproduction and carcass traits in light type of ducks of four conservative flocks over Egg size and post hatch growth of Pekin duck. Arch. Geflügelkunde 56 (1992) 3, 128 131 KSIAZKIEWICZ, J. M.: Comparison of reproduction and carcass traits in light type of ducks of four conservative flocks over eight generations. Arch. Tierz., Dummerstorf 46 (2003) 4, 377-389 , Comparison of reproduction and carcass traits in light type of ducks of four conservative flocks over eight generations. Arch. Tierz., Dummerstorf 46 (2003) 4, 377-389 g g ( ) NIKOLOVA, M.; GERZILOV, V.: Study on egg productivity of Muscovy ducks (Cairina moschata). II. Morphological characteristic. Zhivotnov"dni Nauki 37 (2000) 4, 13-16; Rec. 173 of 758 in CAB Abstracts 2000/08-2002/07. ROMBOLI, I.; GIULOTTI, L.; AVANZI, CF.: Reproductive activity of some varieties of Muscovy duck in successive egg laying cycles. Rivista di Avicoltura, 56 (1987) 12, 73-75, Rec. 201 of 408-CAB Abst. (1987-1989) SELCUK, E.; AKYURT, I.: Duck husbandry. The Ministry of Agriculture and Forestry, publication no: 8, Ankara/Turkey (1986) SHAHIN K A ; SHEMES A R ; ABDALLAH O Y ; SALEH K : SELCUK, E.; AKYURT, I.: Duck husbandry. The Ministry of Agriculture and Forestry, publication no: 8, Ankara/Turkey (1986) SHAHIN, K. A.; SHEMES, A. R.; ABDALLAH, O. Y.; SALEH, K.: Effects of genetic control of subcutaneous fat deposition via using restricted selection indexes on live SHAHIN, K. A.; SHEMES, A. R.; ABDALLAH, O. Y.; SALEH, K.: Effects of genetic control of subcutaneous fat deposition via using restricted selection indexes on live performance and carcass characteristics of Pekin ducks. Arch. Tierz., Dummerstorf. 43 (2000) 1, 69-77 SHAHIN, K. A.; SHEMES, A. R.; ABDALLAH, O. References Y.; SALEH, K.: Effects of genetic control of subcutaneous fat deposition via using restricted selection indexes on live performance and carcass characteristics of Pekin ducks. Arch. Tierz., Dummerstorf. 43 (2000) 1, 69-77 TESTIK, A.: TESTIK, A.: The situation of ducks and geese production in Turkey. 10th European Symp. on waterfowl (1995) proceeding, 43-45 TESTIK, A.: The situation of ducks and geese production in Turkey. 10th European Symp. on waterfowl (1995) proceeding, 43-45 p g TESTIK, A.; SARICA, M.: 100 ISGUZAR: Some egg and hatching traits of local ducks, Turkish Pekins and Muscovy ducks in Isparta/Turkey An investigation on the egg quality traits of Pekin ducks. International congrees of poultry husbandry, Istambul (1991) proceeding, 85-91 TURKOGLU, M.: The investigation on improving of hatching performance of Pekin ducks in Turkey. Ankara University- Agriculture Faculty, publication no: 1288, Ankara/Turkey (1993) An investigation on the egg quality traits of Pekin ducks. International congrees of poultry husbandry, Istambul (1991) proceeding, 85-91 TURKOGLU, M.: The investigation on improving of hatching performance of Pekin ducks in Turkey. Ankara University- Agriculture Faculty, publication no: 1288, Ankara/Turkey (1993) Received: 2003-04-07 Accepted: 2004-12-03 Author’s address Dr. ERGÜL İŞGÜZAR Süleyman Demirel University, Faculty of Agriculture, Depart. of Animal Breeding, Isparta-Turkey
https://openalex.org/W2888912996	http://jrdms.dentaliau.ac.ir/files/site1/user_files_d1a2ed/drarashazizi-A-10-25-1-c5e7726.pdf	English	null	Comparison of Serum Folic Acid Level in Oral Lichen Planus Patients and Healthy Subjects	Journal of research in dental and maxillofacial sciences	2,018	cc-by	3,163	Corresponding author: drarashazizi@yahoo.com J Res Dentomaxillofac Sci J Res Dentomaxillofac Sci J Res Dentomaxillofac Sci http://www.jrdms.dentaiau.ac.ir e(ISSN): 2383 -2754 p(ISSN):2588-4166 Journal of Research in Dental and Maxillofacial Sciences Comparison of Serum Folic Acid Level in Oral Lichen Planus Patients and Healthy Subjects AR Mirzaie1, M Hashemi Shahzadeh2, M Barzegari 3, A Azizi 4 1Dentist , member of dental laser research center of Hamedan university 2 Dentist 3Assistant Professor of dermatology, Tehran university of Medical Sciences,Tehran, Iran. 4Professor ,Oral Medicine Dept,Dental Branch of Tehran, Islamic Azad University, Tehran, Iran. e(ISSN): 2383 -2754 p(ISSN):2588-4166 Journal of Research in Dental and Maxillofacial Sciences 3Assistant Professor of dermatology, Tehran university of Medical Sciences,Tehran, Iran. 4Professor ,Oral Medicine Dept,Dental Branch of Tehran, Islamic Azad University, Tehran, Iran. ABSTRACT ARTICLE INFO Article Type Original Article Article History Received: Nov 2017 Accepted: Dec 2017 ePublished: Jan 2018 Keywords: Oral Lichen Planus, Folic Acid, Antioxidants Background and aim: Oral lichen planus (OLP) is a chronic mucocutaneous in- flammatory disease. Although the etiology of this disease is unknown, some factors including stress, antioxidant deficiency, and folic acid deficiency have been claimed to have a role in its incidence. The purpose of this study was to assess and compare the serum folic acid level in OLP patients and healthy subjects. Materials and methods: in this case-control study, 52 OLP patients (36 females and 16 males) were selected as the case group, and 48 healthy individuals (36 females and 12 males) were assigned to the control group. The serum folic acid level was meas- ured in the two groups. Data were analyzed using T-test with the level of significance set at 0.05. Results: the mean and standard deviation (SD) of serum folic acid level was equal to 8.74±4.11 ng/ml in the case group and 9.12±3.5 ng/ml in the control group with no statistically significant difference (P=0.12). Conclusion: The results of the present study indicated that the serum folic acid level in OLP patients did not have a significant difference with that in healthy subjects. ABSTRACT ARTICLE INFO Article Type Original Article Article History Received: Nov 2017 Accepted: Dec 2017 ePublished: Jan 2018 Keywords: Oral Lichen Planus, Folic Acid, Antioxidants Background and aim: Oral lichen planus (OLP) is a chronic mucocutaneous in- flammatory disease. Although the etiology of this disease is unknown, some factors including stress, antioxidant deficiency, and folic acid deficiency have been claimed to have a role in its incidence. J Res Dentomaxillofac Sci The purpose of this study was to assess and compare the serum folic acid level in OLP patients and healthy subjects. Materials and methods: in this case-control study, 52 OLP patients (36 females and 16 males) were selected as the case group, and 48 healthy individuals (36 females and 12 males) were assigned to the control group. The serum folic acid level was meas- ured in the two groups. Data were analyzed using T-test with the level of significance set at 0.05. Results: the mean and standard deviation (SD) of serum folic acid level was equal to 8.74±4.11 ng/ml in the case group and 9.12±3.5 ng/ml in the control group with no statistically significant difference (P=0.12). Conclusion: The results of the present study indicated that the serum folic acid level in OLP patients did not have a significant difference with that in healthy subjects. ABSTRACT ARTICLE INFO Article Type Original Article Article History Received: Nov 2017 Accepted: Dec 2017 ePublished: Jan 2018 Keywords: Oral Lichen Planus, Folic Acid, Antioxidants Background and aim: Oral lichen planus (OLP) is a chronic mucocutaneous in- flammatory disease. Although the etiology of this disease is unknown, some factors including stress, antioxidant deficiency, and folic acid deficiency have been claimed to have a role in its incidence. The purpose of this study was to assess and compare the serum folic acid level in OLP patients and healthy subjects. Materials and methods: in this case-control study, 52 OLP patients (36 females and 16 males) were selected as the case group, and 48 healthy individuals (36 females and 12 males) were assigned to the control group. The serum folic acid level was meas- ured in the two groups. Data were analyzed using T-test with the level of significance set at 0.05. Results: the mean and standard deviation (SD) of serum folic acid level was equal to 8.74±4.11 ng/ml in the case group and 9.12±3.5 ng/ml in the control group with no statistically significant difference (P=0.12). Conclusion: The results of the present study indicated that the serum folic acid level in OLP patients did not have a significant difference with that in healthy subjects. ABSTRACT ARTICLE INFO Article Type Original Article Article History Received: Nov 2017 Accepted: Dec 2017 ePublished: Jan 2018 Keywords: Oral Lichen Planus, Folic Acid Background and aim: Oral lichen planus (OLP) is a chronic mucocutaneous in- flammatory disease. J Res Dentomaxillofac Sci Although the etiology of this disease is unknown, some factors including stress, antioxidant deficiency, and folic acid deficiency have been claimed to have a role in its incidence. The purpose of this study was to assess and compare the serum folic acid level in OLP patients and healthy subjects. Materials and methods: in this case-control study, 52 OLP patients (36 females and 16 males) were selected as the case group, and 48 healthy individuals (36 females and 12 males) were assigned to the control group. The serum folic acid level was meas- d i h l d i i h h l l f i ifi ABSTRACT ARTICLE INFO Article Type Original Article Article History Received: Nov 2017 Accepted: Dec 2017 ePublished: Jan 2018 Background and aim: Oral lichen planus (OLP) is a chronic mucocutaneous in- flammatory disease. Although the etiology of this disease is unknown, some factors including stress, antioxidant deficiency, and folic acid deficiency have been claimed to have a role in its incidence. The purpose of this study was to assess and compare the serum folic acid level in OLP patients and healthy subjects. Original Article Materials and methods: in this case-control study, 52 OLP patients (36 females and 16 males) were selected as the case group, and 48 healthy individuals (36 females and 12 males) were assigned to the control group. The serum folic acid level was meas- ured in the two groups. Data were analyzed using T-test with the level of significance set at 0.05. Results: the mean and standard deviation (SD) of serum folic acid level was equal to 8.74±4.11 ng/ml in the case group and 9.12±3.5 ng/ml in the control group with no statistically significant difference (P=0.12). Conclusion: The results of the present study indicated that the serum folic acid level in OLP patients did not have a significant difference with that in healthy subjects. Introduction: as the case group, while 48 healthy individuals (36 females and 12 males) were assigned to the control group. Oral lichen planus (OLP) is a chronic mucocu- taneous disease with reticular, popular, atrophic/ erosive, plaque-like, and bullous subtypes.(1) After taking biopsies from the lesions that clinically resembled OLP lesions, the patients without any signs of dysplasia were included in the case group. The exclusion criteria comprised dysplasia in the histological view, pregnancy, immune disorders, lupus erythematosus, hema- tologic diseases, systemic diseases, lack of con- sumption of food supplements such as folic acid by the patients during the last three months, and lichenoid reactions associated with medications or adjacent to dental fillings.(1) This study has been approved by the ethics committee of Islamic Azad University of Tehran (IRCT2014080416090N5). The control group comprised 48 healthy indi- viduals (36 females and 12 males) who had re- ferred to the oral medicine department for peri- odic examinations and did not have any of the exclusion criteria described for the case group. The participants in the case and control groups were matched according to age and gender. The stages of the study were thoroughly explained to the participants, and 5-ml fasting blood samples were collected from both groups after receiving informed consent forms. To assess the serum folic acid level, the samples were transferred to the laboratory of Razi Hospital, Tehran, Iran, for enzyme immunoassay. This method is based on the competition between serum folate and a la- beled folate for a binding protein. This competi- tion between serum folate and the 125I-labeled folate is for binding to the specific protein recep- tor; therefore, the level of binding of folic acid to the labeled folate is inversely proportional to its concentration in the sample.(5) The data were reg- istered in datasheets and were compared between the two groups using T-test at the significance level of P<0.05 in SPSS 18 software (IBM Co., Chicago, IL, USA). Burning sensation and pain are common in atrophic/erosive OLP patients and require treat- ment. Introduction: Although the etiology of this disease is un- known, during the recent years, the role of the immune system in its incidence has been con- firmed.(2) Recently, other factors including stress, antioxidant deficiency, and the deficiency of mi- cronutrients such as folic acid have been claimed to have a role in the etiology of OLP.(3) Folic acid belongs to the group of B vitamins and is neces- sary for cell growth and body metabolism. The word “folate” refers to the folic acid found in natural resources. Folic acid is vital to a healthy nervous system, blood cells, and stem cells. Folic acid protects the body against heart failure, congenital diseases, cancer, and osteomalacia, and is necessary for cell growth and body metabolism.(4) Moreover, folic acid deficiency causes anemia. The mecha- nism of action of folic acid is the production of protein in the body and construction of nucleic ac- ids. The complications associated with long-term folic acid deficiency include anemia, redness and swelling of the tongue, diarrhea, psychological disorders such as depression, and reduced growth in children.(5) Researchers have recently focused on the folic acid deficiency in the blood serum of OLP patients. In one study, a folic acid deficiency was observed in OLP patients,(6) while in another study, this condition was detected in 0.3% of the patients, which was not significant.(7) Consider- ing some shortcomings in this respect and the controversial findings of the previous studies, in this research, we assessed the level of folic acid in OLP patients referring to the dental branch of Islamic Azad University of Tehran and compared it to that in healthy subjects. http://www.jrdms.dentaliau.ac.ir J Res Dent Maxillofac Sci , Vol 3, No 1, Winter 2018 12 Corresponding author: drarashazizi@yahoo.com Please cite this paper as: Mirzaie AR, Hashemi Shahzadeh M, Barzegari M, Azizi A. Comparison of Serum Folic Acid Level in Oral Lichen Planus Patients and Healthy Subjects. J Res Dent Maxillofac Sci. 2018;3(1):12-15. Please cite this paper as: Mirzaie AR, Hashemi Shahzadeh M, Barzegari M, Azizi A. Comparison of Serum Folic Acid Level in Oral Lichen Planus Patients and Healthy Subjects. J Res Dent Maxillofac Sci. 2018;3(1):12-15. Corresponding author: drarashazizi@yahoo.com Serum Folic Acid Level in Oral Lichen Planus Patients and Healthy Subjects http://www.jrdms.dentaliau.ac.ir Journal of Research in Dental and Maxillofacial Sciences, Vol 3, No 1, Winter 2018 Materials and Methods: This case-control study was conducted at the oral medicine department of the dental branch of Islamic Azad University of Tehran. 52 OLP pa- tients (36 females and 16 males) were selected 13 Journal of Research in Dental and Maxillofacial Sciences, Vol 3, No 1, Winter 2018 AR Mirzaie ,et al, Conclusion: The results of the present study indicated that the serum folic acid level in OLP patients did not have a significant difference with that in healthy subjects. Table 1: Patient information cc c Female Male Age (year) Groups 36 16 42.4±3.8 Case 36 12 43.5±2.5 Control LP is often associated with immunological reactions that microscopically mimic hyper- sensitivity reactions. Cell-mediated immune responses degenerate the basal layer of the epi- thelium. The histopathological characteristic of LP comprises the attraction of the lymphocytes to the epithelium-connective tissue interface. (12) Considering the immunologic course of this disease, it seems that folic acid alone does not have a role in the incidence of LP but has a therapeutic role according to the study by Nos- ratzehi.(8) Folic acid reduces the symptoms of LP through the regulation of the activity of the immune system.(13,14) Reticular mucosal involvement was detect- ed in 44% of the patients, followed by the ero- sive subtype (30%), the popular subtype (16%), the plaque-like subtype (8%), and the bullous subtype (2%). Discussion: The purpose of the present study was to compare the level of folic acid in OLP patients with that in healthy subjects. Numerous factors have been considered as the etiology of LP. To date, few studied have been performed on the comparison of serum folic acid in OLP patients and healthy subjects; therefore, the present study is of special value. The results of our study indicated that the se- rum folic acid level in OLP patients did not sig- nificantly differ from that in healthy subjects. This finding is contrary to the results reported by Nosratzehi et al.(6) In an overview study, Nosratzehi showed that folic acid is effective in the treatment of LP.(8) The probable reason for the difference between the results of our study and those of the previous articles can be attrib- uted to the probable differences in the diet of the participants. It can also be attributed to the method of folic acid measurement; for a pre- Aknowledgement: This article is based on General Dentistry The- sis ,No: 11087. Also, we are thankful to Mr. Naser Valaei regarding to his cooperation in preparing of statistical analysis. Result: cise measurement, it is recommended to assess the level of folic acid in red blood cells. In the overview study by Nosratzehi, it has been stat- ed that consumption of folic acid reduces the symptoms of LP; therefore, it may be conclud- ed that vitamins have a therapeutic role rather than an etiologic role.(8) The results of the pre- sent study are similar to those reported by Chi- ang et al as they stated that folic acid deficiency was observed in 0.3% of the patients and is not a common finding.(7) Also, folic acid deficiency has been proposed as one of the risk factors of depression and psychological stresses.(9) Since stress is one of the factors that can cause LP, (10) folic acid deficiency may be associated with this disease. Although the main cause of LP is unclear, it has been understood that T-cell infil- tration (CD4 and particularly CD8) at the epi- thelium-connective tissue interface is the main cause of this disease.(11) The mean and standard deviation (SD) of the age was equal to 42.4±3.8 years in the case group and 43.5±2.5 years in the control group with no statistically significant difference (P=0.4). The mean and SD of serum folic acid level was equal to 8.74±4.11 ng/ml in the case group and 9.12±3.5 ng/ml in the control group with no statistically significant difference (P=0.12). There were no statistically significant differ- ences between the two groups in respect to the gender (P=0.9). Patient information is present- ed in Table 1. J Res Dent Maxillofac Sci , Vol 3, No 1, Winter 2018 14 http://www.jrdms.dentaliau.ac.ir References: 1- Jones KB, Jordan R. White lesions in the oral cav- ity: clinical presentation, diagnosis, and treatment. Semin Cutan Med Surg. 2015 Dec;34(4):161-70. J Res Dent Maxillofac Sci , Vol 3, No 1, Winter 2018 14 http://www.jrdms.dentaliau.ac.ir 2- Lu R, Zhang J, Sun W, Du G, Zhou G. Inflam- mation-related cytokines in oral lichen planus: an overview. J Oral Pathol Med. 2015 Jan;44(1):1-14. 14- Patearroyo T, Ubeda N, Montero A, Achon M, Varela-Moreiras G. Vitamin B(12) and folic acid imbalance modifies NK cy- totoxicity, lymphocytes B and lymphopro- lipheration in aged rats. Nutrients. 2013 Nov 26;5(12):4836-48. 3- Azizi A, Farshchi F. Comparison of salivary and plasma antioxidant levels in lichen planus patients and healthy subjects. J Oral Pathol Med. 2012 Aug;41(7):524-6. 4- Morris MS, Jacques PF, Rosenberg IH, Selhub J. Folate and vitamin B-12 status in relation to anemia, macrocytosis, and cognitive impairment in older Americans in the age of folic acid fortification. Am J Clin Nutr. 2007 Jan;85(1):193-200. 5- Chang JY, Chen IC, Wang YP, Wu YH, Chen HM, Sun A. Anemia and hematinic deficiencies in gastric parietal cell antibody-positive and antibody- negative erosive oral lichen planus patients with thyroid antibody positivity. J Formos Med Assoc. 2016 Nov;115(11):1004-11. 6- Nosratzehi T, Ansari-Moghaddam A, Arbabi-Ka- lati F, Maleki L, Amiriyan A. Effect of the serum vi- tamin B12 and folic acid levels in oral lichen planus. J Res Dent Sci. 2013;9(4):187-91. 7- Chiang CP, Yu-Fong Chang J, Wang YP, Wu YH, Lu SY, Sun A. Oral lichen planus - Differential diag- noses, serum autoantibodies, hematinic deficiencies, and management. J Formos Med Assoc. 2018 Feb 19. pii: S0929-6646(18)30052-4. 7- Chiang CP, Yu-Fong Chang J, Wang YP, Wu YH, Lu SY, Sun A. Oral lichen planus - Differential diag- noses, serum autoantibodies, hematinic deficiencies, and management. J Formos Med Assoc. 2018 Feb 19. pii: S0929-6646(18)30052-4. 8- Nosratzehi T. Oral lichen planus: an overview of potential risk factors, biomarkers and treatments. Asian Pac J Cancer Prev. 2018 May 26;19(5):1161- 67. p ( ) 8- Nosratzehi T. Oral lichen planus: an overview of potential risk factors, biomarkers and treatments. Asian Pac J Cancer Prev. 2018 May 26;19(5):1161- 67. 9- Kemse NG, Kale AA, Joshi SR. A combined sup- plementation of omega-3 fatty acids and micronutri- ents (folic acid, vitamin B12) reduces oxidative stress markers in a rat model of pregnancy induced hyper- tension. PloS One. 2014 Nov18;9(11):e111902. 10- Kalkur C, Sattur AP, Guttal KS. Journal of Research in Dental and Maxillofacial Sciences, Vol 3, No 1, Winter 2018 References: Role of depres- sion, anxiety and stress in patients with oral lichen planus: a pilot study. Indian J Dermatol. 2015 Sep- Oct;60(5):445-9. 11- Azizi A, Lawaf S. The comparison of efficacy of adcortyl ointment and topical tacrolimus in treat- ment of erosive oral lichen planus. J Dent Res Dent Clin Dent Prospects. 2007 Fall;1(3):99-102.l 12-Vered M, Furth E, Shaley Y, Dayan D. Inflamma- tory cells of immunosuppressive phenotypes in oral lichen planus have a proinflammatory pattern of ex- pression and are associated with clinical parameters. Clin Oral Investig. 2013 Jun;17(5):1365-73. g ( ) 13- Li S, Zhi L, Liu Y, Shen J, Liu L, Yao J, et al. Effect of in ovo feeding of folic acid on the folate metabolism, immune function and epigenetic modi- fication of immune effector molecules of broiler. Br J Nutr. 2016 Feb 14;115(3):411-21. g ( ) 13- Li S, Zhi L, Liu Y, Shen J, Liu L, Yao J, et al. Effect of in ovo feeding of folic acid on the folate metabolism, immune function and epigenetic modi- fication of immune effector molecules of broiler. Br J Nutr. 2016 Feb 14;115(3):411-21. 13- Li S, Zhi L, Liu Y, Shen J, Liu L, Yao J, et al. Effect of in ovo feeding of folic acid on the folate metabolism, immune function and epigenetic modi- fication of immune effector molecules of broiler. Br J Nutr. 2016 Feb 14;115(3):411-21. 15 http://www.jrdms.dentaliau.ac.ir http://www.jrdms.dentaliau.ac.ir
https://openalex.org/W3093248111	https://bmcpulmmed.biomedcentral.com/track/pdf/10.1186/s12890-020-01301-9	English	null	Impact of pulmonary rehabilitation on patients’ health care needs and asthma control: a quasi-experimental study	BMC pulmonary medicine	2,020	cc-by	8,353	Salandi et al. BMC Pulmonary Medicine (2020) 20:267 https://doi.org/10.1186/s12890-020-01301-9 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 https://doi.org/10.1186/s12890-020-01301-9 Open Access Impact of pulmonary rehabilitation on patients’ health care needs and asthma control: a quasi-experimental study Julia Salandi1* , Andrea Icks2,3, Jalal Gholami4, Stefan Hummel5, Konrad Schultz6, Christian Apfelbacher7, Aziz Sheikh8 and Adrian Loerbroks1 Abstract T The Creative Commons Public Domain Dedicati data made available in this article, unless otherw * Correspondence: julia.schreitmueller@uni-duesseldorf.de 1Institute of Occupational, Social, and Environmental Medicine, Centre for Health and Society, Medical Faculty, University of Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany Full list of author information is available at the end of the article * Correspondence: julia.schreitmueller@uni-duesseldorf.de 1Institute of Occupational, Social, and Environmental Medicine, Centre for Health and Society, Medical Faculty, University of Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany Full list of author information is available at the end of the article * Correspondence: julia.schreitmueller@uni-duesseldorf.de 1Institute of Occupational, Social, and Environmental Medicine, Centre for Health and Society, Medical Faculty, University of Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany Full list of author information is available at the end of the article Abstract Background: Pulmonary rehabilitation offers potential benefits to people with asthma. It is however unknown if rehabilitation favourably affects patients’ health care needs. We therefore examined if rehabilitation reduced needs and, in addition, if it improved asthma control. Methods: One hundred fifty patients with asthma were surveyed in three rehabilitation clinics at admission and at discharge. Additionally, we surveyed 78 participants with asthma twice 4 weeks apart. The latter sample (i.e. the control group) was recruited through other pathways than rehabilitation clinics. The Patient Needs in Asthma Treatment (NEAT) questionnaire and the Asthma Control Test (ACT) were completed at baseline and follow-up. Differences between baseline and follow-up and between rehabilitation and control group were examined by t- tests and chi-squared-tests. Univariate ANCOVAS were used to examine if NEAT and ACT follow-up scores differed significantly between groups. Within the rehabilitation group, linear regressions were used to examine if self- reported utilization of more interventions that addressed needs were associated with NEAT scores at follow-up. Results: At baseline, there were no differences between the rehabilitation and the control group regarding needs and asthma control. At follow-up, the rehabilitation group showed reduced needs (t(149) = 10.33, p < 0.01) and increased asthma control (t(130) = -6.67, p < 0.01), whereas members of the control group exhibited no changes. Univariate ANCOVAS showed that unmet follow-up needs (F(1, 212) = 36.46, p < 0.001) and follow-up asthma control (F(1, 195) = 6.97, p = 0.009) differed significantly between groups. In the rehabilitation group, self-reported utilization of more interventions was associated with reduced needs (β = 0.21; p = 0.03). Conclusions: This study provides preliminary evidence suggestion that pulmonary rehabilitation in adults with asthma may reduce asthma-related needs and confirms previous findings that rehabilitation may improve asthma control. Keywords: Asthma, Needs, Patient-reported outcome measures, Rehabilitation Keywords: Asthma, Needs, Patient-reported outcome measures, Rehabilitation Keywords: Asthma, Needs, Patient-reported outcome measures, Rehabilitation © The Author(s). 2020 Open Access This article which permits use, sharing, adaptation, distribut appropriate credit to the original author(s) and changes were made. The images or other third licence, unless indicated otherwise in a credit lin licence and your intended use is not permitted permission directly from the copyright holder. Background those needs could facilitate planning rehabilitation goals. Closely related to this, research has yet not examined if patients’ health care needs are being reduced during re- habilitation. To measure these needs we have previously developed and validated the so-called Patient Needs in Asthma Treatment (NEAT) questionnaire [20, 23]. The NEAT assesses needs related to knowledge regarding asthma pathophysiology, medication, and emergency plans, as well as needs to train breathing techniques or the correct use of the inhaler and communication with health care staff and family or friends. Given the close match of issues covered by both pulmonary rehabilita- tion and the NEAT, it seems plausible that existing health care needs as measured by the NEAT may be ad- dressed and favourably modified during pulmonary re- habilitation. Our previous research suggested that health care needs do not change in the short-term (i.e. 4 weeks) without intervention [20]. We hypothesized for the current study that participation in a specific intervention (i.e. inpatient rehabilitation) would help to reduce pa- tients’ health care needs. Further, we hypothesized that pulmonary rehabilitation will enhance asthma control, as suggested by prior studies [17, 18]. Pulmonary rehabilitation is considered a key factor in the successful long-term care of patients suffering from asthma. This holds particularly true whenever patients experience adverse physical, social or psychological con- sequences due to their asthma despite access to and availability of adequate outpatient medical care [1]. Pul- monary rehabilitation has been conceptualized as a set of multidisciplinary interventions to reduce symptoms, stabilize or improve patients’ health status and to in- crease participation in their health care planning. In this context, the main goals of pulmonary rehabilitation are to attain the best possible level of daily functioning and to enable participation in social and professional life des- pite the presence of a chronic disease [2]. This can be achieved by facilitating patients’ coping with and self- management of the bio-psycho-social sequels of their condition (such as anxiety or reduced physical activity), which are not effectively treated by medication [1, 2]. Guidelines on pulmonary rehabilitation [3–6] and re- search findings [7, 8] highlight the importance of individu- ally tailored health care programmes for patients with asthma [9]. Essential components of pulmonary rehabilita- tion are the diagnosis, optimization of pharmacological treatment regimes, avoidance of allergen exposure, compre- hensive patient education programs, physical training, re- spiratory physiotherapy and psychological interventions [10, 11]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Page 2 of 10 Page 2 of 10 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Background Nonetheless, in rehabilitation practice the focus of treatment varies depending on individual therapeutic goals, which are planned and determined individually for and with every person undergoing rehabilitation [12]. In the era of precision medicine and in order to reduce the already mentioned bio-psycho-social sequeles of asthma, the effect- iveness of pulmonary rehabilitation should not only be measured in terms of physiological outcomes [4, 13] or by the use of health care services [4, 14], but also by patient- reported outcomes (PROs), such as asthma-specific quality of life [4, 13, 15–17] and self-reported asthma control [17, 18]. Research on the effectiveness of pulmonary rehabilita- tion has so far focused mainly on people with chronic ob- structive pulmonary disease (COPD), and the existing research in people with asthma is limited both in terms of medical outcomes and PROs [19]. Methods In order to examine if pulmonary rehabilitation can im- prove patients’ health care needs and asthma control, we collected data among two samples. First, we included pa- tients who participated in an inpatient rehabilitation with specific asthma-related interventions (rehabilitation group). Second, to facilitate a comparison, we included a control group. Asthma education/training We examined to what extent participants in the rehabili- tation group received specific asthma treatment or sup- port interventions during rehabilitation that may be assumed to exert effects on specific domains on health care needs. Therefore, we asked for asthma education and training that reflected needs, surveyed in the NEAT questionnaire. In detail, we asked rehabilitation group participants the following question: “What content was conveyed during asthma education?” Response options were based on a tick-off list, e.g. “information about how to take my asthma drugs” or “practical exercise of breathing techniques, which can help during asthma at- tack”. We collected this data to be able to examine if (specific) PR interventions were associated with reduced needs in a dose-response fashion. Sample 1 – rehabilitation group Between August 2018 and January 2019, we carried out a survey among 150 people undergoing pulmonary re- habilitation in three rehabilitation clinics in Germany, all of which adopted a bio-psycho-social approach with in- dividualized interventions (i.e. selection of treatment ac- cording to patients’ needs). All people were at least 18 years old, German speaking, and had physician- diagnosed asthma, but did not have COPD. Participants were recruited through attending physicians during ad- mission and, if inclusion criteria were met, were asked to complete the survey (by paper-and-pencil) at admis- sion (before the treatment period) and at discharge (after the treatment period, thus at the end of rehabilitation). This means that the time between baseline and follow- up differed between participants, depending on the dur- ation of rehabilitation. All patients filled in the question- naire at the respective rehabilitation clinic. Participants in the rehabilitation group received the usual scope of Patient-centeredness in asthma care also includes con- sideration of patients’ health care needs. Addressing un- met health care needs may improve patients’ adherence and health outcomes such as asthma control [7, 20]. In this context, the term need should not be understood as an objective need or a need recommended by guidelines or the physician (i.e. normative need), but instead as a perceived health care need that is felt by a given patient and remains unmet (e.g. information or support needs) [21, 22]. To date, no standardized screening of health care needs is used in asthma care. However, addressing Page 3 of 10 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 care that is common in the respective rehabilitation clinic. Sample 2 – control group Between January and July 2018, we carried out a survey among 78 adults (≥18 years) who reported physician- diagnosed asthma. Participants were recruited by various pathways, e.g. by support groups or pulmonary sport groups, and were asked to complete the questionnaire twice (either web-based or by paper-and-pencil) 4 weeks apart. The participants did not have to meet any asthma-related inclusion criteria (e.g. being stable) and could fill in the questionnaire at home. The data from this sample has already been published elsewhere [20] in the context of assessing the reproducibility of the NEAT (i.e. test-retest reliability). For the present study, that sample was considered as control group which allows for the comparison of the NEAT and Asthma Control Test (ACT) scores between patients who received a spe- cific asthma intervention (i.e. pulmonary rehabilitation) and patients who likely did not. Demographics d d In order to describe and compare both samples appro- priately, we present baseline data on gender, age, school education, reported allergy (based on a tick-off list) and follow-up time. Whereas health care needs, asthma control and demo- graphic information were gathered in the rehabilitation and the control group, questions on specific asthma edu- cation and training during rehabilitation were only asked in the rehabilitation group. Patients’ health care needs Unmet needs were captured by the NEAT questionnaire [23, 24]. This 13-item instrument measured needs by four subscales (i.e. consideration of patient expertise by physicians [4 items]; information on drug effects [3 items]; information and training related to handling of drugs [3 items]; responding to exacerbations [3 items]). Items were phrased as questions with three response op- tions: “Yes, I would like this”, “This need has already been met”; and “No, I do not need this” [23]. In line with our previous work [20, 23–25], we coded responses as 1 = yes versus 0 = no (“This need hast already been met”/ “I do not need this”). We calculated mean scores across all items with higher scores reflecting more unmet needs. Details on the NEAT’s adequate psychometric properties can be found elsewhere [20, 23]. Statistical analyses ll Initially, comparisons between rehabilitation and control group regarding demographics as well as NEAT and ACT scores were carried out by unpaired t-tests (age, follow-up period, NEAT and ACT scores) and chi- squared-tests (gender, school education, reported aller- gies) to examine if the samples’ measured characteristics were sufficiently comparable. In both samples, we examined the potential changes of the NEAT total scores and subscale-specific scores by pre- post-score comparisons and tested for statistical signifi- cance based on paired t-tests. In addition, we examined the respective potential changes of the ACT sum scores also by running paired t-tests. We hypothesized that the NEAT scores would decline (i.e. indicating reduced needs) and that the ACT scores would increase (i.e. reflecting better asthma control) during the follow-up period only in the rehabilitation, but not in the control group. Asthma control Asthma control In addition to health care needs, we assessed asthma control, measured by the well-established Asthma Con- trol Test (ACT). The ACT is a 5-item instrument asses- sing asthma symptoms, use of rescue medications, and the effect of asthma on daily functioning (e.g. “In the past 4 weeks, how much of the time did your asthma keep you from getting as much done at work, school or at home?”). The ACT’s potential score ranges from 5 (very poorly controlled) to 25 (completely controlled) with higher scores indicating better control. A score of 19 or less has been defined as a cut-off score suggesting poor control [26]. Furthermore, we ran univariate ANCOVAS with group membership (1 = rehabilitation, 2 = control) as independ- ent variable and NEAT and ACT follow-up scores as dependent variables. To consider the potentially dissimi- lar demographic characteristics of the rehabilitation and the control group, we included gender, age, educational level, number of allergies, follow-up period and the re- spective baseline score (NEAT or ACT) as covariates. Page 4 of 10 Page 4 of 10 Page 4 of 10 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Finally, to examine if pulmonary interventions were as- sociated with reduced health care needs in a dose- response fashion, we ran linear regression models with the total sum score of self-reported received asthma education/training during rehabilitation that addressed needs as independent variable and the NEAT total change score as dependent variable. Furthermore, we calculated subscale-specific scores capturing the type of received asthma education/training, which reflect subscale-specific topics of the NEAT. For instance, if breathing techniques and correct use of the inhaler were taught to a given patient, a reduction of the correspond- ing NEAT score on the subscale exacerbations was ex- pected; however, if a patient reported not to have learned about patient-physician communication, a re- duction of the NEAT patient expertise subscale score was not to be expected. Linear regressions were con- trolled for gender, age, educational level, duration of stay as well as for a variable reflecting the three different re- habilitation clinics. 93%). Asthma control We excluded an additional 16 participants either because they completed the baseline questionnaire more than 3 days after admission and had therefore possibly received some interventions prior to that or because they filled out the follow-up questionnaire more than 3 days before discharge and interventions were thus potentially still pending. The final sample for our analysis com- prised thus 150 participants. In the control group, 112 people with asthma partici- pated in the first assessment and 78 of those provided data on both occasions (response rate = 67%). Table 1 shows characteristics of the population of sample 1 (rehabilitation) and sample 2 (control). In both samples, more women than men were surveyed (re- habilitation: 59%, control: 83%). In the rehabilitation group, participants were, on average, in their early-to- mid-fifties and had attained more frequently intermedi- ate levels of school education. In the control group, pa- tients were in their mid-forties and, on average, 8 years younger than participants in the rehabilitation group. In the latter sample, participants had achieved mostly inter- mediate or high levels of school education. The mean follow-up period in the rehabilitation group was about 23 days (standard deviation [SD] = 5.00, Min = 17, Max = 51) and in the control group about 31 days (SD = 5.25, Min = 25, Max = 48). g e Reports of allergy to either foods, pollen, dust mites, fur, insect venom, drugs or allergic contact eczema in response to cosmetics, l f Defined as a score ≥20 on the Asthma Control Test g b ACT Asthma Control Test (score range: 5–25) c Comparison between sample 1 and 2 regarding baseline demographics and NEAT and ACT scores, carried out by unpaired t tests (age, follow-up period, NEAT and ACT scores) and chi-squared-tests (gender, school education, reported allergies) d High = general qualification for university entrance (“Abitur”) or entrance qualification limited to universities of applied sciences (“Fachhochschulreife”); intermediate = secondary school level I certificate (“Mittlere Reife”); low = secondary modern school qualification (“Haupt−/Volksschulabschluss”) or no formal school degree e Reports of allergy to either foods, pollen, dust mites, fur, insect venom, drugs or allergic contact eczema in response to cosmetics, lotions and alike f Defined as a score ≥20 on the Asthma Control Test a NEAT Needs in Asthma Treatment Questionnaire (score range: 0–13) b Demographics In the rehabilitation group, 178 participants completed the baseline questionnaire at admission and 166 filled in the follow-up questionnaire at discharge (response rate = Table 1 Characteristics of the study populations and comparison between sample 1 and 2 regarding baseline demographics and NEAT a and ACT b scores Variables Sample 1 –rehabilitation group n = 150 Sample 2 - control group n = 78 p c Female Gender, n (%) 88 (58.67) 65 (83.33) < 0.001 Age (years), mean (SD) 52.87 (11.50) 45.26 (12.19) < 0.001 School education d, n (%) < 0.001 Low 46 (30.67) 11 (14.10) Middle 67 (44.67) 31 (39.74) High 37 (24.67) 34 (43.59) Any allergy e, n (%) 117 (78.00) 70 (89.74) 0.05 NEAT a total score baseline, mean (SD) 5.73 (3.39) 5.72 (3.93) 0.99 NEAT a total score follow-up, mean (SD) 3.13 (2.94) 5.93 (3.77) < 0.001 ACT b total score baseline, mean (SD) 17.39 (4.61) 16.99 (4.81) 0.42 ACT b total score follow-up, mean (SD) 19.55 (4.17) 17.56 (5.36) 0.01 Adequate asthma control at baseline f, n (%) 52/139 (34.66) 28/78 (35.90) 0.94 Adequate asthma control at follow-up f, n (%) 84/139 (56.00) 34/78 (43.59) 0.03 Follow-up period, mean days (SD) 22.83 (5.00) 30.85 (5.25) < 0.001 a NEAT Needs in Asthma Treatment Questionnaire (score range: 0–13) b ACT Asthma Control Test (score range: 5–25) c Comparison between sample 1 and 2 regarding baseline demographics and NEAT and ACT scores, carried out by unpaired t tests (age, follow-up period, NEAT and ACT scores) and chi-squared-tests (gender, school education, reported allergies) d High = general qualification for university entrance (“Abitur”) or entrance qualification limited to universities of applied sciences (“Fachhochschulreife”); intermediate = secondary school level I certificate (“Mittlere Reife”); low = secondary modern school qualification (“Haupt−/Volksschulabschluss”) or no formal school degree e Reports of allergy to either foods, pollen, dust mites, fur, insect venom, drugs or allergic contact eczema in response to cosmetics, lotions and alike f Defined as a score ≥20 on the Asthma Control Test of the study populations and comparison between sample 1 and 2 regarding baseline demographics and Page 5 of 10 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 (Fig. 2). Table 2 shows paired t tests of NEAT total and subscale scores and ACT sum scores at baseline and follow-up in both samples. Univariate ANCOVAS Univariate ANCOVAS Univariate ANCOVAS (Table 3) showed that unmet needs at follow-up (F(1, 212) = 36.46, p < 0.001), and the level of asthma control at follow-up (F(1, 195) = 6.97, p = 0.009), differed significantly between the two groups (1 = rehabili- tation, 2 = control). These observations imply that the re- habilitation group showed significantly less health care needs and better asthma control at follow-up (and after pulmonary rehabilitation) compared to the control group. Asthma outcomes At baseline, there were no differences between both samples regarding health care needs, measured by the NEAT total mean score (5.73 in the rehabilitation vs. 5.72 in the control group). In contrast, at follow-up, we found a significantly lower NEAT total mean score in the rehabilitation as compared to the control group (3.13 in the rehabilitation vs. 5.93 in the control group; p < 0.01). This was explained by a significant reduction of unmet needs in the rehabilitation group (total need score: t(149) = 10.33; p < 0.001) compared to their base- line score whereas the score in the control group remained stable (Fig. 1). Demographics Samples differed significantly in terms of gender (58.67% vs. 83.33% female; p < 0.001), age (52.87 vs. 45.26 years on average; p < 0.001), school education (30.67% vs. 14.10% low school education; p < 0.001), re- ported allergies (78% vs. 89.74% any allergy; p = 0.05) and follow-up period (22.83 vs. 30.85 days on average; p < 0.001). Linear regression analysis In the rehabilitation group we ran additional linear re- gression models to examine if self-reported utilization of more interventions that address needs, surveyed in the NEAT questionnaire, were associated with reduced health care needs in a dose-response fashion (Table 4). We found that a total score of those interventions dur- ing pulmonary rehabilitation was significantly associated with reduced needs, as measured by the NEAT total change score (β = 0.21; p = 0.03) as well as measured by the change scores of the NEAT subscales patient expert- ise (β = 0.31; p = 0.009) and exacerbations (β = 0.20; p = 0.04). Further, received interventions that addressed pa- tient expertise were significantly associated with reduced needs, measured by the change score of the NEAT sub- scale patient expertise (β = 0.25; p = 0.01) and received interventions addressing exacerbations tended to be sig- nificantly associated with reduced needs, measured by the change score of the NEAT subscale exacerbations (β = 0.20; p = 0.052). In contrast, there was no evidence Furthermore, in both samples asthma control was comparably poor at baseline in light of the fact that a score of 19 or lower defines poor control (mean score of 17.39 in the rehabilitation vs. 16.99 in the control group). In contrast, at follow-up, the rehabilitation group showed significantly higher ACT mean scores compared to the control group (19.55 in the rehabilitation vs. 17.56 in the control group, p = 0.01). We found that in the re- habilitation group asthma control significantly increased during follow-up period to an rather appropriate level (well controlled ≥ 20) (t(130) = −6.67, p < 0.001), whereas in the control group asthma control remained poor and exhibited little and non-significant change Fig. 1 Mean change of the Patient Needs in Asthma Treatment (NEAT) questionnaire in rehabilitation and control group between baseline and follow-up Fig. 1 Mean change of the Patient Needs in Asthma Treatment (NEAT) questionnaire in rehabilitation and control group between baseline and follow-up Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Page 6 of 10 Fig. Linear regression analysis 2 Mean change of the Asthma Control Test (ACT) in rehabilitation and control group between baseline and follow-up significantly associated with reduced needs, measured by the change score of the NEAT subscale drug effects (β = 0.24; p = 0.02) and received interventions addressing treat- ment in drug effects were significantly associated with re- duced needs, measured by the change score of the NEAT subscale exacerbations (β = 0.18; p = 0. 04). of associations between received interventions address- ing drug effects and the change score of the NEAT sub- scale drug effects as well as between experienced interventions addressing how to handle drugs and the change score of the NEAT subscale handling drugs. Furthermore, received interventions addressing educa- tion in handling drugs were significantly associated with reduced needs, measured by the change score of the NEAT subscale patient expertise (β = 0.23; p = 0.01). Also, received interventions addressing exacerbations were a NEAT Needs in Asthma Treatment Questionnaire b Discussion BMC Pulmonary Medicine (2020) 20:267 Table 3 Univariate ANCOVAS with group membership (rehabilitation or control) as independent variable, NEAT a and ACT b follow- up scores as dependent variables and sex, age, educational level, numbers of allergies, follow-up period and the respective baseline i t Table 3 Univariate ANCOVAS with group membership (rehabilitation or control) as independent variable, NEAT a and ACT b follow- up scores as dependent variables and sex, age, educational level, numbers of allergies, follow-up period and the respective baseline score as covariates up scores as dependent variables and sex, age, educational level, numbers of allergies, follow-up period and the respective baseline score as covariates Variables Mean – Follow-up (95% CI c) Mean difference – Follow-up (95% CI c) p value for difference Rehabilitation (n = 141) Control (n = 73) NEAT a 0.25 (0.21–0.28) 0.44 (0.38–0.51) −0.19 (−0.26 - -0.12) < 0 .001 Rehabilitation (n = 124) Control (n = 73) ACT b 19.60 (18.88–20.29) 17.48 (16.24–18.65) 2.12 (0.79–3.42) 0.009 a NEAT Needs in Asthma Treatment Questionnaire b ACT Asthma Control Test c CI confidence interval less health care needs in the NEAT subscale exacerba- tions. Similarly, information on drug effects, side effects and interactions of drugs led to reduced health care needs regarding exacerbations. Thereby, we can assume that not only patient education referring to what to do in case of an exacerbation, but also better knowledge of drug effects may help to reduce needs regarding infor- mation on what to do in case of exacerbations. Also, re- ceived interventions addressing exacerbations were associated with reduced health care needs in the NEAT subscale drug effects. Thereby we can assume that more skills and knowledge regarding possible asthma attacks may lead to a decrease in health care needs regarding drug effects. relation to health care needs and confirms prior research on asthma control. Initially, we found that unmet health care needs were reduced during rehabilitation and that this effect was as- sociated with the utilization of suitable asthma interven- tions in a dose-response fashion. To our knowledge, there exist no studies examining the association between pulmonary rehabilitation and (unmet) health care needs and we therefore provide novel evidence. To gain a better understanding, we additionally calculated subscale specific scores on received asthma education/training, which reflect subscale specific topics of the NEAT. Our results indicate that better physician-patient- communication, e.g. Discussion This study provides initial evidence suggestion the bene- fits of pulmonary rehabilitation in adults with asthma in This study provides initial evidence suggestion the bene- fits of pulmonary rehabilitation in adults with asthma in Table 2 Paired t tests of NEAT a total and subscale scores and ACT b sum scores at baseline and follow-up in both samples Variables T1c: mean (SD) T2d: mean (SD) t df p Rehabilitation group NEAT a total score 0.46 (0.27) 0.24 (0.23) 10.33 149 < 0.001 Patient expertise e 0.44 (0.37) 0.29 (0.33) 4.77 132 < 0.001 Drug effects e 0.65 (0.36) 0.37 (0.39) 8.16 145 < 0.001 Handling drugs e 0.19 (0.30) 0.08 (0.20) 3.97 142 0.006 Exacerbations e 0.55 (0.39) 0.21 (0.30) 11.17 142 < 0.001 ACT b sum score 17.39 (4.61) 19.68 (4.12) −6.67 130 < 0.001 Control Group NEAT a total score 0.44 (0.30) 0.43 (0.29) 0.27 77 0.76 Patient expertise e 0.47 (0.39) 0.45 (0.37) 0.59 75 0.56 Drug effects e 0.57 (0.37) 0.54 (0.38) 0.79 77 0.44 Handling drugs e 0.23 (0.32) 0.23 (0.33) 0.00 74 1.00 Exacerbations e 0.48 (0.41) 0.51 (0.40) −0.97 77 0.33 ACT b sum score 16.99 (4.81) 17.56 (5.36) −1.485 77 0.14 a NEAT N d i A h T Q i i Page 7 of 10 Salandi et al. a NEAT Needs in Asthma Treatment Questionnaire b b Adjusted for sex, age, educational level and rehabilitation clinic c NEAT subscales Discussion physician made more time available in case of special requests or considered personal cir- cumstances during treatment, but also practical training and information on how to use asthma drugs were asso- ciated with less health care needs regarding the NEAT subscale patient expertise. These findings suggest that not only a better exchange of information between pa- tient and physician, but also a more appropriate hand- ling of drugs may reduce the need for being an expert for one’s own disease. Furthermore, received interven- tions addressing topics like information on what to do in case of an asthma attack or practical training of breath- ing techniques tended to be significantly associated with Given the findings from our previous study on psycho- metric properties [20], we have to conclude that the average improvement of the total NEAT score in the re- habilitation group (mean change score of 2.69 unmet needs) is less than the minimal important change score (MIC = 4 unmet needs) we have estimated before. The MIC refers to the smallest amount of change, which is considered important by patients [27, 28]. However, even if the MIC was not reached, the reduction of needs is still considerable, given the short time period of 23 days (SD = 5.00) on average between baseline and follow-up. j c NEAT subscales Discussion Table 4 Sample 1: Linear regression with achieved asthma treatment total and subscale scores as independent variables and NEAT a total and subscale change scores as dependent variables Variables NEAT a total change score b Patient expertise change score b c Drug effects change score b c Handling Drugs change score b c Exacerbations change score b c b β p b β p b β p b β p b β p Asthma treatment total score 0.13 0.21 0.03 0.03 0.31 0.009 0.02 0.15 0.13 0.01 0.05 0.60 0.02 0.20 0.04 Treatment in patient expertisec 0.01 0.08 0.37 0.06 0.25 0.01 0.02 0.10 0.25 0.02 0.08 0.42 0.00 0.02 0.86 Treatment in drug effectsc 0.04 0.15 0.08 0.04 0.12 0.20 0.06 0.14 0.12 0.00 0.01 0.91 0.07 0.18 0.04 Treatment in handling drugsc 0.02 0.11 0.21 0.06 0.23 0.01 −0.02 −0.07 0.47 −0.00 −0.01 0.96 0.04 0.16 0.09 Treatment in exacerbationsc 0.05 0.20 0.06 0.04 0.12 0.27 0.09 0.24 0.02 0.02 0.06 0.60 0.07 0.20 0.05 a NEAT Needs in Asthma Treatment Questionnaire b Adjusted for sex, age, educational level and rehabilitation clinic c Table 4 Sample 1: Linear regression with achieved asthma treatment total and subscale scores as independe total and subscale change scores as dependent variables Page 8 of 10 Page 8 of 10 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 the mean control improved from 14.76 (SD = 3.39, Min = 6, Max = 19) to 18.26 (SD = 4.14, Min = 6, Max = 25) and thus the mean score changed by 3.65 points. This means that this improvement is probably relevant for patients with asthma. If only participants with very poorly controlled asthma at admission (ACT≤15) were included in the analysis, the change was even more pro- nounced (baseline: mean = 12.00, SD = 2.61; follow-up: mean = 16.81, SD = 4.43; mean change score = 4.88). In addition, we ran an ad hoc univariate ANCOVA (poorly versus well controlled asthma as independent variable and ACT change score as dependent variable). In those analysis, the effect of pulmonary rehabilitation on the follow-up ACT score differed significantly between well- controlled and poorly controlled patients (F(1, 88) = 44.65, p < 0.001). This finding could indicate that espe- cially the latter could benefit from rehabilitation. Discussion In summary, we can assume that a) interventions dur- ing rehabilitation are associated with reduced health care needs in a dose-response fashion and b) very specific treatment elements may reduce specific health care needs during rehabilitation. Compared to the rehabilita- tion group, we did not find any improvement of health care needs in patients who most likely did not receive any kind of new intervention (control group). By using ANCOVA we were able to ensure that this result is due to participation in rehabilitation and not merely to demographic differences such as age, gender or educa- tional level. Results of the ANCOVA showed that mem- bers of the control group had significant more health care needs at follow-up than members of the rehabilita- tion group. Thus, one can be assumed that unmet health care needs in the rehabilitation group did not decrease merely due to demographic characteristics. Based on these results, our study should be under- stood as an initial step to provide evidence. Experimental studies that are based on the randomized allocation of pulmonary rehabilitation and that maximize the likeli- hood of adequate control groups (i.e. randomized con- trolled trials, RCTs) are needed to provide more valid insights into the causal relations between rehabilitation and health care needs. Although conducting an RCT on this matter remains challenging, as people with consider- able health problems cannot be withheld from rehabilitation. The improvement of asthma control between baseline and follow-up was not observed in adults with asthma who most likely did not receive any kind of new inter- vention during follow-up period: control remained poor in this sample. Building on this finding, we calculated a univariate ANCOVA (with the covariates age, sex, edu- cational level, number of allergies, mean follow-up period and the respective baseline score), which also showed that members of the control group had signifi- cantly less asthma control at follow-up than members of the rehabilitation group. Overall, our results regarding asthma control support previous findings on the impact of pulmonary rehabilitation on patients’ health care [17, 18]. To our knowledge, there is only one waiting-list RCT of pulmonary rehabilitation in patients with uncon- trolled asthma and this RCT demonstrated that rehabili- tation is effective, e.g. in terms of asthma control [17]. This study shows that rehabilitation improves asthma control (measured by the ACT) with clinically relevant effect sizes. Discussion In the present study, we could confirm these results and found that individuals with asthma who par- ticipated in pulmonary rehabilitation displayed signifi- cant improvement in terms of asthma control. Whereas mean control was poor at admission, at discharge partic- ipants showed an ACT mean score of 20, indicating ra- ther adequate levels of control. While at admission only 35% of participants showed well-controlled asthma, at discharge 56% reported an ACT score of 20 or more. On average, the ACT score improved by 2.29 points, which is slightly less than the identified MIC, which equals 3 points [29]. The slightly more limited improve- ment of the ACT in this study could be explained by relatively high ACT mean scores at admission (17.39, SD = 4.61). While ACT scores between 5 to 15 indicate a very poorly controlled asthma, scores between 16 to 19 suggest at least a somewhat controlled asthma. This hy- pothesis is supported by the examination of the charac- teristics of only those patients who did not display controlled asthma at admission (ACT≤19). In this group, Strengths and limitations Importantly, our results are not based on data from an RCT, but we employed a quasi-experimental design. A RCT is considered the gold standard to examine effect- iveness of interventions and its defining feature is randomization. Randomization implies that participants are randomly allocated to either the intervention or the control group which should, if the trial is sufficiently large, yield two samples that are comparable in terms of measured and unmeasured confounders [30]. A quasi- experimental design, by contrast, compares two natural groups without random allocation of participants. Thereby, only limited causal conclusions are possible [31, 32]. Our samples did not originate from the same population and demographics were not equivalent. Dif- ferences in the mode of surveying (paper-and-pencil during rehabilitation versus mainly online in the control group) may explain these demographic differences. Thus, some distortion even after control for confound- ing in the analysis (i.e. residual confounding) cannot be excluded. Furthermore, unfortunately no information on medication was collected. Therefore, we are unable to Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Page 9 of 10 examine a possible link between pulmonary rehabilita- tion and medication (e.g. reduction of doses). study) to evaluate the interventions and to examine if important cornerstones are met from patients’ point of view. It is reassuring though that there were no baseline dif- ferences in terms of our primary outcomes (NEAT and ACT) between rehabilitation and control group. Further- more, despite using age, gender educational level, num- ber of allergies and follow-up period as covariates, members of the control group had significant more health care needs and less asthma control at follow-up than members of the rehabilitation group. Also, we in- cluded individuals from three rehabilitation clinics and thereby collected data in a multicentre design. Thus, we can assume that the improvement of health care needs and asthma control is not limited to an intervention in a specific clinic (i.e. single centre study); rather it seems that rehabilitation by itself and regardless of the clinic or its slightly different style of intervention administration may improve these outcomes. Implications Th EAT h The NEAT has been found to be a reliable and valid in- strument to predict treatment satisfaction [20] and therefore may facilitate the planning of interventions at the beginning of rehabilitation according to shared decision-making between patient and physician through the delivery of patient-centered care. Also, one may speculate that administration of our participant- completed questionnaire may increase awareness among patients. Therefore, they may pay greater attention to their unmet health care needs and the fulfillment of these needs during their treatment. Similarly, it could be very helpful to administer the NEAT at the beginning and the end of rehabilitation (such as in the present Funding g This study was funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, grant number: LO 1730/2–3). The funder had no say related to the study design, the collection, analysis, and interpretation of data; the writing of the manuscript; and in the decision to submit the manuscript for publication. Open Access funding enabled and organized by Projekt DEAL. A special strength of both samples is that we attained good response rate at follow-up (rehabilitation: 93%, control: 67%). Furthermore, responders and non- responders provided similar background data at baseline, except for reported allergies in the control group, which were more common in responders (90% vs. 78%, p = 0.03). Also, there existed no significant differences be- tween responders and non-responders in both samples regarding NEAT and ACT scores at baseline. In sum- mary, we can assume that major selection bias across the follow-up is rather unlikely. Conclusions This study provides preliminary evidence suggestion of the benefits of pulmonary rehabilitation in adults with asthma in reducing patients’ unmet health care needs and confirms previous findings that rehabilitation may improve asthma control. Authors’ contributions JS, AL – made substantial contributions to conception and design, acquisition, analysis and interpretation of data; drafted the article, agree to be accountable for all aspects of the work related to its accuracy or integrity. AI, CA, AS - made substantial contributions to analysis and interpretation of data; reviewed the article critically, gave final approval of the version to be published, agree to be accountable for all aspects of the work related to its accuracy or integrity. JG, SH, KS - made substantial contributions to acquisition of data, reviewed the article critically, gave final approval of the version to be published, agree to be accountable for all aspects of the work related to its accuracy or integrity. The author(s) read and approved the final manuscript. Unfortunately, we cannot know with certainty that participants in the control group did not receive any new intervention (like elements of a disease management program) during the 31-day-follow-up period. However, since the period was short, we can assume that no re- habilitation took place. Furthermore, unfortunately no information on medication was collected. Therefore , we are unable to examine a possible link between pulmon- ary rehabilitation and medication (e.g. reduction of dose). acquisition of data, reviewed the article critically, gave final approval of the version to be published, agree to be accountable for all aspects of the work related to its accuracy or integrity. The author(s) read and approved the final manuscript. Competing interests Three of the co-authors are (or were at the time of the survey) hospital direc- tors in the clinics where the survey was conducted: Jalal Gholami (Nordseek- linik Borkum), Dr. Stefan Hummel: (MEDIAN Klinik Heiligendamm), Dr. Konrad Schultz (Klinik Bad Reichenhall). Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate All surveys received approval from the Institutional Review Board of the Medical Faculty of the University of Düsseldorf. Written informed consent was obtained from all participants. Abbreviations ACT: Asthma Control Test; COPD: Chronic Obstructive Pulmonary Disease; NEAT: The Patient Needs in Asthma Treatment; PRO: Patient reported outcome References Allergy. 2007;62(6):668–74. 30. de Vet H, Terwee CB, Mokkink LB, Knol DL. Measurement in Medicine. Practical Guides to Biostatistics and Epidemiology. Cambridge: Cambridge University Press; 2011. p. 150–201. 30. de Vet H, Terwee CB, Mokkink LB, Knol DL. Measurement in Medicine. Practical Guides to Biostatistics and Epidemiology. Cambridge: Cambridge University Press; 2011. p. 150–201. 8. Worth A, Hammersley V, Knibb R, Flokstra-de-Blok B, DunnGalvin A, Walker S, et al. Patient-reported outcome measures for asthma: a systematic review. NPJ Primary Care Respiratory Med. 2014;24(1):1–8. 8. Worth A, Hammersley V, Knibb R, Flokstra-de-Blok B, DunnGalvin A, Walker S, et al. Patient-reported outcome measures for asthma: a systematic review. NPJ Primary Care Respiratory Med. 2014;24(1):1–8. 31. Harmon RJ, Morgan GA, Gliner JA, Harmon RJ. Quasi-experimental designs. J Am Acad Child Adolesc Psychiatry. 2000;39(6):794–6. 31. Harmon RJ, Morgan GA, Gliner JA, Harmon RJ. Quasi-experimental designs. J Am Acad Child Adolesc Psychiatry. 2000;39(6):794–6. 9. Schultz K, Taube K. Rehabilitation bei COPD und Asthma bronchiale: Erwachsene. In: Lingner H, Schwartz FW, Schultz K, editors. Volkskrankheit Asthma/COPD. Berlin: Springer; 2007. p. 371–84. 32. Hallberg K, Eno J. Quasi-Experimental Designs. In: Wright J, editor. International Encyclopedia of the Social & Behavioral Sciences, 2nd ed. Amsterdam: Elsevier; 2015. p. 742–6. 32. Hallberg K, Eno J. Quasi-Experimental Designs. In: Wright J, editor. International Encyclopedia of the Social & Behavioral Sciences, 2nd ed. Amsterdam: Elsevier; 2015. p. 742–6. 10. AWMF, editor.S2k-Leitlinie zur Diagnostik und Therapie von Patienten mit Asthma. 2017. https://www.awmf.org/uploads/tx_szleitlinien/020-009l_S2k_ Asthma_Diagnostik_Therapie_2017-11_1.pdf. Accessed 15 May 2019. Received: 6 April 2020 Accepted: 24 September 2020 Received: 6 April 2020 Accepted: 24 September 2020 Received: 6 April 2020 Accepted: 24 September 2020 Received: 6 April 2020 Accepted: 24 September 2020 22. Bradshaw J. Taxonomy of social need. In: McLachlan G, editor. Problems and progress in medical care : essays on current research, 7th series. London: Oxford University Press; 1972. p. 71–82. 23. Loerbroks A, Leucht V, Keuneke S, Apfelbacher CJ, Sheikh A, Angerer P. Patients’ needs in asthma treatment: development and initial validation of the NEAT questionnaire. J Asthma. 2016;53(4):427–37. Publisher’s Note 11. Schultz K, Müller C, Schwiersch M. Rehabilitation bei Asthma bronchiale - Stellenwert und Verfahren. Allergo J. 2002;11(4):258–71. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 12. Schultz K. Atemwege und Lunge. In: Bundesarbeitsgemeinschaft für Rehabilitation e.V., editor. Rehabilitation. Vom Antrag bis zur Nachsorge – für Ärzte, Psychotherapeuten und andere Gesundheitsberufe. Berlin: Springer; 2018. p. 57–67. 13. Cambach W, Chadwick-Straver R, Wagenaar R, Van Keimpema A, Kemper H. The effects of a community-based pulmonary rehabilitation programme on exercise tolerance and quality of life: a randomized controlled trial. Eur Respir J. 1997;10(1):104–13. 13. Cambach W, Chadwick-Straver R, Wagenaar R, Van Keimpema A, Kemper H. The effects of a community-based pulmonary rehabilitation programme on exercise tolerance and quality of life: a randomized controlled trial. Eur Respir J. 1997;10(1):104–13. 14. Griffiths TL, Burr ML, Campbell IA, Lewis-Jenkins V, Mullins J, Shiels K, et al. Results at 1 year of outpatient multidisciplinary pulmonary rehabilitation: a randomised controlled trial. Lancet. 2000;355(9201):362–8. 14. Griffiths TL, Burr ML, Campbell IA, Lewis-Jenkins V, Mullins J, Shiels K, et al. Results at 1 year of outpatient multidisciplinary pulmonary rehabilitation: a randomised controlled trial. Lancet. 2000;355(9201):362–8. 15. Haave E, Hyland M. Different short-term and longitudinal results on perceived health status for asthma and COPD patients after pulmonary rehabilitation. Patients living alone have the largest improvements in perceived quality of life. Chronic Respiratory Dis. 2008;5(2):69–73. 15. Haave E, Hyland M. Different short-term and longitudinal results on perceived health status for asthma and COPD patients after pulmonary rehabilitation. Patients living alone have the largest improvements in perceived quality of life. Chronic Respiratory Dis. 2008;5(2):69–73. 16. Farin E, Opitz U, Jäckel W, Gallenmüller K, Schwiersch M, Schultz K. Pneumologische Rehabilitation: Langzeitdaten zur Lebensqualität und Prädiktoren der Behandlungseffekte. Physikalische Medizin Rehabilitationsmedizin, Kurortmedizin. 2009;19(02):75–84. 16. Farin E, Opitz U, Jäckel W, Gallenmüller K, Schwiersch M, Schultz K. Pneumologische Rehabilitation: Langzeitdaten zur Lebensqualität und Prädiktoren der Behandlungseffekte. Physikalische Medizin Rehabilitationsmedizin, Kurortmedizin. 2009;19(02):75–84. 17. Schultz K, Wittmann M, Wagner R, Lehbert N, Schwarzkopf L, Szentes B, et al. Effectiveness of pulmonary rehabilitation for patients with asthma: EPRA-RCT. Eur Respir J. 2018;52(suppl 62):OA1620. 17. Schultz K, Wittmann M, Wagner R, Lehbert N, Schwarzkopf L, Szentes B, et al. Effectiveness of pulmonary rehabilitation for patients with asthma: EPRA-RCT. Eur Respir J. 2018;52(suppl 62):OA1620. 18. Author details 1 1Institute of Occupational, Social, and Environmental Medicine, Centre for Health and Society, Medical Faculty, University of Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany. 2Institute of Health Services Research and Health Economics, Centre for Health and Society, Medical Faculty, University of Düsseldorf, Moorenstraße 5, 40225 Düsseldorf, Germany. 3Institute of Health Services Research and Health Economics, German Diabetes Center, Medical Faculty, University of Düsseldorf, Auf’m Hennekamp 65, 40225 Düsseldorf, Germany. 4Nordseeklinik Borkum der DRV Rheinland, Bubertstraße 4, 26757 Borkum, Germany. 5MEDIAN Klinik Heiligendamm, Kinderstrand 1, 18209 Bad Page 10 of 10 Page 10 of 10 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Salandi et al. BMC Pulmonary Medicine (2020) 20:267 Doberan, Germany. 6Klinik Bad Reichenhall der DRV Bayern Süd, Salzburger Str. 8-11, 83435 Bad Reichenhall, Germany. 7Institute of Social Medicine and Health Systems Research (ISMHSR), Otto von Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany. 8Usher Institute, University of Edinburgh, Old Medical School, Teviot Place, Edinburgh EH8 9AG, UK. 20. Schreitmüller J, Apfelbacher C, Sheikh A, Loerbroks A. The Patient Needs in Asthma Treatment (NEAT) questionnaire: Further evidence on its psychometric properties. Allergy. 2019;74(8):1511–21. 21. Faller H, Weis J, Koch U, Brähler E, Härter M, Keller M, et al. Perceived need for psychosocial support depending on emotional distress and mental comorbidity in men and women with cancer. J Psychosom Res. 2016;81:24– 30. References 1. Bundesärztekammer (BÄK), editor. Nationale VersorgungsLeitlinie Asthma – Langfassung, 3. Auflage. Version 1. 2018. https://www.aezq.de/mdb/ downloads/nvl/asthma/archiv/asthma-3aufl-konsultation-llr.pdf. Accessed 15 May 2019. 24. Loerbroks A, Sheikh A, Leucht V, Apfelbacher CJ, Icks A, Angerer P. Determinants of patients’ needs in asthma treatment: a cross-sectional study. NPJ Primary Care Respiratory Med. 2016;26:16044. 2. Menz G, Kronenberger H, Lecheler J, Schultz K. Rehabilitation bei Asthma bronchiale. Pneumologie. 2007;61(11):710–18. 25. Schreitmüller J, Loerbroks A. The role of self-efficacy and locus of control in asthma-related needs and outcomes: a cross-sectional study. J Asthma. 2018;57(2):196–204. 3. Morgan MDL. Pulmonary rehabilitation. Thorax. 2001;56(11):827–34. 4. Ries AL, Bauldoff GS, Carlin BW, Casaburi R, Emery CF, Mahler DA, et al. Pulmonary rehabilitation: joint ACCP/AACVPR evidence-based clinical practice guidelines. Chest. 2007;131(5):4S–42S. 26. Schatz M, Sorkness CA, Li JT, Marcus P, Murray JJ, Nathan RA, et al. Asthma control test: reliability, validity, and responsiveness in patients not previously followed by asthma specialists. J Allergy Clin Immunol. 2006;117(3):549–56. 5. Fischer J, Schnabel M, Sitter H. Rehabilitation von Patienten mit chronisch obstruktiver Lungenerkrankung (COPD). Pneumologie. 2007;61(04):233-48. 27. Revicki DA, Cella D, Hays RD, Sloan JA, Lenderking WR, Aaronson NK. Responsiveness and minimal important differences for patient reported outcomes. Health Qual Life Outcomes. 2006;4(1):70. 27. Revicki DA, Cella D, Hays RD, Sloan JA, Lenderking WR, Aaronson NK. Responsiveness and minimal important differences for patient reported outcomes. Health Qual Life Outcomes. 2006;4(1):70. 6. Deutsche Rentenversicherungen Bund, editor. Leitlinien der Deutschen Gesellschaft für Sozialmedizin und Prävention: Chronisch obstruktive Lungenkrankheiten (COPD) und Asthma bronchiale. Berlin: AWMF online; 2003. 28. McGlothlin AE, Lewis RJ. Minimal clinically important difference: defining what really matters to patients. Jama. 2014;312(13):1342–3. 28. McGlothlin AE, Lewis RJ. Minimal clinically important difference: defining what really matters to patients. Jama. 2014;312(13):1342–3. 29. Schatz M, Kosinski M, Yarlas AS, Hanlon J, Watson ME, Jhingran P. The minimally important difference of the Asthma Control Test. J Allergy Clin Immunol. 2009;124(4):719–23 e1. 29. Schatz M, Kosinski M, Yarlas AS, Hanlon J, Watson ME, Jhingran P. The minimally important difference of the Asthma Control Test. J Allergy Clin Immunol. 2009;124(4):719–23 e1. 7. Canonica G, Baena-Cagnani C, Blaiss M, Dahl R, Kaliner M, Valovirta E, et al. Unmet needs in asthma: global asthma physician and patient (GAPP) survey: global adult findings. Allergy. 2007;62(6):668–74. 7. Canonica G, Baena-Cagnani C, Blaiss M, Dahl R, Kaliner M, Valovirta E, et al. Unmet needs in asthma: global asthma physician and patient (GAPP) survey: global adult findings. Publisher’s Note Lingner H, Ernst S, Groβhennig A, Djahangiri N, Scheub D, Wittmann M, et al. Asthma control and health-related quality of life one year after inpatient pulmonary rehabilitation: the ProKAR study. J Asthma. 2015;52(6): 614–21. 18. Lingner H, Ernst S, Groβhennig A, Djahangiri N, Scheub D, Wittmann M, et al. Asthma control and health-related quality of life one year after inpatient pulmonary rehabilitation: the ProKAR study. J Asthma. 2015;52(6): 614–21. 19. Foster S. Thomas Hr. pulmonary rehabilitation in lung disease other than chronic obstructive pulmonary disease. Am Rev Respir Dis. 1990;141(3):601– 4.
https://openalex.org/W2031600726	https://discovery.dundee.ac.uk/ws/files/52358177/fnsyn_05_00003.pdf	English	null	Leptin regulation of neuronal morphology and hippocampal synaptic function	Frontiers in synaptic neuroscience	2,013	cc-by	7,899	Leptin regulation of neuronal morphology and hippocampal synaptic function Harvey Jenni Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal Citation for published version (APA): Harvey, J. (2013). Leptin regulation of neuronal morphology and hippocampal synaptic function. Frontiers in Synaptic Neuroscience, 5(AUG), Article 3. https://doi.org/10.3389/fnsyn.2013.00003 General rights Copyright and moral rights for the publications made accessible in Discovery Research Portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Citation for published version (APA): Harvey, J. (2013). Leptin regulation of neuronal morphology and hippocampal synaptic function. Frontiers in Synaptic Neuroscience, 5(AUG), Article 3. https://doi.org/10.3389/fnsyn.2013.00003 University of Dundee Leptin regulation of neuronal morphology and hippocampal synaptic function Harvey, Jenni Published in: Frontiers in Synaptic Neuroscience DOI: 10.3389/fnsyn.2013.00003 Publication date: 2013 Licence: CC BY Document Version Publisher's PDF, also known as Version of record Link to publication in Discovery Research Portal Citation for published version (APA): Harvey, J. (2013). Leptin regulation of neuronal morphology and hippocampal synaptic function. Frontiers in Synaptic Neuroscience, 5(AUG), Article 3. https://doi.org/10.3389/fnsyn.2013.00003 University of Dundee General rights i h d Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 24. Oct. 2024 REVIEW ARTICLE published: 06 August 2013 doi: 10.3389/fnsyn.2013.00003 SYNAPTIC NEUROSCIENCE REVIEW ARTICLE published: 06 August 2013 doi: 10.3389/fnsyn.2013.00003 REVIEW ARTICLE LEPTIN RECEPTOR SIGNALING The endocrine hormone, leptin is a 167 amino acid protein that is mainly produced by adipocytes and circulates in the plasma at levels closely correlated with body fat content (Maffei et al., 1995). Although leptin acts on a number of peripheral tissues, leptin also readily targets the CNS, following its transport across the blood brain barrier. Several lines of evidence indicate the expression of leptin mRNA and protein in a number of brain regions, thereby raising the possibility that leptin is also synthesized and released locally in the CNS. The most well documented roles for leptin focus on its actions in the hypothalamus where it participates in the regulation of energy homeostasis, bone formation as well as reproductive function (Spiegelman and Flier, 2001; Karsenty, 2006). However the central actions of leptin are not conﬁned to the hypothalamus. Indeed, numerous studies have detected high levels of leptin receptor expression in extra-hypothalamic brain regions and evidence is growing that leptin has widespread actions throughout the CNS. Frontiers in Synaptic Neuroscience Jenni Harvey* Division of Neuroscience, Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dund Division of Neuroscience, Medical Research Institute, Ninewells Hospital and Medical School, Univer The central actions of the hormone leptin in regulating energy homeostasis via the hypothalamus are well documented. However, evidence is growing that this hormone can also modify the structure and function of synapses throughout the CNS. The hippocampus is a region of the forebrain that plays a crucial role in associative learning and memory and is an area also highly vulnerable to neurodegenerative processes. Recent studies indicate that leptin is a potential cognitive enhancer as it modulates the cellular processes underlying hippocampal-dependent learning and memory including dendritic morphology, glutamate receptor trafﬁcking and activity-dependent synaptic plasticity. Here, we review the recent evidence implicating the hormone leptin as a key regulator of hippocampal synaptic function and discuss the role of leptin receptor-driven lipid signaling pathways involved in this process. Edited by: Akira Yoshii, Massachusetts Institute of Technology, USA Reviewed by: Jason D. Shepherd, University of Utah, USA Akiko Taguchi, University of Miyazaki, Japan Correspondence: Jenni Harvey, Division of Neuroscience, Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK e-mail: j.z.harvey@dundee.ac.uk Leptin regulation of neuronal morphology and hippocampal synaptic function Jenni Harvey Edited by: Edited by: Akira Yoshii, Massachusetts Institute of Technology, USA Reviewed by: Jason D. Shepherd, University of Utah, USA Akiko Taguchi, University of Miyazaki, Japan *Correspondence: Jenni Harvey, Division of Neuroscience, Medical Research Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee DD1 9SY, UK e-mail: j.z.harvey@dundee.ac.uk Keywords: leptin, synaptic plasticity, morphology, receptor trafﬁcking, hippocampus, AMPA receptor, PTEN Keywords: leptin, synaptic plasticity, morphology, receptor trafﬁcking, hippocampus, AMPA receptor, PTEN INTRODUCTION LEPTIN RECEPTOR SIGNALING JAK2 activation, JAK2 associates with and promotes phosphory- lation of tyrosine residues within the C-terminal domain. The phosphorylated tyrosine residues enable recruitment and acti- vation of various downstream signaling pathways including the STAT (signal transducers and activators of transcription) fam- ily of transcription factors, phosphoinositide 3-kinase as well as adaptor proteins associated with the Ras-Raf-MAPK (mitogen activated protein kinase) pathway. Leptin is capable of activat- ing all of these ObR-driven signaling in central neurons (Harvey, 2003). LEPTIN RECEPTOR EXPRESSION IN THE CNS In accordance with its role in regulating energy balance, high lev- els of leptin receptor expression have been detected on speciﬁc hypothalamic nuclei involved in energy homeostasis (Schwartz et al., 1996). Leptin receptors are also expressed throughout the CNS with high levels of expression reported in the amyg- dala, brainstem and cerebellum. High levels of ObR mRNA and immunoreactivity have also been detected in hippocampal CA1, CA3, and dentate gyrus regions (Mercer et al., 1996; Hâkansson et al., 1998). In hippocampal cultures, ObR expression is local- ized to principal neurons and glial cells. Moreover, dual labeling studies indicate that ObR is expressed at somato-dendritic regions and also in close proximity to NMDA receptors (Shanley et al., 2002b; O’Malley et al., 2007). Although the main source of leptin is from peripheral tissues, leptin readily gains access to the brain via a saturable transport process at the blood brain barrier (Banks et al., 1996). Short ObR isoforms expressed on brain microves- sels are implicated in this process. High levels of ObRa are also expressed at the choroid plexus thereby supporting a possible role for the cerebrospinal ﬂuid in transporting leptin to the CNS. Direct diffusion of leptin to the hypothalamus may also occur as the main leptin-sensitive hypothalamic neurons are located close to the median eminence. g The biological actions of leptin are mediated by leptin recep- tors; members of the class I cytokine receptor superfamily (Tartaglia et al., 1995). The leptin receptor (ObR) is encoded by the diabetes gene (db) and alternative splicing of this gene generates six leptin receptor isoforms (ObRa-f) with identical N-terminal domains but vary in the length of their C-terminal region. All the isoforms, except ObRe, have a membrane span- ning region consisting of 34 amino acids. ObRe is thought to buffer free circulating leptin levels in the plasma. The mem- brane spanning ObRs all contain an intracellular proline-rich box which enables association with janus tyrosine kinases, in particular JAK2. However, only the long leptin receptor isoform (ObRb) contains additional intracellular signaling motifs nec- essary for full JAK-STAT (signal transducer and activation of transcription) signaling. Following leptin binding to ObRb and August 2013 \| Volume 5 \| Article 3 \| 1 www.frontiersin.org www.frontiersin.org www.frontiersin.org Leptin regulates hippocampal synaptic function Harvey Harvey the experimental approach or age of tissue used. LEPTIN-INDUCED LTD Although NMDA receptors contribute little to basal synap- tic transmission, activation of NMDA receptors is essential for the induction of hippocampal LTP (Collingridge et al., 1983). Like other hormones and growth factors, leptin potently enhances NMDA receptor function which contributes to its ability to facili- tate LTP. Indeed, leptin is capable of enhancing Ca2+ inﬂux via native NMDA receptor channels in hippocampal cultures and NMDA evoked currents in Xenopus oocytes expressing recom- binant NMDA receptors (Shanley et al., 2001; Harvey et al., 2005). Conversely, other studies have reported either no effect or attenuation of NMDA responses by leptin in hippocampal neu- rons (Oomura et al., 2006) which may be due to differences in Two main forms of LTD occur at mammalian central synapses that are generated by the synaptic activation of NMDA and metabotropic glutamate receptors, respectively (Massey and Bashir, 2007). Several studies have shown that hormones and growth factors are also capable of inducing novel forms of LTD. Similarly, under conditions of enhanced excitability, exposure to leptin results in the induction of a novel form of NMDA receptor-dependent hippocampal LTD (Durakoglugil et al., 2005). Leptin-induced LTD shares similar expression mechanisms to activity-dependent LTD as LTD induced by low frequency stim- ulation (LFS) occludes leptin-induced LTD (Durakoglugil et al., FIGURE 1 \| Divergent effects of leptin on hippocampal synaptic function. Schematic representation of the key signaling pathways that contributes to the diverse effects of leptin in the hippocampus. Activation of leptin receptors triggers PI 3-kinase stimulation which results in AMPA receptor exocytosis and a sustained increased in synaptic efﬁcacy (leptin-induced LTP). Leptin driven stimulation of PI 3-kinase also enhances GluN2A activity which in turn promotes AMPA receptor delivery to synapses and subsequent LTP at adult hippocampal CA1 synapses. In contrast, at early stages of postnatal development, leptin dependent activation of the ERK (MAPK) signaling cascade facilitates GluN2B-mediated responses resulting in either persistent (LTD) or transient depressions in excitatory synaptic transmission. Leptin is also capable of depotentiating hippocampal synapses via a process involving the activation of calcineurin and subsequent endocytosis of GluA2-lacking AMPA receptors. Rapid alterations in dendritic morphology are also evoked by leptin that are mediated by the MAPK (ERK) signaling cascade. FIGURE 1 \| Divergent effects of leptin on hippocampal synaptic function postnatal development leptin dependent activation of the ERK (MAPK) FIGURE 1 \| Divergent effects of leptin on hippocampal synaptic function. LEPTIN RECEPTOR EXPRESSION IN THE CNS Nevertheless, more recent studies support a crucial role for NMDA receptors, as distinct GluN2 subunits play a key role in leptin’s effects on excitatory synaptic transmission at different stages of postnatal development and aging (Moult and Harvey, 2011). In addition, divergent signaling pathways are implicated in the age-dependent effects of leptin such that the ERK pathway mediates the synap- tic depressions evoked by leptin early in postnatal development whereas leptin-driven increases in excitatory synaptic strength in adult are PI 3-kinase dependent (Moult and Harvey, 2011). Previous studies revealed that distinct signaling pathways link leptin receptors to facilitation of molecularly distinct NMDA receptors in cerebellar granule cells (Irving et al., 2006). Thus, early in postnatal development it is possible that leptin driven ERK activation facilitates GluN2B-mediated responses thereby resulting in either persistent or transient depressions in excitatory synaptic transmission. Conversely in adult hippocampus, leptin increases GluN2A-mediated responses via PI 3-kinase which in turn promotes a long lasting increase in synaptic efﬁcacy (Moult and Harvey, 2011; Figure 1). LEPTIN REGULATION OF HIPPOCAMPAL SYNAPTIC FUNCTION PTIN REGULATION OF HIPPOCAMPAL SYNAPTIC FUNCTIO The hippocampus is a key brain area involved in learning and memory. Activity-dependent forms of hippocampal synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD) are key cellular events underlying learning, memory and habituation (Bliss and Collingridge, 1993). Recent studies indicate that leptin can modify excitatory synaptic trans- mission at hippocampal CA1 synapses (Shanley et al., 2001; Xu et al., 2008). Various forms of activity-dependent hippocampal synaptic plasticity are also regulated by this hormone. Indeed, electrophysiological studies have detected impairments in both LTP and LTD at hippocampal CA1 synapses in slices from leptin-insensitive rodents (fa/fa rats; db/db mice; Li et al., 2002). Defects in hippocampal memory tasks have also been observed in leptin-insensitive rodents (Li et al., 2002; Farr et al., 2006). Administration of leptin directly into the hippocampus not only enhances LTP but also boosts hippocampal-dependent learning and memory (Wayner et al., 2004). Cellular studies performed by Shanley et al. (2001) established that exposure to leptin promotes the conversion of short-term potentiation (STP) into LTP in acute hippocampal slices. LEPTIN REVERSES ESTABLISHED LTP It is known that LTP can be readily reversed shortly after induc- tion via a process known as depotentiation. This phenomenon is thought to boost the capacity of neuronal networks to store information by preventing saturation of potentiated synapses. Several studies indicate that application of theta burst stimuli or LFS can reverse LTP at hippocampal CA1 synapses (Bashir and Collingridge, 1994). Recent evidence indicates that leptin can also reverse established LTP, in a time and concentration-dependent manner (Moult et al., 2009). The ability of leptin to depotenti- ate hippocampal CA1 synapses is not associated with any change in paired-pulse ratio or coefﬁcient of variance (CV), suggesting a postsynaptic expression mechanism. Leptin-induced depotentia- tion also requires the activation of NMDA receptors as blockade of NMDA receptors with D-AP5 prevented the effects of leptin. A decrease in the rectiﬁcation properties of synaptic AMPA recep- tors accompanied leptin- induced depotentiation. Moreover the effects of leptin were mirrored by application of philanthotoxin, a selective inhibitor of GluA2-lacking AMPA receptors, indicating that a reduction in the density of GluA2-lacking AMPA receptors at hippocampal synapses underlies leptin-induced depotentiation (Moult et al., 2009). The involvement of AMPA receptor inter- nalization in leptin-induced depotentiation displays parallels to other studies as removal of AMPA receptors from synapses also contributes to the reversal of hippocampal LTP by LFS, mGluRs, and neuregulin (Zho et al., 2002; Kwon et al., 2005). Activation of NMDA receptors is pivotal for the induction of hippocampal LTP (Collingridge et al., 1983) as well as promoting AMPA receptor trafﬁcking to synapses during LTP (Collingridge et al., 2004). Recent evidence indicates that hippocampal synaptic plasticity is also associated with alterations in the subunit con- tent of AMPA receptors as synaptic activity increases the density of synaptic GluA2-lacking AMPA receptors (Isaac et al., 2007). Alterations in the molecular composition of AMPA receptors also accompany leptin-induced LTP as AMPA receptor rectiﬁcation increases after exposure to leptin whereas application of philan- thotoxin to block GluA2-lacking AMPA receptors reverses the leptin-induced increase in synaptic efﬁcacy (Moult et al., 2010). The surface expression of GluA1, but not GluA2, subunits is also elevated in hippocampal slices and cultured neurons treated with low nanomolar concentrations of leptin. LEPTIN-INDUCED LTD postnatal development, leptin dependent activation of the ERK (MAPK) signaling cascade facilitates GluN2B-mediated responses resulting in either persistent (LTD) or transient depressions in excitatory synaptic transmission. Leptin is also capable of depotentiating hippocampal synapses via a process involving the activation of calcineurin and subsequent endocytosis of GluA2-lacking AMPA receptors. Rapid alterations in dendritic morphology are also evoked by leptin that are mediated by the MAPK (ERK) signaling cascade. 1 \| Divergent effects of leptin on hippocampal synaptic func Schematic representation of the key signaling pathways that contributes to the diverse effects of leptin in the hippocampus. Activation of leptin receptors triggers PI 3-kinase stimulation which results in AMPA receptor exocytosis and a sustained increased in synaptic efﬁcacy (leptin-induced LTP). Leptin driven stimulation of PI 3-kinase also enhances GluN2A activity which in turn promotes AMPA receptor delivery to synapses and subsequent LTP at adult hippocampal CA1 synapses. In contrast, at early stages of August 2013 \| Volume 5 \| Article 3 \| 2 Frontiers in Synaptic Neuroscience www.frontiersin.org www.frontiersin.org Leptin regulates hippocampal synaptic function Harvey Harvey depression of excitatory synaptic transmission (Shanley et al., 2001; Xu et al., 2008). Conversely, in adult leptin evokes a long lasting increase in hippocampal excitatory synaptic strength (leptin-induced LTP) which is sustained following washout of leptin (Moult et al., 2010). Activation of leptin receptors is crucial for leptin-induced LTP as leptin has no effect in slices from Zucker fa/fa rats, whereas robust LTP is observed in Zucker lean animals. NMDA receptor activation is also essential as NMDA recep- tor blockade with D-AP5 prevents leptin-induced LTP (Moult et al., 2010). The ability of leptin to induce LTP in adult is likely to be mediated by GluN2A containing NMDA receptors as the leptin-driven increase in synaptic efﬁcacy is completely blocked by NVP-AAM077, an NMDA receptor antagonist with preferen- tial selectivity for GluN2A subunits (Auberson et al., 2002), but is unaffected by inhibition of GluN2B subunits with ifenprodil (Moult and Harvey, 2011; Figure 1). 2005). Moreover, like de novo LTD, leptin-induced LTD has a postsynaptic locus of expression as no alteration in the paired pulse ratio was observed during leptin-induced LTD. Moreover, inhibition of PI 3-kinase increased the magnitude of leptin- induced LTD suggesting that PI 3-kinase negatively regulates this process. LEPTIN-INDUCED LTD Similarly, inhibitors of serine/threonine protein phos- phatase 1/2A, but not 2B, also enhanced the synaptic depression induced by leptin, indicating that leptin-induced LTD is nega- tively regulated by phosphatase 1/2A (Durakoglugil et al., 2005). LEPTIN REVERSES ESTABLISHED LTP PI 3-kinase, which catalyses phosphorylation of PtdIns(45)P2 into PtdIns(3,4,5)P3, is not only an integral part of neuronal leptin receptor-driven signaling but it is also critical for traf- ﬁcking AMPA receptors to hippocampal synapses during LTP (Man et al., 2003). Similarly, the ability of leptin to increase GluA1 surface expression involves activation of PI 3-kinase as an increase in PtdIns(3,4,5)P3 levels accompanied the effects of leptin and the leptin-driven increase in both GluA1 expres- sion and PtdIns(3,4,5)P3 levels were blocked by selective PI 3-kinase inhibitors. However, PtdIns(3,4,5)P3 levels are also controlled by PTEN, a phosphatase that promotes conversion of PtdIns(3,4,5)P3 to PtdIns(4,5)P2, and thus antagonizes PI 3-kinase activity. In support of a role for PTEN, the leptin-driven increase in GluA1 surface expression is combined with an eleva- tion in P366-PTEN staining in hippocampal cultures. Moreover, application of leptin to hippocampal slices resulted in a robust increase in the phosphorylation of PTEN (Moult et al., 2010). In accordance with studies revealing that casein kinase 2 (CK2) phosphorylates PTEN, inhibition of CK2 prevented the effects of leptin on GluA1 surface expression, PTEN phosphorylation and excitatory synaptic transmission. Thus, it is feasible that leptin activates CK2 which phosphorylates and inhibits PTEN thereby increasing PtdIns(3,4,5)P3 levels and in turn driving alterations in AMPA receptor trafﬁcking and excitatory synaptic strength. Previous studies have identiﬁed a role for the stress-activated protein kinase JNK (c-Jun N-terminal kinase) in LFS-induced depotentiation (Zhu et al., 2005). In contrast, however, treatment of slices with selective inhibitors of JNK failed to alter the mag- nitude of leptin-induced depotentiation suggesting involvement of a JNK-independent process (Moult et al., 2009). However, calcineurin (protein phosphatase 2B) is implicated in NMDA receptor-driven removal of GluA2-lacking AMPA receptors from synapses (Beattie et al., 2000). In accordance with this, leptin- induced depotentiation is blocked by selective inhibitors of cal- cineurin, indicating a role for PP2B in this process (Figure 1). As dephosphorylation of GluA1 on Ser845 is pivotal for NMDA receptor-driven AMPA receptor endocytosis (Man et al., 2007), it is likely that leptin promotes the activation of calcineurin and subsequent dephosphorylation of GluR1 which in turn results in removal of GluA2-lacking AMPA receptors from hippocampal synapses. LEPTIN EVOKES A NOVEL FORM OF LTP IN ADULT HIPPOCAMPUS Application of leptin to acute hippocampal slices from juvenile (2–3 weeks old) rats results in a transient and readily reversible LEPTIN EVOKES A NOVEL FORM OF LTP IN ADULT HIPPOCAMPUS In further support of a role for PTEN, expression of dominant-negative PTEN mutants (C124S or G129E) in neurons Application of leptin to acute hippocampal slices from juvenile (2–3 weeks old) rats results in a transient and readily reversible August 2013 \| Volume 5 \| Article 3 \| 3 Frontiers in Synaptic Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Leptin regulates hippocampal synaptic function Harvey oppose to extrasynaptic NMDA receptors is pivotal for the mor- phological changes induced by leptin. It is known that PI 3-kinase and MAPK are two of the major signaling pathways activated by neuronal leptin receptors (Niswender et al., 2001; Harvey, 2003; Irving et al., 2006). Moreover, the ability of leptin to enhance NMDA receptor function in the hippocampus is medi- ated by both PI 3-kinase and MAPK (Shanley et al., 2001). However, only the MAPK signaling cascade is required for the rapid structural changes induced by leptin as inhibitors of MEK but not PI 3-kinase attenuated the effects of leptin on den- dritic morphology (Figure 1). Our previous studies indicate that actin ﬁlament reorganization underlies the activation of large- conductance Ca2+-activated K+ (BK) channels by leptin (Shanley et al., 2002a; O’Malley et al., 2005). However, a PI 3-kinase, rather than MAPK- dependent process mediates actin ﬁlament disrup- tion and subsequent activation of BK channels by leptin (Shanley et al., 2002a). Activation of PI 3-kinase by leptin has been shown to trigger localized elevations in phosphatidylinositol- 3,4,5-triphosphate [PtdIns(3,4,5)P3; (O’Malley et al., 2005)] lev- els in close proximity to the plasma membrane. Thus, it is likely that leptin-driven reorganization of actin ﬁlaments via this pathway occurs in a highly compartmentalized manner. In con- trast, however, activation of MAPK by leptin may promote more extensive alterations in the actin cytoskeleton as numerous down- stream targets for MAPK are widely expressed in dendritic and somatic regions in hippocampal neurons (Thomas and Huganir, 2004). or pharmacological inhibition of PTEN with the phosphatase inhibitor bisperoxovanadium (Bpv) mirror and occlude the effects of leptin on GluA1 trafﬁcking and hippocampal synaptic function (Moult et al., 2010). As inhibition of PTEN leads to an elevation in PtdIns(3,4,5)P3 levels, it is likely that alterations in the levels of this inositol lipid are crucial for modifying AMPA receptor trafﬁcking processes. Recent evidence supports this possibility as the availability of PtdIns(3,4,5)P3 is reported to maintain AMPA receptor clustering and synaptic function at hippocampal synapses (Arendt et al., 2010). LEPTIN PROMOTES MORPHOLOGICAL CHANGES IN HIPPOCAMPAL DENDRITES Alterations in the morphology of dendrites and spines are reported to occur after hippocampal activity-dependent synaptic plasticity and these structural changes are thought to contribute to modiﬁcations in excitatory synaptic strength (Maletic-Savatic et al., 1999). In addition, several hormones are reported to induce rapid morphological changes in neurons which can enable fur- ther regulation of neuronal connectivity and synaptic strength. Likewise, exposure of hippocampal neurons to leptin results in marked changes in the density and motility of dendritic ﬁlopodia within a few minutes (O’Malley et al., 2007). Time lapse con- focal microscopy studies also reveal that leptin rapidly increases the density and motility of dendritic ﬁlopodia in hippocampal neurons transfected with a cytosolic EGFP construct (O’Malley et al., 2007). The actin cytoskeleton is reported to play a key role in the morphological changes that occur during synaptic plastic- ity (Matus, 2000; Smart and Halpain, 2000). Thus, as leptin can rapidly alter the structure of actin ﬁlaments in hippocampal neu- rons and hypothalamic cell lines (O’Malley et al., 2005; Ning et al., 2006) it is feasible that leptin-driven formation of new ﬁlopo- dia involves alterations in actin cytoskeletal dynamics. Indeed, the leptin-dependent increase in dendritic ﬁlopodia is accompa- nied by a signiﬁcant reduction in polymerized actin staining in proximal dendrites, which is consistent with leptin driven redis- tribution of actin ﬁlaments from the dendritic shaft to dendritic ﬁlopodia. During the earliest stages of synaptogenesis there is increased movement and extension of dendritic ﬁlopodia (Fiala et al., 1998; Munno and Syed, 2003). Dendritic ﬁlopodia are also thought to play an active role in initiating synaptic contacts during synap- togenesis (Ziv and Smith, 1996). Thus, it is feasible that leptin, by increasing the density and motility of dendritic ﬁlopodia, alters the number of synaptic connections. Indeed, O’Malley et al. (2007) evaluated if leptin altered synaptic connectivity using immunocytochemical approaches to assay the relative number of presynaptic terminals. Exposure of hippocampal cultures to leptin for 30 min resulted in elevations in the density of actin- rich spines and also synapsin-1-positive puncta, which is consis- tent with leptin increasing the number of hippocampal synapses (O’Malley et al., 2007). The structural changes induced by leptin display many par- allels to the morphological changes observed following activity- dependent synaptic plasticity. LEPTIN EVOKES A NOVEL FORM OF LTP IN ADULT HIPPOCAMPUS Although the exact role of PtdIns(3,4,5)P3 remains to be determined, it is likely that PtdIns(3,4,5)P3 inﬂuences AMPA receptor trafﬁcking by either altering actin cytoskeletal dynamics (Zhou et al., 2001) or promoting Akt-driven inhibition of glycogen synthase kinase 3 (GSK-3; Peineau et al., 2007). Frontiers in Synaptic Neuroscience LEPTIN PROMOTES MORPHOLOGICAL CHANGES IN HIPPOCAMPAL DENDRITES Indeed, increases in the density of actin-rich spines and dendritic ﬁlopodia occur following hippocampal LTP, which parallels the morphological changes induced by leptin in hippocampal neurons (Maletic-Savatic et al., 1999; Fukazawa et al., 2003). However, the appearance of dendritic ﬁlopodia has also been linked to the induction of hippocampal LTD (Bourne and Harris, 2007). Thus, the pos- sibility that leptin-driven alterations in dendritic ﬁlopodia play a role in the persistent synaptic depressions induced by lep- tin cannot be excluded. The time course of the morphological changes induced by leptin is similar to those associated with LTP (Maletic-Savatic et al., 1999), and like LTP, the rapid growth of dendritic ﬁlopodia induced by leptin requires the synaptic activation of NMDA receptors (Maletic-Savatic et al., 1999). It has been shown that activation of synaptic NMDA recep- tors stimulates the appearance of dendritic protrusions (Maletic- Savatic et al., 1999) and neuronal exposure to glutamate regulates the formation and motility of dendritic ﬁlopodia (McKinney et al., 1999; Fischer et al., 2000). In accordance with these stud- ies, the synaptic activation of NMDA receptors is pivotal for the effects of leptin as the morphology of dendrites is unaffected by leptin in neurons treated with either tetrodotoxin or the com- petitive NMDA receptor antagonist, D-AP5. However, blockade of GluN2B containing NMDA receptors with ifenprodil failed to alter the leptin-driven structural changes indicating the likely involvement of GluN2A containing NMDA receptors (O’Malley et al., 2007). As GluN2A subunits are predominantly expressed at synaptic loci, it is likely that the activation of synaptic, as August 2013 \| Volume 5 \| Article 3 \| 4 www.frontiersin.org www.frontiersin.org www.frontiersin.org Leptin regulates hippocampal synaptic function Harvey Activation of MAPK is implicated in the structural changes associated with activity-dependent synaptic plasticity as NMDA receptor activation promotes increases in MAPK activity and den- dritic spine density in hippocampal neurons (Goldin and Segal, 2003), whereas new hippocampal dendritic spines and ﬁlopo- dia are formed after sustained activation of MAPK (Wu et al., 2001). Similarly, a role for MAPK is also thought to under- lie the effects of leptin as leptin failed to induce structural changes following blockade of MAPK activation (O’Malley et al., 2007). leptin and insulin in the hippocampus, it is likely that there is also cross-talk between the hormonal driven signaling path- ways in this brain region. LEPTIN PROMOTES MORPHOLOGICAL CHANGES IN HIPPOCAMPAL DENDRITES This possibility is supported by studies in hypothalamic neurons as insulin not only mimics the abil- ity of leptin to activate ATP-sensitive K+ (KATP) channels but channel activation by both hormones involves a PI 3-kinase- dependent process (Harvey et al., 2000; Spanswick et al., 2000). PI3-Kinase is also implicated in the activation of hypothalamic large-conductance Ca2+-activated K+ (BK) channels by leptin and insulin (Yang et al., 2010). Thus, modulation of PI3K signal- ing may act as a point of convergence for the regulation of hip- pocampal synaptic function by the leptin and insulin hormonal systems. In addition to regulating hippocampal neuron morphology, recent evidence indicates that leptin has the capacity to alter the morphology of neuronal populations. Thus, leptin promotes neurite outgrowth from hypothalamic (Bouret et al., 2004) and cerebellar purkinje neurons (Oldreive et al., 2008). Exposure to leptin also alters the size of axonal growth cones in cortical neu- rons (Valerio et al., 2006). Studies in leptin deﬁcient (ob/ob) mice have identiﬁed signiﬁcant alterations in the density of both excitatory and inhibitory synapses compared to wild type mice (Pinto et al., 2004). Furthermore, these alterations are normal- ized within 6 h of systemic administration of leptin to ob/ob mice. However, the morphological changes induced by leptin in hypothalamic, cerebellar, and cortical neurons occur on a much slower time scale than in the hippocampus, as structural changes were only evident after treatment with leptin for several hours (Bouret et al., 2004; Valerio et al., 2006; Oldreive et al., 2008). Both the PI 3-Kinase and the Ras/Raf/MEK signaling pathways are activated by other class I cytokines, including interleukin 6 (IL-6). Moreover, as evidence is growing that IL-6 is a potent reg- ulator of hippocampal synaptic function (Tancredi et al., 2000; Nelson et al., 2012), the possibility that there is also convergence between leptin and IL-6 driven signaling cascades in hippocam- pal neurons cannot be excluded. It is also feasible that other hormonal systems indirectly inﬂuence leptin-driven regulation of hippocampal synaptic function by modulating leptin levels. In support of this possibility, insulin-like growth factor-1 (IGF-1) is reported to increase the expression of leptin in organotypic hippocampal slices (Marwarha et al., 2011). Although there is some evidence supporting potential interactions between lep- tin and other hormonal systems in the hippocampus, it is clear that a greater understanding of the interplay between different hormones is required. LEPTIN PROMOTES MORPHOLOGICAL CHANGES IN HIPPOCAMPAL DENDRITES In particular it is key that the com- plex cellular events underlying synaptic plasticity and how such processes are modulated by hormonal systems are more fully understood. POTENTIAL CROSS-TALK BETWEEN LEPTIN AND OTHER HORMONAL SYSTEMS IN THE HIPPOCAMPUS Numerous studies indicate that leptin is an important modula- tor of activity-dependent synaptic plasticity at different stages of postnatal development and aging. However, although numerous growth factors and hormones are capable of regulating hip- pocampal synaptic function, it remains to be determined if the effects of leptin are inﬂuenced by other hormonal systems. It is feasible that there is not only convergence of function but also potential cross-talk between hormonal systems at the level of the signal transduction pathways. Indeed, the endocrine hor- mone insulin that is secreted by pancreatic beta cells in response to elevated glucose levels displays many parallels to leptin in terms of its central actions and also the signaling pathways that it activates. Thus, in a manner similar to leptin, insulin reduces food intake when administered centrally (Schwartz et al., 1992). Insulin derived from peripheral sources is also readily transported into the brain where it can regulate hippocam- pal synaptic function. Indeed, at hippocampal CA1 synapses, insulin promotes endocytosis of GluA2 and subsequent induc- tion of a novel form of NMDA receptor-dependent LTD (Man et al., 2000; Ahmadian et al., 2004; Huang et al., 2004). Like leptin, insulin is capable of facilitating NMDA responses and increasing NMDA receptor exocytosis (Liu et al., 1995; Skeberdis et al., 2001). Furthermore, exposure of hippocampal neurons to insulin stimulates an increase in the cell surface expression of GluA1 (Man et al., 2000; Passafaro et al., 2001), which paral- lels the actions of leptin on GluA1 trafﬁcking processes (Moult et al., 2010). Thus, given the overlapping functional effects of Frontiers in Synaptic Neuroscience REFERENCES H., and Thompson, S. M. (1999). Miniature synaptic events maintain dendritic spines via AMPA receptor activation. Nat. Neurosci. 2, 44–49. Fukazawa, Y., Saitoh, Y., Ozawa, F., Ohta, Y., Mizuno, K., and Inokuchi, Fukazawa, Y., Saitoh, Y., Ozawa, F., Ohta, Y., Mizuno, K., and Inokuchi, K. (2003). Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP mainte- nance in vivo. Neuron 38, 447–460. Li, X. L., Aou, S., Oomura, Y., Hori, N., Fukunaga, K., and Hori, T. (2002). Impairment of long-term potentiation and spatial memory in leptin receptor-deﬁcient rodents. Neuroscience 113, 607–615. K. (2003). Hippocampal LTP is accompanied by enhanced F-actin content within the dendritic spine that is essential for late LTP mainte- nance in vivo. Neuron 38, 447–460. Banks, W. A., Kastin, A. J., Huang, W., Jaspan, J. B., and Maness, L. M. (1996). Leptin enters the brain by a saturable system independent of insulin. Peptides 17, 305–311. doi: 10.1016/0196-9781(96)00025-3 Mercer, J. G., Hoggard, N., Williams, L. M., Lawrence, C. B., Hannah, L. T., and Trayhurn, P. (1996). Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypotha- lamus and adjacent brain regions by in situ hybridization. FEBS Lett. 387, 113–116. Goldin, M., and Segal, M. (2003). Protein kinase C and ERK involve- ment in dendritic spine plastic- ity in cultured rodent hippocam- pal neurons. Eur. J. Neurosci. 17, 2529–2539. Liu, L., Brown, J. C. 3rd., Webster, W. W., Morrisett, R. A., and Monaghan, D. T. (1995) Insulin potentiates N- methyl-D-aspartate receptor activ- ity in Xenopus oocytes and rat hip- pocampus. Neurosci. Lett. 192, 5–8. Bashir, Z. I., and Collingridge, G. L. (1994). An investigation of depoten- tiation of long-term potentiation in the CA1 region of the hippocampus. Exp. Brain Res. 100, 437–443. doi: 10.1007/BF02738403 Hâkansson, M. L., Brown, H., Ghilardi, N., Skoda, R. C., and Meister, B. (1998). Leptin receptor immunore- activity in chemically deﬁned tar- get neurons of the hypothalamus. J. Neurosci. 18, 559–572. Maffei, M., Halaas, J., Ravussin, E., Pratley, R. E., Lee, G. H., Zhang, Y., et al. (1995). Leptin levels in human and rodent: measurement of plasma leptin and ob RNA in obese and weight-reduced sub- jects. Nat. Med. 1, 1155–1161. doi: 10.1038/nm1195-1155 Moult, P. R., Cross, A., Santos, S. D., Carvalho, A. L., Lindsay, Y., Connolly, C. N., et al. (2010). Leptin regulates AMPA receptor trafﬁcking via PTEN inhibition. J. REFERENCES insertion during LTP of mEPSCs in cultured hippocampal neu- rons. Neuron 38, 611–624. doi: 10.1016/S0896-6273(03)00228-9 via a mitogen-activated pro- tein kinase-dependent process in cerebellar granule cells. Neuroscience 138, 1137–1148. doi: 10.1016/j.neuroscience.2005.11.042 Durakoglugil, M., Irving, A. J., and Harvey, J. (2005). Leptin induces a novel form of NMDA receptor- dependent long-term depression. J. Neurochem. 95, 396–405. Ahmadian, G., Ju, W., Liu, L., Wyszynski, M., Lee, S. H., Dunah, A. W., et al. (2004). Tyrosine phos- phorylation of GluR2 is required for insulin-stimulated AMPA receptor endocytosis and LTD. EMBO J. 23, 1040–1050. Marwarha, G., Prasanthi, J. R., Schommer, J., Dasari, B. and Ghribi, O. (2011). Molecular inter- play between leptin, insulin-like growth factor-1, and β-amyloid in organotypic slices from rabbit hippocampus. Mol. Neurodegener. 6, 41 doi: 10.1186/1750-1326-6-41 Farr, S. A., Banks, W. A., and Morley, J. E. (2006). Effects of leptin on memory processing. Peptides 27, 1420–1425. doi: 10.1016/j.peptides.2005.10.006 Isaac, J. T., Ashby, M. C., and McBain, C. J. (2007). The role of the GluR2 subunit in AMPA receptor function and synaptic plasticity. Neuron 54, 859–871. Arendt, K. L., Royo, M., Fernández- Monreal, M., Knafo, S., Petrok, C. N., Martens, J. R., et al. (2010). PIP3 controls synaptic function by maintaining AMPA receptor clus- tering at the postsynaptic mem- brane. Nat. Neurosci. 13, 36–44. doi: 10.1038/nn.2462 Karsenty, G. (2006). Convergence between bone and energy home- ostases: leptin regulation of bone mass. Cell Metab. 4, 341–348. doi: 10.1016/j.cmet.2006.10.008 Fiala, J. C., Feinberg, M., Popov, V., and Harris, K. M. (1998). Synaptogenesis via dendritic ﬁlopo- dia in developing hippocampal area CA1. J. Neurosci. 18, 8900–8911. Massey, P. V., and Bashir, Z. I. (2007). Long-term depression: multiple forms and implica- tions for brain function. Trends Neurosci. 30, 176–184. doi: 10.1016/j.tins.2007.02.005 Fischer, M., Kaech, S., Wagner, U., Brinkhaus, H., and Matus, A. (2000). Glutamate receptors reg- ulate actin-based plasticity in dendritic spines. Nat. Neurosci. 3, 887–894. Kwon, O. B., Longart, M., Vullhorst, D., Hoffman, D. A., and Buonanno, A. (2005). Neuregulin-1 reverses long-term potentiation at CA1 hippocampal synapses. J. Neurosci. 25, 9378–9383. doi: 10.1523/JNEUROSCI.2100-05.2005 Auberson, Y. P., Allgeier, H., Bischoff, S., Lingenhoehl, K., Moretti, R., and Schmutz, M. (2002). 5-Phosphono- methylquinoxalinediones as com- petitive NMDA receptor antagonists with a preference for the human 1A/2A, rather than 1A/2B receptor composition. Bioorg. Med. Chem. Lett. 12, 1099–1102. Matus, A. (2000). Actin-based plastic- ity in dendritic spines. Science 290, 754–758. McKinney, R. A., Capogna, M., Durr, R., Gahwiler, B. REFERENCES Neurosci. 30, 4088–4101. Beattie, E. C., Carroll, R. C., Yu, X., Morishita, W., Yasuda, H., von Zastrow, M., et al. (2000). Regulation of AMPA receptor endo- cytosis by a signaling mechanism shared with LTD. Nat. Neurosci. 3, 1291–1300. doi: 10.1038/81823 Harvey, J. (2003). Novel actions of leptin in the hippocampus. Ann. Med. 35, 197–206. doi: 10.1080/07853890310008251 Moult, P. R., and Harvey, J. (2011). NMDA receptor subunit compo- sition determines the polarity of leptin-induced synaptic plasticity. Neuropharmacology 61, 924–936. doi: 10.1016/j.neuropharm.2011.06.021 Maletic-Savatic, M., Malinow, R., and Svoboda, K. (1999). Rapid dendritic morphogenesis in CA1 hippocam- pal dendrites induced by synap- tic activity. Science 283, 1923–1927. doi: 10.1126/science.283.5409.1923 Bliss, T. V., and Collingridge, G. L. (1993). A synaptic model of mem- ory: long-term potentiation in the hippocampus. Nature 361, 31–39. doi: 10.1038/361031a0 Harvey, J., McKay, N. G., Walker, K. S., Van der Kaay, J., Downes, C. P., and Ashford, M. L. (2000). Essential role of phosphoinosi- tide 3-kinase in leptin-induced K(ATP) channel activation in the rat CRI-G1 insulinoma cell line. J. Biol. Chem. 275, 4660–4669. doi: 10.1074/jbc.275.7.4660 Man, H. Y., Lin, J. W., Ju, W. H., Ahmadian, G., Liu, L., Becker, L. E., et al. (2000). Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization. Neuron 25, 649–662. doi: 10.1016/S0896-6273 (00)81067-3 Bouret, S. G., Draper, S. J., and Simerly, R. B. (2004). Trophic action of leptin on hypothalamic neurons that regulate feeding. Science 304, 108–110. Moult, P. R., Milojkovic, B., and Harvey, J. (2009). Leptin reverses long-term potentiation at hippocampal CA1 synapses. J. Neurochem. 108, 685–696. doi: 10.1111/j.1471-4159.2008.05810.x Harvey, J., Shanley, L. J., O’Malley, D., and Irving, A. J. (2005). Leptin: a potential cognitive enhancer? Biochem. Soc. Trans. 33, 1029–1032. Bourne, J., and Harris, K. M. (2007). Do thin spines learn to be mush- room spines that remember? Curr. Opin. Neurobiol. 17, 381–386. Munno, D. W., and Syed, N. I. (2003). Synaptogenesis in the CNS: an odyssey from wiring together to ﬁr- ing together. J. Physiol. 552, 1–11. Man, H. Y., Sekine-Aizawa, Y., and Huganir, R. L. (2007). Regulation of {alpha}-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptor trafﬁcking through PKA phosphorylation of the Glu recep- tor 1 subunit. Proc. Natl. Acad. Sci. U.S.A. 104, 3579–3584. doi: 10.1073/pnas.0611698104 Huang, C. C., Lee, C. C., and Hsu, K. S. (2004). An investigation into signal transduction mecha- nisms involved in insulin-induced long-term depression in the CA1 region of the hippocampus. J. Neurochem. CONCLUSION It is well established that the endocrine hormone leptin regulates many central processes including energy homeostasis. However, evidence is growing that the structure and function of hippocam- pal CA1 synapses is also markedly inﬂuenced by leptin. Recent studies indicate that leptin has cognitive enhancing properties as it rapidly alters glutamate receptor trafﬁcking, dendritic mor- phology and different forms of activity-dependent hippocampal synaptic plasticity. Regulation of NMDA receptor activity by lep- tin appears to be key for its ability to inﬂuence multiple aspects of hippocampal synaptic function, although it is not entirely clear how leptin-driven activation of NMDARs leads to such oppos- ing effects on hippocampal synaptic function. However, emerging studies indicate not only that distinct NMDA receptor subunits are pivotal for leptin’s effects on excitatory synaptic transmission, but also that distinct signaling pathways couple leptin receptors to molecularly distinct NMDA receptors at different developmen- tal stages. Although it is established that leptin plays a pivotal role in normal brain function, disruption of the leptin system is also linked to neurodegenerative disorders, like Alzheimer’s disease. Thus, the ability of leptin to regulate neuronal morphol- ogy and synaptic efﬁcacy is likely to have important implications not only in health but also in diseases associated with leptin dysfunction. August 2013 \| Volume 5 \| Article 3 \| 5 Frontiers in Synaptic Neuroscience www.frontiersin.org Leptin regulates hippocampal synaptic function Harvey REFERENCES Identiﬁcation of targets of leptin action in rat hypothalamus. J. Clin. Invest. 98, 1101–1106. doi: 10.1172/JCI118891 Tartaglia, L. A., Dembski, M., Weng, X., Deng, N., Culpepper, J., Devos, R., et al. (1995). Identiﬁcation and expression cloning of a leptin recep- tor, OB-R. Cell 83, 1263–1271. doi: 10.1016/0092-8674(95)90151-5 Zhou, Q., Xiao, M., and Nicoll, R. A. (2001). Contribution of cytoskeleton to the internalization of AMPA receptors. Proc. Natl. Acad. Sci. U.S.A. 98, 1261–1266. doi: 10.1073/pnas.98.3.1261 Oldreive, C. E., Harvey, J., and Doherty, G. H. (2008). Neurotrophic effects of leptin on cerebellar Purkinje but not granule neurons in vitro. Neurosci. Lett. 438, 17–21. Shanley, L. J., Irving, A. J., and Harvey, J. (2001). Leptin enhances NMDA receptor function and mod- ulates hippocampal synaptic plastic- ity. J. Neurosci. 21:RC186. Thomas, G. M., and Huganir, R. L. (2004). MAPK cascade signalling and synaptic plasticity. Nat. Rev. Neurosci. 5, 173–183. Zhu, Y., Pak, D., Qin, Y., McCormack, S. G., Kim, M. J., Baumgart, J. P., et al. (2005). Rap2-JNK removes synap- tic AMPA receptors during depo- tentiation. Neuron 46, 905–916. doi: 10.1016/j.neuron.2005.04.037 O’Malley, D., Irving, A. J., and Harvey, J. (2005). Leptin-induced dynamic alterations in the actin cytoskeleton mediate the activation and synaptic clustering of BK channels. FASEB J. 19, 1917–1919. Shanley, L. J., Irving, A. J., Rae, M. G., Ashford, M. L., and Harvey, J. (2002a). Leptin inhibits rat hip- pocampal neurons via activation of large conductance calcium- activated K+ channels. Nat. Neurosci. 5, 299–300. Valerio, A., Ghisi, V., Dossena, M., Tonello, C., Giordano, A., Frontini, A., et al. (2006). Leptin increases axonal growth cone size in devel- oping mouse cortical neurons by convergent signals inactivating glycogen synthase kinase-3beta. J. Biol. Chem. 281, 12950–12958. doi: 10.1074/jbc.M508691200 Ziv, N. E., and Smith, S. J. (1996). Evidence for a role of dendritic ﬁlopodia in synaptogenesis and spine formation. Neuron 17, 91–102. O’Malley, D., MacDonald, N., Mizielinska, S., Connolly, C. N., Irving, A. J., and Harvey, J. (2007). Leptin promotes rapid dynamic changes in hippocam- pal dendritic morphology. Mol. Cell. Neurosci. 35, 559–572. doi: 10.1016/j.mcn.2007.05.001 Shanley, L. J., O’Malley, D., Irving, A. J., Ashford, M. L., and Harvey, J. (2002b). Leptin inhibits epileptiform-like activity in rat hippocampal neurones via PI 3-kinase-driven activation of BK channels. J. Physiol. 545, 933–944. Wayner, M. J., Armstrong, D. L., Phelix, C. F., and Oomura, Y. (2004). Wayner, M. J., Armstrong, D. L., Phelix, C. F., and Oomura, Y. (2004). REFERENCES 89, 217–231 doi: 10.1111/j.1471-4159.2003.02307.x Collingridge, G. L., Isaac, J. T., and Wang, Y. T. (2004). Receptor traf- ﬁcking and synaptic plasticity. Nat. Rev. Neurosci. 5, 952–962. doi: 10.1038/nrn1556 Nelson, T. E., Olde Engberink, A., Hernandez, R., Puro, A., Huitron- Resendiz, S., Hao, C., et al. (2012). Altered synaptic transmission in the hippocampus of transgenic mice with enhanced central nervous systems expression of interleukin- 6. Brain Behav. Immun. 26, 959–971. doi: 10.1016/j.bbi.2012. 05.005 Collingridge, G. L., Kehl, S. J., and McLennan, H. (1983). Excitatory amino acids in synaptic trans- mission in the Schaffer collateral- commissural pathway of the rat hip- pocampus. J. Physiol. 334, 33–46. Irving, A. J., Wallace, L., Durakoglugil, D., and Harvey, J. (2006). Leptin enhances NR2B-mediated N- methyl-D-aspartate responses Man, H. Y., Wang, Q., Lu, W. Y., Ju, W., Ahmadian, G., Liu, L., et al. (2003). Activation of PI3-kinase is required for AMPA receptor August 2013 \| Volume 5 \| Article 3 \| 6 Frontiers in Synaptic Neuroscience www.frontiersin.org Leptin regulates hippocampal synaptic function Harvey Harvey arcuate nucleus feeding circuits by leptin. Science 304, 110–115. arcuate nucleus feeding circuits by leptin. Science 304, 110–115. arcuate nucleus. Am. J. Physiol. Endocrinol. Metab. 298, E193–E201. doi: 10.1152/ajpendo.00155.2009 Ning, K., Miller, L. C., Laidlaw, H. A., Burgess, L. A., Perera, N. M., Downes, C. P., et al. (2006). A novel leptin signalling pathway via PTEN inhibition in hypothalamic cell lines and pancreatic beta-cells. EMBO J. 25, 2377–2387. Tancredi, V., D’Antuono, M., Cafè, C., Giovedì, S., Buè, M. C., D’Arcangelo, G., et al. (2000). The inhibitory effects of interleukin-6 on synaptic plasticity in the rat hippocampus are associated with an inhibition of mitogen-activated protein kinase ERK. J. Neurochem. 75, 634–643. Schwartz, M. W., Figlewicz, D. P., Baskin, D. G., Woods, S. C., and Porte, D. Jr. (1992). Insulin in the brain: a hormonal regulator of energy balance. Endocr. Rev. 13, 387–414. Zho, W. M., You, J. L., Huang, C. C., and Hsu, K. S. (2002). The group I metabotropic glutamate receptor agonist (S)-3,5- dihydroxyphenylglycine induces a novel form of depotentiation in the CA1 region of the hippocampus. J. Neurosci. 22, 8838–8849. Niswender, K. D., Morton, G. J., Stearns, W. H., Rhodes, C. J., Myers, M. G. Jr., and Schwartz, M. W. (2001). Intracellular signalling. Key enzyme in leptin-induced anorexia. Nature 413, 794–795. doi: 10.1038/35101657 Schwartz, M. W., Seeley, R. J., Campﬁeld, L. A., Burn, P., and Baskin, D. G. (1996). REFERENCES Orexin-A (Hypocretin-1) and leptin enhance LTP in the dentate gyrus of rats in vivo. Peptides 25, 991–996. doi: 10.1016/j.peptides. 2004.03.018 Conﬂict of Interest Statement: The author declares 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 author declares that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Skeberdis, V. A., Lan, J., Zheng, X., Zukin, R. S., and Bennett, M. V. (2001). Insulin promotes rapid delivery of N-methyl-D- aspar- tate receptors to the cell surface by exocytosis. Proc. Natl. Acad. Sci. U.S.A. 98, 3561–3566. doi: 10.1073/pnas.051634698 Oomura, Y., Hori, N., Shiraishi, T., Fukunaga, K., Takeda, H., Tsuji, M., et al. (2006). Leptin facilitates learn- ing and memory performance and enhances hippocampal CA1 long- term potentiation and CaMK II phosphorylation in rats. Peptides 27, 2738–2749. Wu, G. Y., Deisseroth, K., and Tsein, R. W. (2001). Spaced stimuli stabi- lize MAPK pathway activation and its effects on dendritic morphology. Nat. Neurosci. 4, 151–158. Received: 21 May 2013; accepted: 12 July 2013; published online: 06 August 2013. Citation: Harvey J (2013) Leptin reg- ulation of neuronal morphology and hippocampal synaptic function. Front. Synaptic Neurosci. 5:3. doi: 10.3389/ fnsyn.2013.00003 Received: 21 May 2013; accepted: 12 July 2013; published online: 06 August 2013. Citation: Harvey J (2013) Leptin reg- ulation of neuronal morphology and hippocampal synaptic function. Front. Synaptic Neurosci. 5:3. doi: 10.3389/ fnsyn.2013.00003 Xu, L., Rensing, N., Yang, X. F., Zhang, H. X., Thio, L. L., Rothman, S. M., et al. (2008). Leptin inhibits 4-aminopyridine- and pentylenetetrazole-induced seizures and AMPAR-mediated synaptic transmission in rodents. J. Clin. Invest. 118, 272–280. doi: 10.1172/JCI33009 Passafaro, M., Piëch, V., and Sheng, M. (2001). Subunit-speciﬁc tempo- ral and spatial patterns of AMPA receptor exocytosis in hippocampal neurons. Nat. Neurosci. 4, 917–926. doi: 10.1038/nn0901-917 Smart, F. M., and Halpain, S. (2000). Regulation of dendritic spine stability. Hippocampus 10, 542–554. Copyright © 2013 Harvey. 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 origi- nal author(s) or licensor are credited and that the original publication in this jour- nal is cited, in accordance with accepted academic practice. REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2013 Harvey. 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 origi- nal author(s) or licensor are credited and that the original publication in this jour- nal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Spanswick, D., Smith, M. A., Mirshamsi, S., Routh, V. H., and Ashford, M. L. (2000). Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats. Nat. Neurosci. 3, 757–758. doi: 10.1038/77660 Peineau, S., Taghibiglou, C., Bradley, C., Wong, T. P., Liu, L., Lu, J., et al. (2007). LTP inhibits LTD in the hippocampus via regulation of GSK3beta. Neuron 53, 703–717. doi: 10.1016/j.neuron.2007.01.029 Yang, M. J., Wang, F., Wang, J. H., Wu, W. N., Hu, Z. L., Cheng, J., et al. (2010). PI3K integrates the effects of insulin and leptin on large-conductance Ca2+-activated K+ channels in neuropeptide Y neurons of the hypothalamic Spiegelman, B. M., and Flier, J. S. (2001). Obesity and the regula- tion of energy balance. Cell 104, 531–543. Pinto, S., Roseberry, A. G., Liu, H., Diano, S., Shanabrough, M., Cai, X., et al. (2004) Rapid rewiring of August 2013 \| Volume 5 \| Article 3 \| 7 Frontiers in Synaptic Neuroscience www.frontiersin.org
https://openalex.org/W4221150162	https://ir.cwi.nl/pub/31979/31979.pdf	English	null	GPU-Accelerated Parallel Gene-pool Optimal Mixing in a Gray-Box Optimization Setting	arXiv (Cornell University)	2,022	cc-by	9,768	1 INTRODUCTION In a Gray-Box Optimization (GBO) setting that allows for partial evaluations, the fitness of an individual can be updated efficiently after a subset of its variables has been modified. This enables more efficient evolutionary optimization with the Gene-pool Optimal Mixing Evolutionary Algorithm (GOMEA) due to its key strength: Gene-pool Optimal Mixing (GOM). For each solution, GOM per- forms variation for many (small) sets of variables. To improve effi- ciency even further, parallel computing can be leveraged. For EAs, typically, this comprises population-wise parallelization. However, unless population sizes are large, this offers limited gains. For large GBO problems, parallelizing GOM-based variation holds greater speed-up potential, regardless of population size. However, this potential cannot be directly exploited because of dependencies between variables. We show how graph coloring can be used to group sets of variables that can undergo variation in parallel with- out violating dependencies. We test the performance of a CUDA implementation of parallel GOM on a Graphics Processing Unit (GPU) for the Max-Cut problem, a well-known problem for which the dependency structure can be controlled. We find that, for suffi- ciently large graphs with limited connectivity, finding high-quality solutions can be achieved up to 100 times faster, showcasing the great potential of our approach. Evolutionary computation has been used to tackle many real-world problems in the past, including engineering problems [12], vehicle routing [17, 18], and even the treatment of cancer [9, 21]. Though Evolutionary Algorithms (EAs) are historically most often consid- ered as Black-Box Optimization (BBO) algorithms, many real-world problems exist where some domain knowledge about the problem, or even the entire problem definition, is known, eliminating the necessity of tackling the problem in a BBO setting. Instead, the problem can then be optimized in a Gray-Box Optimization (GBO) setting, where (a limited amount of) domain knowledge is used to improve the effectiveness or the efficiency of the optimization algo- rithm. Having such information available does not mean that the problem is not complex and can be solved using simple heuristics or convex optimization techniques. Acknowledging this, EAs have, for example, previously used custom recombination operators [13], problem decompositions [11], or partial evaluations [6], to achieve great success where other optimization methods failed. GPU-Accelerated Parallel Gene-Pool Optimal Mixing in a Gray-Box Optimization Setting Peter A.N. Bosman Centrum Wiskunde & Informatica Amsterdam, The Netherlands Peter.Bosman@cwi.nl Anton Bouter Centrum Wiskunde & Informatica Amsterdam, The Netherlands Anton.Bouter@cwi.nl 1 INTRODUCTION In this paper, we specifically consider a GBO setting where partial evaluations, by which we mean that evaluating the change in fitness after only a few variables have changed, can be done (proportionally) more efficiently than when a full evaluation is performed. For the optimization of many real-world problems, it is further- more important that the total computation time is minimized, e.g., in the case where a patient is waiting to be treated for cancer [21]. For this reason, and because EAs naturally lend themselves to par- allelization due to their population-based approach, meaning that the entire population can be evaluated in parallel, much successful research has been done into the application of parallel computing techniques and EAs [1, 26]. More recently, much of this research has been focused on large-scale parallelization using Graphics Process- ing Units (GPUs) [20, 31], because the clock frequencies of Central Processing Units (CPUs) are nearing their physical limits [2, 8], meaning that it is more effective to increase the number of parallel cores rather than increasing the frequency of each of these cores. • Mathematics of computing →Evolutionary algorithms. • Mathematics of computing →Evolutionary algorithms. KEYWORDS Parallel Computing, CUDA, Gray-Box Optimization, Optimal Mix- ing ACM Reference Format: ACM Reference Format: Anton Bouter and Peter A.N. Bosman. 2022. GPU-Accelerated Parallel Gene- Pool Optimal Mixing in a Gray-Box Optimization Setting. In Genetic and Evolutionary Computation Conference (GECCO ’22), July 9–13, 2022, Boston, MA, USA. ACM, New York, NY, USA, 10 pages. https://doi.org/10.1145/ 3512290.3528797 In comparison to parallelization on a number of CPUs, the appli- cation of GPUs is generally required to be much more fine-grained, however, because of the thousands of cores present in modern GPUs, and the Single Instruction, Multiple Data (SIMD) computa- tional model used, which requires groups of threads on the GPU to perform the same (computational) instructions simultaneously. Therefore, unless population sizes are vast, only evaluating the population in parallel will not have a high enough degree of paral- lelizability to fully utilize the computational resources of a GPU. GECCO ’22, July 9–13, 2022, Boston, MA, USA © 2022 Copyright held by the owner/author(s). ACM ISBN 978-1-4503-9237-2/22/07. https://doi.org/10.1145/3512290.3528797 This work is licensed under a Creative Commons Attribution International 4.0 License. GECCO ’22, July 9–13, 2022, Boston, MA, USA © 2022 Copyright held by the owner/author(s). ACM ISBN 978-1-4503-9237-2/22/07. https://doi.org/10.1145/3512290.3528797 This work is licensed under a Creative Commons Attribution International 4.0 License. In particular, the Gene-pool Optimal Mixing Evolutionary Algo- rithm (GOMEA) [28] has excellent synergy with a GBO setting, be- cause the variation steps of GOMEA, performed with the Gene-pool 675 GECCO ’22, July 9–13, 2022, Boston, MA, USA Anton Bouter and Peter A.N. Bosman Optimal Mixing (GOM) variation operator, are applied to subsets of variables, and are only accepted if they do not decrease the fitness of the parent. Moreover, often, many relatively small such subsets are used. In a GBO setting, such modifications to subsets of variables can be efficiently evaluated using partial evaluations. Such a GBO setting can be further exploited by using the fact that separate steps in GOM, which consider different subsets, are independent when the respective sets of modified variables are mutually independent. Therefore, if such dependence information is known, a higher de- gree of parallelizability can be achieved by performing conditionally independent partial evaluations in parallel. This means that, for each solution in the population, a large number of variation steps can be evaluated in parallel, as long as these subsets of variables are conditionally independent. ACM Reference Format: This was previously showcased in the domain of continuous optimization and applied to the real-world problem of deformable image registration, where these independent subsets could be manually identified and hard-coded a priori [7]. the notation 𝒙𝒀with 𝑌⊆I to denote the subset of variables of 𝒙restricted to the indices in 𝑌, i.e., 𝒙𝒀= [𝑥𝑌1,𝑥𝑌2, . . . ,𝑥𝑌\|𝑌\|] with 𝒀= [𝑌1,𝑌2, . . . ,𝑌\|𝑌\|]. The set 𝕀= [𝕀1, 𝕀2, . . . , 𝕀𝑞] is given by the problem definition, and determines which variables are required as input for each of the subfunctions. Consequently, each subfunction 𝑓𝑖, defined as 𝑓𝑖: Z\|𝕀𝑖\| →R, is assumed to be non-separable, and is furthermore treated as a black box. The GBO fitness function 𝑓is then defined as an aggregation over all subfunctions, as follows: 𝑓(𝑥) = 𝑔 𝑓1(𝒙𝕀1) ⊕𝑓2(𝒙𝕀2) ⊕· · · ⊕𝑓𝑞(𝒙𝕀𝑞) , (1) (1) 𝑓(𝑥) = 𝑔 𝑓1(𝒙𝕀1) ⊕𝑓2(𝒙𝕀2) ⊕· · · ⊕𝑓𝑞(𝒙𝕀𝑞) , with ⊕a binary commutative operator that has a known inverse ⊖, e.g., addition or multiplication, and 𝑔: R →R, named the mapping function, any (possibly non-linear) function aggregating the output of all subfunctions to the domain of the fitness function. Note that the domain of the output of each subfunction, and that of the input of the mapping function 𝑔, are not required to be the continuous domain R. Instead, it could be any (possibly high-dimensional) domain D𝜅, or a product of different domains, as long as the output domain of each subfunction is identical to the domain of the input of the mapping function 𝑔, , i.e., 𝑓𝑖: Zℓ→D𝜅 and 𝑔: D𝜅→R. In this paper, for the sake of simplicity, and the fact that it applies to most (real-world) GBO problems, we restrict the definition of GBO to the domain where the output of each subfunction is in the continuous domain. i In this paper, we introduce the parallel application of GOM in the discrete domain. Moreover, we introduce a general method using graph coloring to identify conditionally independent subsets of variables, removing the need to hard-code these for a specific problem instance a priori. 2.2 Partial Evaluationsi After the modification of a variable 𝑥𝑖of a solution 𝒙, the fitness of the modified solution 𝒙′ can be efficiently computed through a partial evaluation. This partial evaluation requires the computation of all subfunctions 𝑓𝑗that require 𝑥𝑖as an input variable, i.e., all 𝑓𝑗 for which 𝑖∈𝕀𝑗. If the mapping function 𝑔is the identity function, updating the fitness value of the solution is done by subtracting (more generally, using the ⊖operator) the previous contribution of the subfunction to the fitness, and adding (more generally, using the ⊕operator) the current contribution of the subfunction to the fitness. If 𝑔is not the identity function, it is required to keep track of the sum of all subfunctions, i.e., 𝑓1(𝒙𝕀1) ⊕𝑓2(𝒙𝕀2) ⊕· · ·⊕𝑓𝑞(𝒙𝕀𝑞), for each individual in the population. The subtractions and additions of subfunctions are then applied to this sum, and used as input for the mapping function to find the fitness value. ACM Reference Format: Such conditionally independent subsets of variables can be found by applying graph coloring to the Variable Interaction Graph (VIG) [29], i.e., the graph that describes problem variables as vertices and mutual dependencies between pairs of problem variables as edges, which can be derived from any problem to which partial evaluations can be applied. We create a CUDA [24] implementation of parallel GOM, and apply it to the well-known NP- hard Max-Cut problem [19], because the graph describing a MaxCut instance directly translates to the VIG, making it a problem with an easily controllable dependency structure. However, everything we described here that is needed to make the CUDA implementation work, may be applied to any problem that allows for a GBO setting where partial evaluations can be applied. We analyze the benefit of parallel GOM for graphs with different structures, and compare it to the original non-parallelized GOMEA. 2.1 Decomposable Fitness Functionsi We define the (discrete) optimization function 𝑓(𝒙) : Zℓ→R as the optimization function of interest that is subject to maximization. This optimization function maps a solution 𝒙= [𝑥1,𝑥2, . . . ,𝑥ℓ], i.e., an instantiation of the set of all ℓproblem variables 𝑿= [𝑋1,𝑋2, . . . ,𝑋ℓ], to a fitness value 𝑓(𝒙) ∈R. The set of problem variables is indexed through I = [1, 2, . . . , ℓ].i 2.3 Variable Interaction Graph The structure of (variable interactions in) an optimization problem can be captured in the VIG [29]. This graph VIG = (𝑽VIG, 𝑬VIG) consists of a set of vertices 𝑽VIG, one for each problem variable, and a set of edges 𝑬VIG. Each edge (𝑢, 𝑣) ∈𝑬VIG denotes that variables 𝑋𝑢and 𝑋𝑣are dependent, i.e., a subfunction 𝑓𝑖∈𝑭exists that requires both 𝑋𝑢and 𝑋𝑣as input. An example of a VIG of a 5-dimensional problem is displayed in Figure 1. In this paper, we specifically consider a GBO setting that allows for partial evaluations, previously defined for a continuous opti- mization setting [5]. This means that the fitness of a solution can be efficiently updated after a modification to a small number of variables has been made. For partial evaluations to be performed, it must be known (from domain knowledge or otherwise) how the fit- ness function is constructed from any number of subfunctions. For this purpose, we define the set of 𝑞subfunctions 𝑭= {𝑓1, 𝑓2, . . . , 𝑓𝑞} that compose the fitness function 𝑓. Each subfunction 𝑓𝑖∈𝑭is a function of a subset of problem variables of 𝒙, where the indices of 𝒙that this subfunction is restricted to, are defined by 𝕀𝑖. We use 1 2 3 4 5 Figure 1: Example of a VIG of a 5-dimensional problem. 1 2 3 4 5 Figure 1: Example of a VIG of a 5-dimensional problem. 676 GECCO ’22, July 9–13, 2022, Boston, MA, USA GPU-Accelerated Parallel Gene-Pool Optimal Mixing in a Gray-Box Optimization Setting Algorithm 1 GOMEA Algorithm 1 GOMEA 1: procedure GOMEA(𝑛) 2: P ←InitializePopulation(𝑛) 3: while not TerminationCriterionSatisfied() do 4: F ←LearnLinkageModel(P) 5: O ←∅ 6: for 𝒙∈P do ⊲Iterate over solutions 7: 𝒐←clone(𝒙) 8: for F𝑖∈F do ⊲Random order 9: 𝒅←SelectDonorFromPopulation(P, F𝑖) 10: 𝒐←GOM(𝒐, 𝒅, F𝑖) 11: 𝒐←ApplyForcedImprovement(𝒐) 12: O ←O ∪𝒐 13: P ←O : procedure GOMEA(𝑛) Due to the fact that GPUs contain a large number of computational units, and the fact that CPU cores are close to reaching their physical limits [2, 8], GPUs are well known to be among the state of the art for high-performance computing, accelerating and enabling wide-spread use of deep learning. Also in the field of evolutionary computation, the large-scale parallel computing power of GPUs has previously been used [20, 31]. In this paper, we use NVIDIA GPUs, for which code is developed in CUDA. These GPUs consist of a large number, up to approx- imately 10,000 in modern GPUs, of computational units, named CUDA cores. Parallel computing on NVIDIA GPUs is done with the Single Instruction/Multiple Data (SIMD) computational model, which limits the flexibility of the computations compared to a reg- ular, serial computational model. Executing computations on the GPU is done through kernels, which are functions that are executed in parallel for a large number of threads organized in a grid. Within such a grid, threads are distributed into thread blocks, for which the size must be specified in the CUDA code. Each of these threads execute the exact same code, but may access different data by using the thread ID, i.e., the location of the thread in the grid. Algorithm 2 Gene-pool Optimal Mixing Algorithm 2 Gene-pool Optimal Mixing Algorithm 2 Gene-pool Optimal Mixing 1: procedure GOM(𝒐, 𝒅, F𝑗) 2: 𝒐′ ←𝒅[F𝑗] ⊲Variables of 𝒅restricted to indices in F𝑗 3: Δ𝑓′𝑜←PartialEvaluation(𝒐′, 𝒐, 𝑓𝑥, F𝑗) 4: 𝑓′𝑜←𝑓𝑜+ Δ𝑓′𝑜 5: acceptVariation ←False 6: if 𝑓′𝑜> 𝑓𝑜then 7: acceptVariation ←True 8: else if 𝑓′𝑜== 𝑓𝑜and not EqualGenotype(𝒐, 𝒙elitist) then 9: acceptVariation ←True 10: if acceptVariation then 11: 𝑓𝑜←𝑓𝑜+ Δ𝑓′𝑜 ⊲Update fitness of 𝒐 12: 𝒐[F𝑗] ←𝒐′ ⊲Update genotype for indices in F𝑗 1: procedure GOM(𝒐, 𝒅, F𝑗) It is beyond the scope of this paper to provide more details of GPU architectures and computation models for which we refer the interested reader to relevant literature [23, 24]. 4 GENE-POOL OPTIMAL MIXING EVOLUTIONARY ALGORITHM The Gene-pool Optimal Mixing Evolutionary Algorithm (GOMEA) [28] is a Model-Based Evolutionary Algorithm (MBEA), of which the main strength is the Gene-pool Optimal Mixing (GOM) vari- ation operator. This variation operator uses an explicit linkage model, describing interactions between problem variables in terms of groups of variables called linkage sets, to perform crossover with groups of highly dependent variables, and only accepting variation operations that do not decrease the fitness of the solution. As such, dependencies in the optimization problem are exploited, because important building blocks are not disrupted, yet mixed well. the current linkage set, and continues to the next iteration. The ApplyForcedImprovement function applies the Forced Improve- ment (FI) [4] procedure to solutions for which no variation step of GOM in the current generation was accepted, or for which the fitness has not improved for a certain number of generations. Dur- ing the FI procedure, the respective solution 𝒐undergoes variation with GOM for each linkage model. However, instead of randomly selecting a donor from the population, the elitist solution is used as the donor. Moreover, if any such variation step of GOM improves the fitness of 𝒐, the FI procedure is terminated. If instead, at the end of the FI procedure, no improvement was made to 𝒐, a copy of the elitist solution 𝒙elitist takes its place in the population. A population P of size 𝑛is maintained by GOMEA, which is typically initialized uniformly at random, although problem-specific information can be leveraged here as well. During each generation of GOMEA, GOM is applied to each individual in the population, for each linkage set in the linkage model. Pseudo-code for GOMEA is displayed in Algorithm 1. 3 GENERAL PURPOSE GRAPHICS PROCESSING UNITS Algorithm 1 GOMEA 5 PARALLEL GENE-POOL OPTIMAL MIXING Unweighted Pair Grouping Method with Arithmetic mean (UP- GMA) [15] clustering algorithm, continuously merging the pairs of linkage sets that are considered to be the most strongly depen- dent. Dependence between sets is defined as the pairwise average notion of dependence between all variables in one set and all vari- ables in the other set. Formally stated, for each linkage set F𝑖∈F of size larger than 1, there exists exactly one pair of linkage sets F𝑗, F𝑘∈F such that F𝑗∩F𝑘= ∅and F𝑗∪F𝑘= F𝑖. An example of an LT is shown in Figure 2. Note that the linkage set containing all problem variables is not included in the LT, because using this set as a crossover mask would simply result in the copying of the donor solution. Unweighted Pair Grouping Method with Arithmetic mean (UP- GMA) [15] clustering algorithm, continuously merging the pairs of linkage sets that are considered to be the most strongly depen- dent. Dependence between sets is defined as the pairwise average notion of dependence between all variables in one set and all vari- ables in the other set. Formally stated, for each linkage set F𝑖∈F of size larger than 1, there exists exactly one pair of linkage sets F𝑗, F𝑘∈F such that F𝑗∩F𝑘= ∅and F𝑗∪F𝑘= F𝑖. An example of an LT is shown in Figure 2. Note that the linkage set containing all problem variables is not included in the LT, because using this set as a crossover mask would simply result in the copying of the donor solution. When GOM is applied to a specified subset of variables F𝑖, a partial evaluation is required that depends only on a subset of variables. In particular, it depends on all variables in F𝑖, and any other variable for which a connection exists in the VIG to a variable in F𝑖. It does not depend on any other variables. Therefore, a number of such partial evaluations may be performed in parallel. When the map- ping function 𝑔in Equation 1 is the identity function, the decision whether each of these variation steps needs to be accepted, is also independent, allowing them to be performed in parallel as well. Moreover, this means that two applications of GOM that do not share any dependent variables, are completely independent, and can therefore be performed in parallel. 5.2 Operationalizing Parallelization Potential p g In order to divide the linkage model into the smallest possible number of mutually independent sets, we first create a graph L similar to the VIG of the optimization problem. In contrast to the VIG, the graph L shows interactions between linkage sets instead of interactions between variables. We therefore name this graph the Linkage Model Interaction Graph (LMIG). We define the graph L = (𝑽, 𝑬) with 𝑽= [1, 2, . . . , \|F \|] and 𝑬such that there exists an edge (𝑖, 𝑗) ∈𝑬iff linkage sets F𝑖and F𝑗are dependent. Two linkage sets F𝑖and F𝑗are dependent when F𝑖∩F𝑗≠∅, or an edge (𝑢, 𝑣) exists in the VIG such that 𝑢∈F𝑖and 𝑣∈F𝑗. The problem of finding the minimum number of mutually independent sets within the linkage model F is now equivalent to finding the minimum graph coloring of L. The application of graph coloring for this purpose within GOMEA was previously suggested in [7], and was, in a similar way, applied to a parallel hill climber for the optimization of NK-landscapes [14]. Because graph coloring is an NP-complete problem, finding the optimal minimum graph coloring would be computationally infeasible. Therefore, we use the greedy Welsh-Powell algorithm [30] to find a graph coloring. An example of a colored LMIG, given the VIG displayed in Figure 1 and the linkage model displayed in Figure 2, is shown in Figure 3. 5 PARALLEL GENE-POOL OPTIMAL MIXING {1} {3} {2} {4} {5} {1,3} {1,2,3} {4,5} F1 F2 F3 F4 F5 F6 F7 F8 {1} {3} {2} {4} {5} {1,3} {1,2,3} {4,5} F1 F2 F3 F4 F5 F6 F7 F8 Figure 2: An example of an LT for 5 problem variables, where each node indicates that a linkage set exists contain- ing the problem variables with the denoted indices. Figure 2: An example of an LT for 5 problem variables, where each node indicates that a linkage set exists contain- ing the problem variables with the denoted indices. 4.1 Linkage Model The linkage model used by GOMEA is a Family Of Subsets (FOS) F = {F1, F2, . . . , F𝑚}, with F𝑖⊆I for each F𝑖∈F. Each element F𝑖of the linkage model, named a linkage set, defines a subset of variables that is considered to be strongly dependent. Note that, each FOS used to describe a linkage model should be complete, i.e., contain each problem variable in at least one linkage set. In Algorithm 1, the InitializePopulation function initializes a population P of size 𝑛uniformly at random. At the start of each generation, a linkage model is learned based on the population P. In a GBO setting, however, it may be possible to learn a linkage model offline, before optimization, based on domain knowledge, and keeping it constant throughout a run of GOMEA. In this setting, the linkage model learning procedure at the start of each generation would therefore be omitted. The SelectDonorFromPopulation function randomly selects a donor from the population for which the genotype, restricted to the problem variables in the respective linkage set, are not equal to that of the parent solution 𝒐. If no such donor is present in the population, GOM is not performed for Various linkage models exist. The model most commonly adopted, especially in a BBO setting, is the Linkage Tree (LT). An LT can capture hierarchical dependencies and consists of linkage sets of various sizes, from just a single variable, up to a linkage set of at most ℓ−1 variables. An LT firstly consists of all univariate elements, while all larger linkage sets are the union of exactly two linkage sets. This hierarchical model is constructed using the hierarchical 677 GECCO ’22, July 9–13, 2022, Boston, MA, USA Anton Bouter and Peter A.N. Bosman 5 PARALLEL GENE-POOL OPTIMAL MIXING For large-scale problems with relatively sparse VIGs, it is possible that many such applications of GOM are mutually independent, and may therefore be performed in parallel in the procedure that we name parallel GOM. Moreover, since partial evaluations within different individuals in the popula- tion are also independent, there are potentially many parallel steps possible, which fits well with the computing architecture of GPUs. An LT is generally learned at the start of each generation based on mutual information in the population [28], though it is possible to learn an LT prior to the optimization process based on domain knowledge. Such an LT is generally referred to as a Fixed Linkage Tree (FLT). In particular, this is interesting in a GBO setting that allows for partial evaluations, because it is known from the VIG which variables are (in)dependent. Furthermore, an LT may be bounded, in which case, merges of linkage sets that would create linkage sets above the maximum linkage set size would be avoided, and the learning process halts when no more linkage sets with an allowed size can be created. In this case, the LT is referred to as a Bounded Fixed Linkage Tree (BFLT). Prior to performing parallel GOM, we divide all linkage sets into 𝑘groups of mutually independent linkage sets, where we aim to minimize 𝑘in order to maximize the potential for parallelizability. Since each linkage set in such a group is independent of all other linkage sets within the same group, all iterations of GOM with linkage sets within the same group may be performed in parallel. For this purpose, we define the set G = {G1, G2, . . . , G𝑘} with G𝑗⊆F and G𝑖∩G𝑗= ∅for each G𝑖, G𝑗∈G. Furthermore, because all linkage sets are distributed into groups, for each linkage set F𝑗∈F , there exists exactly one G𝑖∈G that contains F𝑗. {1} {3} {2} {4} {5} {1,3} {1,2,3} {4,5} F1 F2 F3 F4 F5 F6 F7 F8 Figure 2: An example of an LT for 5 problem variables, where each node indicates that a linkage set exists contain- ing the problem variables with the denoted indices. 4.2 Gene-pool Optimal Mixing The key strength of GOMEA comes from its use of the GOM varia- tion operator, with which variation is applied to subsets of variables at a time, determined by the linkage model, and variation steps that lead to a deterioration of the fitness are rejected During each gener- ation of GOMEA, the GOM operator is applied to each individual 𝒙 in the population P, using each linkage F𝑖set in the linkage model F . Before applying GOM to the parent solution 𝒙, a donor solution is randomly selected from the population. A requirement for this donor is that its genotype, restricted to the variables included in the linkage set F𝑖, is not identical to that of the parent solution 𝒙. If no individual in the population adheres to this requirement, this iteration of GOM, i.e., with specified linkage set F𝑖for the speci- fied parent 𝒙, is not applied during this generation. When GOM is applied to the parent solution 𝒙using linkage set F𝑖and donor solution 𝒅, all genes specified by the linkage set F𝑖are copied from the donor 𝒅to the parent 𝒙, and the modification is then evalu- ated, if possible using a partial evaluation. Pseudo-code of GOM is displayed in Algorithm 2. 678 GPU-Accelerated Parallel Gene-Pool Optimal Mixing in a Gray-Box Optimization Setting GECCO ’22, July 9–13, 2022, Boston, MA, USA F1 F2 F3 F4 F5 F6 F7 F8 Figure 3: The LMIG of a 5-dimensional problem, given the VIG displayed in Figure 1, and the linkage model displayed in Figure 2. A possible graph coloring is shown that de- fines the distribution of linkage sets into the groups G = {{F1, F4}, {F2, F7}, {F3}, {F5}, {F6}, {F8}}. Note that there is a limited number of linkage sets with the same color, because this example is very small. F1 F2 F3 F4 F5 F6 F7 F8 of these sets of subfunctions, such that each subfunction has a key that uniquely depends on the dependent linkage set and the parent solution. All subfunctions in the list of dependent subfunctions are then evaluated in parallel for all solutions in the offspring, i.e., the solutions to which variation has been applied and are required to be evaluated, and their results stored in a matrix of fitness value contributions. In parallel, for all solutions in the population, the values of all dependent subfunctions are evaluated. 5.3 GPU Implementation Pseudo-code of parallel GOMEA is displayed in Algorithm 3. In the function DetermineAndInsertDonorGenes, firstly, a suitable donor from the population P is selected for each of the 𝑛\|G𝑖\| it- erations of GOM, and inserted into a copy of the offspring O′. The selection of the donor for a specific GOM iteration, i.e., for a specific individual and a specific linkage set, is done by one thread block, because this enables the use of all threads within the thread block to check whether the donor is equal to the par- ent, which is required by GOM. Secondly, once a donor has been found that is not equal to the parent, a copy of its genes, restricted to those in the specified linkage set, are inserted into the parent within a copy of the offspring population, named O′. The partial fitness contributions for each of the 𝑛\|G𝑖\| modifications caused by DetermineAndInsertDonorGenes are then evaluated in paral- lel in the ParallelPartialEvaluations function. For each of the modifications, the fitness of the parent is updated, and the fitness contribution is stored in the 𝑛× \|G𝑖\| matrix 𝚫𝒇𝑶, which stores the contribution of the GOM iteration with linkage set F𝑗applied to in- dividual P[𝑖] at position [𝑖, 𝑗]. This matrix is then used to determine which of the variation steps must be accepted, which is done in the function DetermineImprovements. In this function, the 𝑛× \|G𝑖\| matrix 𝑴IMP is computed, which contains a 1 at position [𝑖, 𝑗] when the GOM iteration linkage set F𝑗applied to individual P[𝑖] must be accepted. This is the case when 𝚫𝒇𝑶′ [𝑖, 𝑗] is larger than 0, or when 𝚫𝒇𝑶′ [𝑖, 𝑗] is equal to 0 and the individual P[𝑖] is not equal to the elitist solution. Otherwise, the GOM iteration must be rejected, and a 0 is placed in position [𝑖, 𝑗] of the matrix 𝑴IMP. Since all variation steps, those that are accepted and those that are rejected, have been applied to O′ in the function DetermineAndInsertDonorGenes, the rejected variation steps now need to be restored to their backup state O, which is done in the function ResetNonImprovements. Similarly, in the function InsertImprovements, all accepted varia- tion steps are applied to the population, meaning that the offspring O and its copy O′ are now exact copies again. 4.2 Gene-pool Optimal Mixing These results are then subtracted from the matrix of fitness value contributions, meaning that this matrix now describes, for each subfunction, the difference in fitness contribution caused by the respective variation step. Note that it is possible to store the fitness contribution values of all subfunctions of the population to avoid their recalculation, though this requires a larger amount of memory. Figure 3: The LMIG of a 5-dimensional problem, given the VIG displayed in Figure 1, and the linkage model displayed in Figure 2. A possible graph coloring is shown that de- fines the distribution of linkage sets into the groups G = {{F1, F4}, {F2, F7}, {F3}, {F5}, {F6}, {F8}}. Note that there is a limited number of linkage sets with the same color, because this example is very small. The thrust library [3] is then used to apply a parallel reduction by key, using the keys matrix and the fitness value contribution matrix. This results in a pair of vectors, one with all occurring keys, and one with the total sum of all fitness contributions for each of the occurring keys. These vectors are transformed to the matrix 𝚫𝒇𝑶such that each element contains the change in fitness caused the one variation step of GOM applied to a particular solution in the population. 5.3 GPU Implementation Algorithm 3 Parallel GOMEA (GBO) 1: procedure Par-GOMEA(𝑛, VIG) 2: P ←InitializePopulation(𝑛) 3: F ←LearnLinkageModel(VIG) 4: G ←GraphColoring(F) 5: while not TerminationCriterionSatisfied() do 6: O ←Clone(P) 7: for G𝑖∈G do ⊲Random order 8: O′ ←DetermineAndInsertDonorGenes(G𝑖, O, P) 9: 𝚫𝒇O′ ←ParallelPartialEvaluations(G𝑖, O, O′) 10: 𝑴IMP ←DetermineImprovements(G𝑖, O′, Δ𝑓O′) 11: O′ ←ResetNonImprovements(G𝑖, O, 𝑴IMP) 12: O ←InsertImprovements(G𝑖, O′, 𝑴IMP) 13: P ←O Algorithm 3 Parallel GOMEA (GBO) 1: procedure Par-GOMEA(𝑛, VIG) 2: P ←InitializePopulation(𝑛) 3: F ←LearnLinkageModel(VIG) 4: G ←GraphColoring(F) 5: while not TerminationCriterionSatisfied() do 6: O ←Clone(P) 7: for G𝑖∈G do ⊲Random order 8: O′ ←DetermineAndInsertDonorGenes(G𝑖, O, P) 9: 𝚫𝒇O′ ←ParallelPartialEvaluations(G𝑖, O, O′) 10: 𝑴IMP ←DetermineImprovements(G𝑖, O′, Δ𝑓O′) 11: O′ ←ResetNonImprovements(G𝑖, O, 𝑴IMP) 12: O ←InsertImprovements(G𝑖, O′, 𝑴IMP) 13: P ←O Algorithm 3 Parallel GOMEA (GBO) Algorithm 3 Parallel GOMEA (GBO) Algorithm 4 Parallel Partial Evaluations , \|O′\|] do 26: ind𝑓←𝑲dep [𝑢] + 𝑣· \|𝑲dep \| 27: 𝚫𝒇𝑶[𝑢, 𝑣] ←𝑭out O′ [ind𝑓] −𝑭out O [ind𝑓] 28: return 𝚫𝒇𝑶[𝑢, 𝑣] 6.1 Overall Set-up All experiments with the serial version of GOMEA are executed on an Intel Xeon CPU E5-2630 v4 core with a clock frequency of 2.20GHz. The experiments performed on a GPU are executed on an NVIDIA Geforce RTX 2080 Ti, which consists of 4352 CUDA cores at a frequency of 1.54 GHz, and 11 GB of global memory. For the population size parameter, we use the Interleaved Multi-start Scheme (IMS) (see Section 6.3). Unless otherwise mentioned, all versions of GOMEA use an LT learned based on the Max-Cut graph with UPGMA using the weights of the graph as a notion of similarity. Furthermore, default parameters are used unless specified. We refer to the CUDA implementation of GOMEA, using parallel GOM, as parallel GOMEA, and the original, serial version of GOMEA, as serial GOMEA. 6 EXPERIMENTS In this section, we benchmark the performance of parallel GOM, and compare it to that of the original, serial, GOMEA. The set-up of these experiments is first described in Section 6.1, followed by the description of the benchmark problems in Section 6.2. We then described various experiments in Sections 6.3 through 6.5. 6.2 Benchmark Problems We consider the design of a procedure akin to FI that is more amenable to parallelization also a topic of future work.f • Set A consists of fully connected graphs with 6 up to 200 vertices, and 15 up to 19,900 edges. • Set B consists of graphs on a 3D torus, i.e., a 2D grid with wrap-around on all edges, with 9 up to 1600 vertices, and 18 up to 3200 edges. For each graph in Set B, the connectivity, i.e., number of connected edges, of each vertex is equal to 4. Any of these changes may have a potential effect on the conver- gence of GOMEA. Therefore, they are evaluated in Section 6.6. Any of these changes may have a potential effect on the conver- gence of GOMEA. Therefore, they are evaluated in Section 6.6. • Set C consists of a selection of graphs from the BIQMAC library [25], named g1, g22, g55, g60, g65, g66, g72, g77, and g81. These graphs contain 800, 2000, 5000, 7000, 8000, 9000, 10000, 14000, and 20000 vertices, respectively. A table of all properties is included in the supplementary material. 6.2 Benchmark Problems We focus on the well-known NP-complete (Weighted) Maximum Cut (Max-Cut) problem [19], because it has a clearly defined depen- dency structure that allows for a clear demonstration of the use of parallel GOM. Furthermore, all techniques introduced in this paper directly apply to other optimization problems for which partial evaluations are possible, and, consequently, the VIG is known. The objective of the Max-Cut problem is, given a weighted graph 𝐺= (𝑉, 𝐸), to assign each vertex in 𝑉to a set S or its complement, and to maximize the total sum of weights of the edges between vertices in complementing sets. Formally, given a graph 𝐺= (𝑉, 𝐸), the weight function 𝑤(𝑖, 𝑗) that defines the weight of the edge between each pair of vertices 𝑖and 𝑗in𝑉, and a binary solution 𝒙of length ℓ= \|𝑉\|, the optimization function, subject to maximization, of the Max-Cut problem is defined as follows: max 𝑓Max-Cut(𝒙) = 1 2 Õ 𝑖,𝑗∈𝑉;𝑖<𝑗 𝑤𝑖𝑗 1 −𝑥𝑖𝑥𝑗 , (2) s.t. 𝑤𝑖𝑗∈R, 𝑖, 𝑗∈𝑉, (3) 𝑥𝑖,𝑥𝑗∈[−1, 1], 𝑖, 𝑗∈𝑉. (4) max 𝑓Max-Cut(𝒙) = 1 2 Õ 𝑖,𝑗∈𝑉;𝑖<𝑗 𝑤𝑖𝑗 1 −𝑥𝑖𝑥𝑗 , (2) s.t. 𝑤𝑖𝑗∈R, 𝑖, 𝑗∈𝑉, (3) 𝑥𝑖,𝑥𝑗∈[−1, 1], 𝑖, 𝑗∈𝑉. (4) (2) Secondly, learning an LT based on the population at the start of each generation is computationally expensive, easily becoming a bottleneck when the amount of computation time dedicated to GOM is largely reduced. Instead, a fixed LT can be learned prior to optimization based on domain knowledge, greatly reducing re- quired computation time. It is, however, possible to parallelize the UPGMA procedure that is used for the construction of the LT [10], which we consider a potential topic of future work. s.t. 𝑤𝑖𝑗∈R, 𝑥𝑖,𝑥𝑗∈[−1, 1], Generally speaking, in a GA, each solution 𝒙has binary variables 𝑥𝑖∈[0, 1], but this is trivially mapped to the domain [−1, 1]. For our experiments, we use three sets of Max-Cut instances with different properties: Set A, Set B, and Set C. Thirdly, the Forced Improvement (FI) procedure is disabled in the parallel version of GOMEA, because it is by nature a serial procedure that halts as soon as one of the steps results in an im- provement. Furthermore, FI is generally applied to only a small number of solutions at once, unlike GOM. Algorithm 4 Parallel Partial Evaluations Algorithm 4 Parallel Partial Evaluations Algorithm 4 Parallel Partial Evaluations 1: procedure ParallelPartialEvaluations(G𝑖, O, O′) 2: 𝑭dep, 𝑲dep, 𝑴dep, 𝚫𝒇𝑶←[] 3: // Determine subfunctions to be evaluated 4: for F𝑗∈G𝑖do 5: 𝑭𝑗←∅ ⊲Dependent subfunctions of F𝑗 6: for 𝑢∈F𝑗do 7: 𝑭𝑗←𝑭𝑗∪DependentSubfunctions(𝑢) 8: for 𝑢∈𝑭𝑗do 9: 𝑭dep.append(𝑢) ⊲Dependent subfunctions 10: 𝑲dep.append(𝑗) ⊲Respective linkage sets 11: // Create matrix of unique keys 12: parfor 𝑢∈[1, 2, . . . , \|𝑲dep \|] do 13: parfor 𝑣∈[1, 2, . . . , \|O′\|] do 14: 𝑴dep [𝑢, 𝑣] ←𝑲dep [𝑢] + 𝑣· \|𝑲dep \| 15: // Evaluate dependent subfunctions 16: parfor 𝑢∈[1, 2, . . . , \|O \|] do ⊲O and O′ have equal sizes 17: parfor 𝑣∈𝑭dep do ⊲Dependent subfunctions 18: 𝑭𝑝𝑎𝑟𝑡 O [𝑢, 𝑣] ←EvaluateSubfunction(O𝑢, 𝑣) 19: 𝑭𝑝𝑎𝑟𝑡 O′ [𝑢, 𝑣] ←EvaluateSubfunction(O′𝑢, 𝑣) 20: // Determine sum of evaluated subfunctions 21: 𝑭out O ←ReduceByKey(𝑭𝑝𝑎𝑟𝑡 O , 𝑴dep) 22: 𝑭out O′ ←ReduceByKey(𝑭𝑝𝑎𝑟𝑡 O′ , 𝑴dep) 23: // Matrix of fitness change for all variation steps 24: parfor 𝑢∈[1, 2, . . . , \|𝑲dep \|] do 25: parfor 𝑣∈[1, 2, . . . , \|O′\|] do 26: ind𝑓←𝑲dep [𝑢] + 𝑣· \|𝑲dep \| 27: 𝚫𝒇𝑶[𝑢, 𝑣] ←𝑭out O′ [ind𝑓] −𝑭out O [ind𝑓] 28: return 𝚫𝒇𝑶[𝑢, 𝑣] Algorithm 4 Parallel Partial Evaluations 1: procedure ParallelPartialEvaluations(G𝑖, O, O′) 2: 𝑭dep, 𝑲dep, 𝑴dep, 𝚫𝒇𝑶←[] 3: // Determine subfunctions to be evaluated 4: for F𝑗∈G𝑖do 5: 𝑭𝑗←∅ ⊲Dependent subfunctions of F𝑗 6: for 𝑢∈F𝑗do 7: 𝑭𝑗←𝑭𝑗∪DependentSubfunctions(𝑢) 8: for 𝑢∈𝑭𝑗do 9: 𝑭dep.append(𝑢) ⊲Dependent subfunctions 10: 𝑲dep.append(𝑗) ⊲Respective linkage sets 11: // Create matrix of unique keys 12: parfor 𝑢∈[1, 2, . . . , \|𝑲dep \|] do 13: parfor 𝑣∈[1, 2, . . . , \|O′\|] do 14: 𝑴dep [𝑢, 𝑣] ←𝑲dep [𝑢] + 𝑣· \|𝑲dep \| 15: // Evaluate dependent subfunctions 16: parfor 𝑢∈[1, 2, . . . , \|O \|] do ⊲O and O′ have equal sizes 17: parfor 𝑣∈𝑭dep do ⊲Dependent subfunctions 18: 𝑭𝑝𝑎𝑟𝑡 O [𝑢, 𝑣] ←EvaluateSubfunction(O𝑢, 𝑣) 19: 𝑭𝑝𝑎𝑟𝑡 O′ [𝑢, 𝑣] ←EvaluateSubfunction(O′𝑢, 𝑣) 20: // Determine sum of evaluated subfunctions 21: 𝑭out O ←ReduceByKey(𝑭𝑝𝑎𝑟𝑡 O , 𝑴dep) 22: 𝑭out O′ ←ReduceByKey(𝑭𝑝𝑎𝑟𝑡 O′ , 𝑴dep) 23: // Matrix of fitness change for all variation steps 24: parfor 𝑢∈[1, 2, . . . , \|𝑲dep \|] do 25: parfor 𝑣∈[1, 2, . . . 5.4 Differences with Serial GOM Due to the serial nature of GOM, and the SIMD architecture of GPUs, some aspects of GOMEA do not translate well to GPUs. Therefore, some of these aspects of GOMEA have been adapted or left out, because they would have a large impact on the paralleliza- tion potential of GOMEA when left unchanged. Firstly, in the serial GOMEA, GOM is applied to one individual with all linkage sets, before moving to the next individual. Because this order of operations would leave the parallelization potential of the population unused, this order is changed in the parallel GOM, opening the possibility that a large number of steps of GOM are performed for each of the solutions in the population in paral- lel. Additionally, because linkage sets are divided into mutually independent groups, the order in which GOM is applied to the population is no longer uniformly at random. Instead, only the order of the groups is uniformly at random, which can potentially introduce a bias. These changes in the order of GOM are, however, unavoidable to enable parallelization. In particular, pseudocode for the ParallelPartialEvaluations is displayed in Algorithm 4. This procedure starts with finding all subfunctions dependent on the linkage sets to which GOM is ap- plied in parallel, and the initialization of a matrix of keys for each 679 GECCO ’22, July 9–13, 2022, Boston, MA, USA Anton Bouter and Peter A.N. Bosman 6.3 Interleaved Multi-start Scheme To prevent having to tune the population size of each of the algo- rithms by hand, we use the Interleaved Multi-start Scheme (IMS) 680 GECCO ’22, July 9–13, 2022, Boston, MA, USA GPU-Accelerated Parallel Gene-Pool Optimal Mixing in a Gray-Box Optimization Setting sizes, only a small increase in performance is observed over serial GOMEA. No optimum was known for graphs with more than 1600 nodes, prohibiting extending the scalability analysis, although from the results it is to be expected that large speed-ups may well be possible for larger graphs, which we consider in the next subsection. [6, 16]. In this scheme, the generations of multiple populations of dif- ferent sizes are performed in an interleaved way, with smaller pop- ulations iterating through generations at a higher frequency. The smallest population in the IMS, denoted P1, starts at the base pop- ulation size 𝑛base, and each newly initialized population is double the size of the previously largest population, i.e., \|P𝑖\| = 2𝑖−1𝑛base. Generations of all populations are performed recursively, such that one generation of population P𝑖is performed as soon as population P𝑖−1 has performed 𝑐IMS generations. 0.001 0.01 0.1 1 10 100 10 100 Time (s) Dimensionality Serial Parallel (a) Set A 0.001 0.01 0.1 1 10 100 10 100 1000 Time (s) Dimensionality Serial Parallel (b) Set B Figure 5: Scalability of serial and parallel GOMEA, showing the median and interdecile range (30 runs) of the time re- quired to find the optimum, for the instances in set A and set B, respectively. 0.001 0.01 0.1 1 10 100 10 100 1000 Time (s) Dimensionality Serial Parallel (b) Set B 0.001 0.01 0.1 1 10 100 10 100 Time (s) Dimensionality Serial Parallel (a) Set A Though 𝑛base and 𝑐IMS are still parameters that may be tuned, their effect on the performance of an EA is much smaller than that of the population size parameter, because a population size that is too small may lead to premature convergence, while this is not the case within the IMS. In this paper, we use the default setting of 𝑐IMS = 4, and we show the difference in performance of parallel GOMEA for 𝑛base in Figure 4. This figure shows convergence for different population sizes on different instances in set C, using a time limit of an hour. For each of the settings, a fixed LT was used. 6.5 Convergence Speed 11400 11450 11500 11550 11600 1 10 100 1000 Fitness Time (s) 8 16 32 64 5000 5100 5200 5300 5400 5500 5600 1 10 100 1000 Fitness Time (s) 8 16 32 64 Figure 4: Median and interdecile range (30 runs) of fitness values achieved of different settings for 𝑛base in the IMS for parallel GOMEA using a fixed LT, for g1 and g65 (Set C), re- spectively. In this section, we analyze how the use of parallel GOMEA influ- ences the speed of convergence of GOMEA and whether on larger graphs, GOMEA with the best settings within our time budget can still be run faster on a GPU. Firstly, for 2 instances, we show con- vergence plots in Figure 6, comparing GOMEA with and without the use of parallel GOM. Convergence plots for the other instances are provided in the supplementary material. 11400 11450 11500 11550 11600 1 10 100 1000 Fitness Time (s) 8 16 32 64 5000 5100 5200 5300 5400 5500 5600 1 10 100 1000 Fitness Time (s) 8 16 32 64 Secondly, in Figure 7, we show speed-ups achieved by using parallel GOMEA, compared to serial GOMEA. This Figure is split, showing speed-ups for the five smallest instances on the left, and speed-ups for the four largest instances on the right. For the smaller instances, a time limit of 1 hour was used for both serial and paral- lel GOMEA. For the larger instances, a time limit of 6 hours was used for serial GOMEA, and 1 hour for parallel GOMEA. On the horizontal axis, we show the (median of 30 runs) amount of time required by parallel GOMEA to reach a certain fitness value. The vertical axis shows the factor by which serial GOMEA required more time (median of 30 runs) than parallel GOMEA, to achieve the same fitness value, i.e., the parallel GOMEA speed-up factor.i Time (s) Time (s) Figure 4: Median and interdecile range (30 runs) of fitness values achieved of different settings for 𝑛base in the IMS for parallel GOMEA using a fixed LT, for g1 and g65 (Set C), re- spectively. 6.3 Interleaved Multi-start Scheme Based on Figure 4, we find that the setting of 𝑛base has only a marginal impact on the performance of parallel GOMEA, with only 𝑛base = 8 giving slightly worse results. Similar results were found for different instances. We therefore use 𝑛base = 16 in the remainder of our experiments. (b) Set B (a) Set A Figure 5: Scalability of serial and parallel GOMEA, showing the median and interdecile range (30 runs) of the time re- quired to find the optimum, for the instances in set A and set B, respectively. 6.4 Scalability In this section, we test the scalability GOMEA with and without the use of parallel GOM. This is done on instances from Set A and Set B, because these instances have a fixed structure, allowing us to scale up the number of vertices while keeping the structure of the instances constant. In Figure 5 we show what the effect of parallel GOM is on the scalability of GOMEA.fi We find that, for large instances, speed-ups up to a factor of 100 can be achieved. Likely, if run even longer and for even larger instances, the speed-up factor could be even larger. On the five smallest instances, parallel GOMEA generally achieves a speed-up at the start of the optimization process, but this speed-up decreases over time. This is likely a consequence of the differences between serial and parallel GOMEA, as discussed in Section 5.4. No efficiency improvement was observed for fully connected graphs (see Figure 5a), because no iterations of GOM may be per- formed in parallel. Though GOM may still be performed in parallel for different individuals in the population, this leaves a large part of the GPU idle, while keeping the overhead from copying mem- ory to and from the GPU device. Therefore, we find that the GPU implementation of GOMEA performs worse than the serial version of GOMEA on fully connected graphs. For graphs with a torus-like structure, as displayed in Figure 5b, many FOS elements can be subjected to GOM in parallel. Concordantly, we observe a clear difference in scalability. Still, within the scope of available problem 6.6 Parallel and Serial GOMEA Differencesf In this section, we analyze the difference in convergence for parallel and serial GOMEA from an algorithmic point of view. That is, all experiments in this Section are run with a serial version of GOMEA, but with changes to some of its operations to reflect algorithmic differences with the GPU parallel version of GOMEA. 681 GECCO ’22, July 9–13, 2022, Boston, MA, USA Anton Bouter and Peter A.N. Bosman 11400 11450 11500 11550 11600 1 10 100 1000 Fitness Time (s) Serial Parallel 5000 5100 5200 5300 5400 5500 5600 1 10 100 1000 Fitness Time (s) Serial Parallel Figure 6: Median and interdecile range (30 runs) of fitness values achieved by serial and parallel GOMEA for g1 and g65 (Set C), respectively. 11400 11450 11500 11550 11600 1 10 100 1000 Fitness Time (s) Serial Parallel 5000 5100 5200 5300 5400 5500 5600 1 10 100 1000 Fitness Time (s) Serial Parallel in Section 5.4, influence performance. Note that these figures are zoomed in to the later stages of convergence (after 100 seconds). We observe that, while the graph-coloring-based group-wise order of processing the FOS needed to realize large-scale parallelization does not appear to have an impact on performance of GOMEA, the FI procedure does, allowing it to converge to better fitness values in the very late stages of the optimization process. This is also the reason why the speed-ups obtained by parallel GOMEA as observed in Figure 7, disappear at a later stage of the search process. Even though the parallel GOMEA then still performs many more evaluations per second than the serial GOMEA, this benefits is outweighed by the algorithmic added value of FI. Figure 6: Median and interdecile range (30 runs) of fitness values achieved by serial and parallel GOMEA for g1 and g65 (Set C), respectively. 1 10 100 1 10 100 1000 Speed-up Time (s) g1 g22 g55 g60 g65 1 10 100 1 10 100 1000 Speed-up Time (s) g66 g72 g77 g81 Figure 7: Median (30 runs) speed-up achieved by parallel GOMEA, compared to serial GOMEA, for set C. 7 DISCUSSION AND CONCLUSION The main purpose of this paper was to show the potential of parallel GOM, for which the Max-Cut problem was selected as a benchmark, because it is a well-known problem with a clear structure that enables a GBO setting. Even so, we note that it is likely that parallel GOM is outperformed by various other methods that are considered state-of-the-art for the Max-Cut problem. Furthermore, though it is not within the scope of this paper, the performance of GOMEA on the Max-Cut problem may be greatly improved by the addition of (Iterated) Local Search ((I)LS). Similar to how graph coloring is applied to find independent sets for the application of parallel GOMEA, this can be done to parallelize (I)LS to create a hybrid parallel GOMEA, which is an interesting direction for future work. 9800 9850 9900 9950 10000 10050 10100 10150 10200 1 10 100 1000 Fitness Time (s) LT FLT BFLT (10) BFLT (100) 5000 5100 5200 5300 5400 5500 5600 1 10 100 1000 Fitness Time (s) LT FLT BFLT (10) BFLT (100) Figure 8: Median and interdecile range (30 runs) of fitness values achieved by serial GOMEA using different linkage models for g55 and g65 (Set C), respectively. 9800 9850 9900 9950 10000 10050 10100 10150 10200 1 10 100 1000 Fitness Time (s) LT FLT BFLT (10) BFLT (100) p g In this paper, we showed how the GOM variation operator of the state-of-the-art model-based EA known as GOMEA may be applied in a large-scale parallel manner to apply variation steps to a large number of non-trivial-sized subsets of problem variables for each individual in the population. Using a CUDA implementation of the so-constructed parallel GOMEA on a GPU, we were able to speed up the performance of GOMEA on the well-known MaxCut problem up to a factor of 100. Likely, larger speed-up factors are possible if even larger graph instances would be used. Moreover, we identified additional potential improvements. Altogether, this paper contributes to the body of empirical evidence that shows that the use of GPUs holds vast potential to accelerate modern, powerful EAs on contemporary computing hardware and have an important impact to the field of EC in general, similar to how they propelled the field of deep learning to new heights [22]. 6.6 Parallel and Serial GOMEA Differencesf 1 10 100 1 10 100 1000 Speed-up Time (s) g66 g72 g77 g81 1 10 100 1 10 100 1000 Speed-up Time (s) g1 g22 g55 g60 g65 11460 11480 11500 11520 11540 11560 11580 11600 100 1000 Fitness Time (s) FI no FI parOrder parOrder,no FI 5300 5320 5340 5360 5380 5400 5420 5440 100 1000 Fitness Time (s) FI no FI parOrder parOrder,no FI Figure 9: Median and interdecile range (30 runs) of fitness values achieved by serial GOMEA using a random FOS or- der, or one dictated by parallel GOM (parOrder), and with FI enabled or disabled, for g1 and g65 (Set C), respectively. 11460 11480 11500 11520 11540 11560 11580 11600 100 1000 Fitness Time (s) FI no FI parOrder parOrder,no FI 5300 5320 5340 5360 5380 5400 5420 5440 100 1000 Fitness Time (s) FI no FI parOrder parOrder,no FI Figure 7: Median (30 runs) speed-up achieved by parallel GOMEA, compared to serial GOMEA, for set C. Figure 9: Median and interdecile range (30 runs) of fitness values achieved by serial GOMEA using a random FOS or- der, or one dictated by parallel GOM (parOrder), and with FI enabled or disabled, for g1 and g65 (Set C), respectively. Firstly, we test the impact of using different linkage models. This includes a Linkage Tree (LT) learned from the population using mutual information at the start of each generation, an FLT learned using UPGMA with weights of the Max-Cut graph as a similarity measure, and BFLTs bounded by 10 and 100. Convergence results pertaining to different linkage models are shown in Figure 8. This Figure shows that, in particular for instance g65, using a static linkage tree may lead to premature convergence, as using the linkage tree that is learned at the start of every generation clearly performs better, which is in-line with existing literature that showed better performance using learned LTs versus fixed LTs on linkage benchmark problems [27]. However, better results are obtained much later in terms of time due to the need to, every generation, estimate a large mutual information matrix and, based on this, create an LT. Results on instance g55 show that the added value of learning an LT during search may come even later in the search process, as within our time limit it led to the worst results. REFERENCES [29] R. Tintos, D. Whitley, and F. Chicano. 2015. Partition crossover for pseudo- boolean optimization. In Proceedings of the 2015 ACM Conference on Foundations of Genetic Algorithms XIII. ACM, 137–149. [1] Enrique Alba. 2006. Parallel evolutionary computations. Vol. 22. springer. [1] Enrique Alba. 2006. Parallel evolutionary computations. Vol. 22. sp [2] Krste Asanovic, Ras Bodik, Bryan Christopher Catanzaro, Joseph James Gebis, Parry Husbands, Kurt Keutzer, David A Patterson, William Lester Plishker, John Shalf, Samuel Webb Williams, et al. 2006. The landscape of parallel computing research: A view from Berkeley. (2006). f g [30] Dominic J A Welsh and Martin B Powell. 1967. An upper bound for the chromatic number of a graph and its application to timetabling problems. Comput. J. 10, 1 (1967), 85–86. [31] Man-Leung Wong, Tien-Tsin Wong, and Ka-Ling Fok. 2005. Parallel evolutionary algorithms on graphics processing unit. In 2005 IEEE Congress on Evolutionary Computation, Vol. 3. IEEE, 2286–2293. y ( ) [3] N. Bell and J. Hoberock. 2011. Thrust: A productivity-oriented library for CUDA. GPU computing gems Jade edition 2 (2011), 359–371. p g g J ( ) P. A. N. Bosman and D. Thierens. 2012. Linkage neighbors, optimal p g g [4] P. A. N. Bosman and D. Thierens. 2012. Linkage neighbors, optimal mixing and forced improvements in genetic algorithms. In Proc. GECCO. ACM, 585–592. [5] Bouter, A., Tanja Alderliesten, Arjan Bel, Cees Witteveen, and Peter A N Bosman. 2018. Large-scale parallelization of partial evaluations in evolutionary algorithms for real-world problems. In Proc. GECCO. ACM, 1199–1206. [6] Bouter, A., Tanja Alderliesten, and Peter A. N. Bosman. 2021. Achieving highly scalable evolutionary real-valued optimization by exploiting partial evaluations. Evolutionary computation 29, 1 (2021), 129–155. y p [7] Bouter, A., Tanja Alderliesten, and Peter A N Bosman. 2021. GPU-Accelerated Parallel Gene-pool Optimal Mixing applied to Multi-Objective Deformable Image Registration. In 2021 IEEE Congress on Evolutionary Computation (CEC). IEEE, 2539–2548. [8] Andre R Brodtkorb, Christopher Dyken, Trond R Hagen, Jon M Hjelmervik, and Olaf O Storaasli. 2010. State-of-the-art in heterogeneous computing. Scientific Programming 18, 1 (2010), 1–33. g g ( ) [9] Guillermo Cabrera, Matthias Ehrgott, Andrew Mason, and Andy Philpott. 2014. Multi-objective optimisation of positively homogeneous functions and an appli- cation in radiation therapy. Operations Research Letters 42, 4 (2014), 268–272. [10] Yu-Rong Chen, Che Lun Hung, Yu-Shiang Lin, Chun-Yuan Lin, Tien-Lin Lee, and Kual-Zheng Lee. 2012. Parallel UPGMA algorithm on graphics processing units using CUDA. REFERENCES In 2012 IEEE 14th International Conference on High Performance Computing and Communication & 2012 IEEE 9th International Conference on Embedded Software and Systems. IEEE, 849–854. [11] F. Chicano, D. Whitley, G. Ochoa, and R. Tinós. 2017. Optimizing one million variable NK landscapes by hybridizing deterministic recombination and local search. In Proceedings of the Genetic and Evolutionary Computation Conference. ACM, 753–760. [12] Dipankar Dasgupta and Zbigniew Michalewicz. 2013. Evolutionary algorithms in engineering applications. Springer Science & Business Media. engineering applications. Springer Science & Business Media. [13] Kalyanmoy Deb and Christie Myburgh. 2016. Breaking the billion-variable barrier in real-world optimization using a customized evolutionary algorithm. In Proc. GECCO 2016. ACM, 653–660. [14] Bilel Derbel and Lorenzo Canonne. 2021. A graph coloring based parallel hill climber for large-scale NK-landscapes. In Proceedings of the Genetic and Evolu- tionary Computation Conference. 216–224. y p f [15] I. Gronau and S. Moran. 2007. Optimal implementations of UPGMA and other common clustering algorithms. Information Processing Letters 104, 6 (2007), 205–210. [16] G.R. Harik and F.G. Lobo. 1999. A parameter-less genetic algorithm. In Proc. GECCO. Morgan Kaufmann Publishers Inc., 258–265. g [17] E Jabir, Vinay V Panicker, and R Sridharan. 2015. Multi-objective optimization model for a green vehicle routing problem. Procedia-Social and Behavioral Sciences 189 (2015), 33–39. ( [18] Nicolas Jozefowiez, Frédéric Semet, and El-Ghazali Talbi. 2008. Multi-objective vehicle routing problems. European journal of operational research 189, 2 (2008), 293–309. [19] Richard M Karp. 1972. Reducibility among combinatorial problems. In Complexity of computer computations. Springer, 85–103. [20] Sung-Chi Li and Tian-Li Yu. 2017. Speeding Up DSMGA-II on CUDA Platform. In Proceedings of the Genetic and Evolutionary Computation Conference (Berlin, Germany) (GECCO ’17). ACM, New York, NY, USA, 809–816. [21] Ngoc Hoang Luong, Tanja Alderliesten, Arjan Bel, Yury Niatsetski, and Peter A N Bosman. 2018. Application and benchmarking of multi-objective evolution- ary algorithms on high-dose-rate brachytherapy planning for prostate cancer treatment. Swarm and Evolutionary Computation 40 (2018), 37–52. y p [22] Sparsh Mittal and Shraiysh Vaishay. 2019. A survey of techniques for optimizing deep learning on GPUs. Journal of Systems Architecture 99 (2019), 101635. 22] Sparsh Mittal and Shraiysh Vaishay. 2019. A survey of techniques f [23] NVIDIA Corporation. 2017. NVIDIA Tesla V100 GPU architecture : The world’s most advanced data center GPU. [24] NVIDIA Corporation. 2018. CUDA C Programming guide v9.1.85. [25] Franz Rendl, Giovanni Rinaldi, and Angelika Wiegele. 2010. 7 DISCUSSION AND CONCLUSION 5000 5100 5200 5300 5400 5500 5600 1 10 100 1000 Fitness Time (s) LT FLT BFLT (10) BFLT (100)i Figure 8: Median and interdecile range (30 runs) of fitness values achieved by serial GOMEA using different linkage models for g55 and g65 (Set C), respectively. Secondly, in Figure 9, we show how the order of GOM and the FI procedure, which are different in the parallel GOMEA as discussed 682 GECCO ’22, July 9–13, 2022, Boston, MA, USA GPU-Accelerated Parallel Gene-Pool Optimal Mixing in a Gray-Box Optimization Setting GECCO ’22, July 9–13, 2022, Boston, MA, USA REFERENCES Solving max-cut to optimality by intersecting semidefinite and polyhedral relaxations. Mathematical Programming 121, 2 (2010), 307–335. [26] Dirk Sudholt. 2015. Parallel evolutionary algorithms. In Springer Handbook of Computational Intelligence. Springer, 929–959. p g p g [27] Dirk Thierens and Peter Bosman. 2012. Predetermined versus learned linkage models. In Proc. GECCO. ACM, 289–296. models. In Proc. GECCO. ACM, 289–296. [28] D. Thierens and P. A. N. Bosman. 2011. Optimal mixing evolutionary algorithms. In Proc. GECCO. ACM, 617–624. 683
https://openalex.org/W2913007890	https://europepmc.org/articles/pmc6542715?pdf=render	English	null	Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC	Oncogene	2,019	cc-by	15,272	Abstract Ovarian cancer (OvCa) exhibits a speciﬁc predilection for metastasis to the omentum. Our earlier studies highlighted the tumour-suppressor effect of secreted protein acidic and rich in cysteine (SPARC) in OvCa through multi-faceted roles inhibiting cancer cell interactions within the peritoneal milieu. The goal of this study is to investigate the role of SPARC in OvCa interactions with omental adipocytes and its role in OvCa colonization in the omentum. We employed multi-pronged approach using primary omental adipocytes from Sparc knockout mice, genetically engineered human omental adipocytes in 3D co-cultures with OvCa cells, as well as treatment with recombinant SPARC protein. We show that SPARC suppresses multistep cascade in OvCa omental metastasis. SPARC inhibited in vivo and adipocyte- induced homing, proliferation, and invasion of OvCa cells. SPARC suppressed metabolic programming of both adipocytes and OvCa cells and exerted an inhibitory effect of adipocyte differentiation and their phenotypic switch to cancer-associated phenotype. Mechanistic studies revealed that this effect is mediated through inhibition of cEBPβ-NFkB-AP-1 transcription machinery. These ﬁndings deﬁne a novel and functionally important role of SPARC in OvCa and not only bridge the knowledge gap but highlight the need to consider SPARC protein expression in therapeutic development. 3 Department of Urology, Wake Forest University School of Medicine, Winston-Salem, NC, USA 4 Present address: Surgical Pathology, University of Kentucky College of Medicine, Lexington, KY, USA 5 Present address: Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY 40202, USA Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC Bincy John1 ●Christine Naczki1 ●Chirayu Patel1 ●Alia Ghoneum1 ●Shadi Qasem2,4 ●Ziyan Salih2,5 ●Neveen Said1,2,3 Bincy John1 ●Christine Naczki1 ●Chirayu Patel1 ●Alia Ghoneum1 ●Shadi Qasem2,4 ●Ziyan Received: 23 July 2018 / Revised: 19 January 2019 / Accepted: 19 January 2019 © The Author(s) 2019. This article is published with open access Oncogene Oncogene Oncogene https://doi.org/10.1038/s41388-019-0728-3 https://doi.org/10.1038/s41388-019-0728-3 ARTICLE ARTICLE ARTICLE Introduction The role of omental adipocytes in promoting OvCa metas- tasis to the omentum has been recently established as they represent a signiﬁcant source of factors that substantially promote OvCa-omental tropism, and colonization [3–8]. Adipocytes adjacent to cancer cells have been named cancer-associated adipocytes (CAAs) as they not only promote tumour growth, survival, and chemo-resistance [9], but they revert from mature, differentiated adipocytes to pre-adipocytes stage releasing their lipids to provide energy to cancer cells [3]. This phenotypic plasticity is controlled Epithelial ovarian cancer (OvCa) is the leading cause of death from gynecologic malignancies in the United States [1]. More than 75% of patients are diagnosed at late stages with high mortality rates [2]. The main site of OvCa metastasis within the abdomen is the omentum, a vascular and adipocyte-rich tissue that drapes abdominal organs [2]. 1 Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA * Ziyan Salih ziyan.salih@louisville.edu * Neveen Said nsaid@wakehealth.edu 2 Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC, USA SPARC inhibits OvCa cell homing to the omentum in vivo and in vitro To determine whether SPARC inhibits ID8 cells preferential homing to omental adipocytes, we injected ID8-GFP cells intraperitoneally (ip) in SP–/– and SP+/+ mice [5] and determined adherent ID8 cells harvested omenta (Fig. 1a) by measuring A488 ﬂuorescence of green ﬂuorescent protein (GFP)-labeled cells. We found that homing of ID8-GFP cells to SP–/– omenta was signiﬁcantly higher than to the SP+/+ at 60–120 min. To determine whether this increased homing was SPARC dependent, we injected recombinant murine SPARC (rSPARC 5 µg/100 µl phosphate buffered saline (PBS)) ip 30 min prior to ID8 injection. We found that SPARC inhibited ID8 homing to the omentum starting at 60 min post ID8 injection and mitigated the increased ID8- GFP adhesion to SP–/– omenta (Fig. 1a). To clearly distin- guish the role of omental adipocyte-SPARC, independent of other sources of SPARC in the complex peritoneal milieu, we constructed three-dimensional (3D) omental adipocyte culture composed of freshly isolated primary SP–/– and SP +/+ omental adipocytes (Supplement Figure 1) embedded in reduced growth factor matrigel and co-cultured them with ID8-GFP cells as illustrated in Fig. 1b. We ﬁrst determined the effect adipocyte-SPARC on ID8-GFP cell chemotaxis/ migration or homing towards SP–/– and SP+/+ omental adi- pocytes, and found that ID8 homing to SP–/– omental adi- pocytes was signiﬁcantly higher than to SP+/+ adipocytes (Fig. 1b). We next determined whether difference of homing of ID8 cells to adipocytes was mediated by differences in secreted factors and found that SP–/– omental adipocytes exhibited signiﬁcant increase in the levels of IL-6, CCL2/ MCP1, CCL3/MIP1, VEGF, TNFα, IL-2, and leptin with modest though insigniﬁcant increase in levels of CTACK/ CCL27, and TIMP1 (Supplement Figure. 2A). Neutralizing antibodies of the factors that exhibited signiﬁcant differences between the two genotypes, signiﬁcantly inhibited migra- tion/homing of ID8 cells towards SP–/– and SP+/+ omental adipocytes (Supplement Figure 2B). Of note that homing of ID8 cells to adipocytes isolated from mice bearing ID8 peritoneal tumours (will be referred to as CAA) was sig- niﬁcantly higher than to normal adipocytes (normal Adi) isolated from non-tumour-bearing mice. Homing of ID8 to SP–/– CAA was signiﬁcantly higher than to SP+/+ CAA (Supplement Figure 2C). Furthermore, CAA exhibited sig- niﬁcantly higher levels of the aforementioned inﬂammatory factors than normal adipocytes with SP–/– CAA exhibiting signiﬁcantly higher levels than SP+/+ CAA (Supplement Figure 2D). Results by factors that orchestrate differentiation, inﬂammation, and metabolic reprogramming in metabolic diseases, obesity, and cancer [10–15]. Strong evidence exists for a link between inﬂammation and adipocyte plasticity and their phenotypic switch to CAA; however, little is known about the signaling networks involved. Co-regulation and trans- activation of CCAAT/enhancer-binding protein beta (C/ EBPβ), Nuclear Factor-κB (NFκB) and activating protein-1 (AP-1) have been reported in the context of obesity, insulin resistance, and inﬂammation [16–19] as they are involved in the upregulation of inﬂammatory mediators [20, 21]. In addition, the three transcription factors (TFs) have been reported as crucial factors with transactivation circuitry in inﬂammation and cancer [22]. However, their pivotal role in OvCa-peritoneal dissemination is relatively unexplored. Supplementary information The online version of this article (https:// doi.org/10.1038/s41388-019-0728-3) contains supplementary i l hi h i il bl h i d Supplementary information The online version of this article (https:// doi.org/10.1038/s41388-019-0728-3) contains supplementary t i l hi h i il bl t th i d Supplementary information The online version of this article (https:// doi.org/10.1038/s41388-019-0728-3) contains supplementary material, which is available to authorized users. * Ziyan Salih ziyan.salih@louisville.edu * Neveen Said nsaid@wakehealth.edu 1 Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA 2 Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC, USA Supplementary information The online version of this article (https:// doi.org/10.1038/s41388-019-0728-3) contains supplementary material, which is available to authorized users. material, which is available to authorized users. * Ziyan Salih ziyan.salih@louisville.edu * Neveen Said nsaid@wakehealth.edu 1 Department of Cancer Biology, Wake Forest University School of Medicine, Winston-Salem, NC, USA 2 Department of Pathology, Wake Forest University School of Medicine, Winston-Salem, NC, USA Published online: 14 February 2019 B. John et al. SPARC inhibits OvCa cell homing to the omentum in vivo and in vitro P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adi- pocytes in absence of SPARC. P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. Student’s t-test. e Western blots showing the expression of SPARC after overexpression and knockdown in primary human omental adipocytes (hAdi). f Schema of the in vitro homing assay with GFP-labeled human OvCa cell lines. Bars represent means ± SEM of fold change of OvCa cells that migrated through trans-wells towards genetically engineered hAdi compared with cells migrated to control media (without adipocytes) considered as 1. (n = 4/experi- mental condition. Experiments were repeated three times). p < 0.05 Student’s t-test with multiple comparisons SP+/+,SP–/– adipocytes in absence of SPARC. *P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. Photomicrographs of ﬂuorescent adherent cells (top, 5×). d Schema of adipocyte-induced OvCa invasiveness through trans-well inserts towards omental adi- pocytes in the bottom chamber. Bars represent the means ± SEM of migrated cells counted in ﬁve random ﬁelds/insert, (n = 3). P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adi- pocytes in absence of SPARC. *P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. Student’s t-test. e Western blots showing the expression of SPARC after overexpression and knockdown in primary human omental adipocytes (hAdi). f Schema of the in vitro homing assay with GFP-labeled human OvCa cell lines. Bars represent means ± SEM of fold change of OvCa cells that migrated through trans-wells towards genetically engineered hAdi compared with cells migrated to control media (without adipocytes) considered as 1. (n = 4/experi- mental condition. Experiments were repeated three times). p < 0.05 Student’s t-test with multiple comparisons SPARC inhibits OvCa cell homing to the omentum in vivo and in vitro *P < 0.05 comparing cells with and without SPARC treatment. ns, non- signiﬁcant. b Schema of the in vitro homing/chemotaxis of ID8 cells towards SP+/+ and SP–/– omental adipocytes. Bars represent means ± SEM of ﬂuorescence intensity of ID8 cells that migrated through trans- wells towards adipocytes. Complete growth media were used as controls for migration (n = 4). P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adipocytes in absence of SPARC. *P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. c Schema of the adhesion assay of GFP-OvCa cells over- layed on top of adipocytes for 120 min (left). Bars represent means ± SEM of ﬂuorescence intensity of adherent cells (n = 6/experimental condition). p < 0.05 comparing ID8 migration towards control media, SP+/+,SP–/– adipocytes in abse migration of ID8 cells treated condition in absence of SPA ﬂuorescent adherent cells (top, OvCa invasiveness through tra pocytes in the bottom chamber migrated cells counted in ﬁve ra comparing ID8 migration towar pocytes in absence of SPARC. * cells treated with SPARC with th of SPARC treatment. Student’s expression of SPARC after over human omental adipocytes (hA assay with GFP-labeled human O ± SEM of fold change of OvCa towards genetically engineered h control media (without adipocy mental condition. Experiments Student’s t-test with multiple co Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC GFP) to omental adipocytes was inhibited by exogenous recombinant human and murine SPARC (rSPARC, Fig. 1c). SPARC inhibits adipocyte-induced OvCa cell proliferation in vitro and in vivo Fig. 1 Effect of SPARC on homing of ovarian cancer (OvCa) cells to omental adipocytes. a In vivo homing of ID8-GFP cells to SP+/+ and SP–/– omenta in the presence or absence of prior injection of 5 µg/ml SPARC. Bars represent means ± Standard error of the mean (SEM) of ﬂuorescence intensity of adherent cells to omenta harvested at the indicated time points. P < 0.05 comparing SP+/+ and SP–/–. P < 0.05 comparing cells with and without SPARC treatment. ns, non- signiﬁcant. b Schema of the in vitro homing/chemotaxis of ID8 cells towards SP+/+ and SP–/– omental adipocytes. Bars represent means ± SEM of ﬂuorescence intensity of ID8 cells that migrated through trans- wells towards adipocytes. SPARC inhibits OvCa cell homing to the omentum in vivo and in vitro Bars represent means ± SEM of ﬂuorescence intensity of ID8 cells that migrated through trans- wells towards adipocytes. Complete growth media were used as controls for migration (n = 4). P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adipocytes in absence of SPARC. *P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. c Schema of the adhesion assay of GFP-OvCa cells over- layed on top of adipocytes for 120 min (left). Bars represent means ± SEM of ﬂuorescence intensity of adherent cells (n = 6/experimental condition). p < 0.05 comparing ID8 migration towards control media, Fig. 1 Effect of SPARC on homing of ovarian cancer (OvCa) cells to omental adipocytes. a In vivo homing of ID8-GFP cells to SP+/+ and SP–/– omenta in the presence or absence of prior injection of 5 µg/ml SPARC. Bars represent means ± Standard error of the mean (SEM) of ﬂuorescence intensity of adherent cells to omenta harvested at the indicated time points. P < 0.05 comparing SP+/+ and SP–/–. P < 0.05 comparing cells with and without SPARC treatment. ns, non- signiﬁcant. b Schema of the in vitro homing/chemotaxis of ID8 cells towards SP+/+ and SP–/– omental adipocytes. Bars represent means ± SEM of ﬂuorescence intensity of ID8 cells that migrated through trans- wells towards adipocytes. Complete growth media were used as controls for migration (n = 4). P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adipocytes in absence of SPARC. *P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. c Schema of the adhesion assay of GFP-OvCa cells over- layed on top of adipocytes for 120 min (left). Bars represent means ± SEM of ﬂuorescence intensity of adherent cells (n = 6/experimental condition). p < 0.05 comparing ID8 migration towards control media, SP+/+,SP–/– adipocytes in absence of SPARC. *P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. Photomicrographs of ﬂuorescent adherent cells (top, 5×). d Schema of adipocyte-induced OvCa invasiveness through trans-well inserts towards omental adi- pocytes in the bottom chamber. Bars represent the means ± SEM of migrated cells counted in ﬁve random ﬁelds/insert, (n = 3). SPARC inhibits OvCa cell homing to the omentum in vivo and in vitro Adhesion of GFP-ﬂuorescent human and murine OvCa cell lines SKOV3, OVCAR3, CAOV3, and ID8 (GFP-SKOV3, GFP-OVCAR3, GFP-CAOV3, and ID8- Secreted protein acidic and rich in cysteine (SPARC), also termed osteonectin and BM-40, is an extracellular matrix (ECM) protein that exhibits contextual highly regulated expression in remodeling tissues to maintain tis- sue homeostasis (summarized in [23]). In this respect, SPARC has been shown to regulate the differentiation of mesenchymal, stem cells including adipocytes [24–26]. SPARC has been reported to inhibit adipogenesis as evi- denced by the phenotype of Sparc null mice exhibiting osteoporosis and fatty bone marrow [24–26]. We have earlier reported that SPARC is an OvCa suppressor [5–8]. We reported that SPARC inhibited OvCa cell adhesion to various ECM proteins enriched in the peritoneal tumour microenvironment (TME) and peritoneal mesothelial cells [5–7]. SPARC exhibited an anti-proliferative effect that was attributed to inhibition of integrin- and growth factor- mediated survival signaling pathways [6–8]. We also reported that SPARC normalizes the TME through anti- inﬂammatory properties through suppression of the bi- directional cross-talk between cancer cells and macrophages and mesothelial cells [5–8, 27]. In addition, we reported that in the immunocompetent Sparc knockout mice (will be referred to as SP–/–), the enhanced peritoneal carcinomatosis was characterized by high levels and biological activity of pro-inﬂammatory mediators in tumours and ascitic ﬂuid [6–8, 27]. These pro-inﬂammatory factors are reciprocated by cancer cells and stromal cells [7, 27, 28] and are cor- related with advanced human disease, chemo-resistance, and poor prognosis [28]. Given the speciﬁc predilection of OvCa cells to the omentum and the reported inhibitory effects of SPARC on adipocyte differentiation [25, 29], we sought to investigate the role of SPARC in the bi-directional cross-talk between OvCa cells and omental adipocytes. We present evidence for the ﬁrst time that the tumour- suppressor role of SPARC in OvCa is mediated through inhibition of OvCa cells–adipocytes interactions, the phe- notypic plasticity of omental adipocytes, and metabolic programming Fig. 1 Effect of SPARC on homing of ovarian cancer (OvCa) cells to omental adipocytes. a In vivo homing of ID8-GFP cells to SP+/+ and SP–/– omenta in the presence or absence of prior injection of 5 µg/ml SPARC. Bars represent means ± Standard error of the mean (SEM) of ﬂuorescence intensity of adherent cells to omenta harvested at the indicated time points. P < 0.05 comparing SP+/+ and SP–/–. SPARC inhibits OvCa cell homing to the omentum in vivo and in vitro Complete growth media were used as controls for migration (n = 4). P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adipocytes in absence of SPARC. P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. c Schema of the adhesion assay of GFP-OvCa cells over- layed on top of adipocytes for 120 min (left). Bars represent means ± SEM of ﬂuorescence intensity of adherent cells (n = 6/experimental condition). p < 0.05 comparing ID8 migration towards control media, SP+/+,SP–/– adipocytes in absence of SPARC. *P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. Photomicrographs of ﬂuorescent adherent cells (top, 5×). d Schema of adipocyte-induced OvCa invasiveness through trans-well inserts towards omental adi- pocytes in the bottom chamber. Bars represent the means ± SEM of migrated cells counted in ﬁve random ﬁelds/insert, (n = 3). P < 0.05 comparing ID8 migration towards control media, SP+/+, SP–/– adi- pocytes in absence of SPARC. *P < 0.05 comparing migration of ID8 cells treated with SPARC with the corresponding condition in absence of SPARC treatment. Student’s t-test. e Western blots showing the expression of SPARC after overexpression and knockdown in primary human omental adipocytes (hAdi). f Schema of the in vitro homing assay with GFP-labeled human OvCa cell lines. Bars represent means ± SEM of fold change of OvCa cells that migrated through trans-wells towards genetically engineered hAdi compared with cells migrated to control media (without adipocytes) considered as 1. (n = 4/experi- mental condition. Experiments were repeated three times). p < 0.05 Student’s t-test with multiple comparisons Regulation of the bi directional cross talk between ovarian cancer cells and adipocytes by SPARC Fig. 1 Effect of SPARC on homing of ovarian cancer (OvCa) cells to omental adipocytes. a In vivo homing of ID8-GFP cells to SP+/+ and SP–/– omenta in the presence or absence of prior injection of 5 µg/ml SPARC. Bars represent means ± Standard error of the mean (SEM) of ﬂuorescence intensity of adherent cells to omenta harvested at the indicated time points. P < 0.05 comparing SP+/+ and SP–/–. P < 0.05 comparing cells with and without SPARC treatment. ns, non- signiﬁcant. b Schema of the in vitro homing/chemotaxis of ID8 cells towards SP+/+ and SP–/– omental adipocytes. SPARC inhibits adipocyte-induced OvCa cell proliferation in vitro and in vivo GFP) to omental adipocytes was inhibited by exogenous recombinant human and murine SPARC (rSPARC, Fig. 1c). Furthermore, rSPARC inhibited adipocyte-induced inva- siveness human and murine OvCa cells (Fig. 1d). In addi- tion, overexpression and depletion of SPARC in human adipocytes (hAdi; Fig. 1e) signiﬁcantly inhibited/increased invasiveness of OvCa cells compared with their corre- sponding vector control adipocytes, respectively (Fig. 1f). Together these data highlight the paracrine effect of adipocyte-SPARC in inhibiting homing and invasiveness of OvCa cells through secreted inﬂammatory factors. To further investigate the adipocyte-SPARC on OvCa cell proliferation, we incubated ID8-GFP cells in direct contact with SP–/– and SP+/+ omental adipocytes and found that ID8 proliferation was signiﬁcantly higher (~3-folds) com- pared with those incubated with the SP+/+ as determined by measuring the GFP ﬂuorescence over 72 h. This effect was partially mitigated by treating co-cultures by rSPARC (Fig. 2a, b). Similar results were obtained by parallel experiments B. John et al. Fig. 2 Effect of SPARC on omental adipocyte-induced OvCa cell proliferation. a Schema of the effect of human and murine omental adipocytes on GFP-OvCa cells proliferation. b Line graphs repre- senting means ± SEM of changes in the proliferation (GFP ﬂuores- cence) of ID8-GFP cells over-layed on top of SP+/+ and SP–/– adipocytes in the presence or absence of 5 µg/ml SPARC, over 72 h. c Means ± SEM of changes of tumours volumes after ID8 SC injection either alone or with SP+/+ and SP–/– adipocytes (1:2) in athymic nude mice. p < 0.05, between experimental conditions starting at week 3 post-injection (n = 8/group; two-way ANOVA with Tukey’s multiple comparison test). d Means ± SEM of changes in the proliferation (GFP ﬂuorescence) of GFP-labeled human OvCa cells on top of genetically engineered human adipocytes over 72 h. (n = 6/experimental condi- tion). P < 0.05 comparing hAdi-SP and hAdi-shSP with their corre- sponding vector control at the indicated time points, two-way ANOVA. e Changes in the proliferation (GFP ﬂuorescence) of OvCa cells over-layed on top of adipocytes in presence or absence of 5 µg/ml SPARC over 72h. (n = 6/experimental condition). p < 0.05, P < 0.001, p < 0.0001, comparing proliferation of GFP-cells on uncoated UC, wells (black), matrigel (blue) and adipocytes (red) in presence and absence of SPARC, two-way ANOVA with Tuckey's multiple comparison test ﬂuorescence) of GFP-labeled human OvCa cells on top of genetically engineered human adipocytes over 72 h. SPARC inhibits adipocyte-induced OvCa cell proliferation in vitro and in vivo We found that ID8 cells injected with SP–/– adipocytes produced ~3.5 times larger tumours than those injected with SP+/+ adipocytes (Fig. 2c). Similar effect of adipocyte-SPARC was observed with early passage differentiated primary human omental (pre)adipo- cytes genetically manipulated SPARC expression (by overexpression and depletion, respectively) incubated with in which OvCa cells were separated from adipocytes by 0.4 µm trans-well inserts placed in direct contact with the differentiated adipocytes for 72 h. Adipocytes and OvCa cells were collected, trypsinized, and counted by Trypan blue exclusion at the same time points. Consistent results were observed with OvCa counting and measuring ﬂuor- escent intensity of GFP, whereas the number of viable adipocyte did not exhibit signiﬁcant change during the experiment (data not shown). To further conﬁrm the role of adipocyte-SPARC on ovarian tumour growth in vivo, we injected ID8 cells with SP+/+ and SP–/– omental adipocytes (1:2, cancer cell:adipocyte ratio) subcutaneously in 6-week- old female athymic nude mice. We found that ID8 cells injected with SP–/– adipocytes produced ~3.5 times larger tumours than those injected with SP+/+ adipocytes (Fig. 2c). Similar effect of adipocyte-SPARC was observed with early passage differentiated primary human omental (pre)adipo- cytes genetically manipulated SPARC expression (by overexpression and depletion, respectively) incubated with SPARC inhibits adipocyte-induced OvCa cell proliferation in vitro and in vivo (n = 6/experimental condi- tion). P < 0.05 comparing hAdi-SP and hAdi-shSP with their corre- sponding vector control at the indicated time points, two-way ANOVA. e Changes in the proliferation (GFP ﬂuorescence) of OvCa cells over-layed on top of adipocytes in presence or absence of 5 µg/ml SPARC over 72h. (n = 6/experimental condition). p < 0.05, P < 0.001, *p < 0.0001, comparing proliferation of GFP-cells on uncoated UC, wells (black), matrigel (blue) and adipocytes (red) in presence and absence of SPARC, two-way ANOVA with Tuckey's multiple comparison test Fig. 2 Effect of SPARC on omental adipocyte-induced OvCa cell proliferation. a Schema of the effect of human and murine omental adipocytes on GFP-OvCa cells proliferation. b Line graphs repre- senting means ± SEM of changes in the proliferation (GFP ﬂuores- cence) of ID8-GFP cells over-layed on top of SP+/+ and SP–/– adipocytes in the presence or absence of 5 µg/ml SPARC, over 72 h. c Means ± SEM of changes of tumours volumes after ID8 SC injection either alone or with SP+/+ and SP–/– adipocytes (1:2) in athymic nude mice. p < 0.05, between experimental conditions starting at week 3 post-injection (n = 8/group; two-way ANOVA with Tukey’s multiple comparison test). d Means ± SEM of changes in the proliferation (GFP SKOV3-GFP, OVCAR3-GFP, and CAOV3-GFP (Fig. 2d). Furthermore, exogenous rSPARC inhibited adipocyte- induced OvCa cell proliferation (Fig. 2e). It is noteworthy that OvCa cell proliferation was signiﬁcantly higher when plated on adipocyte/matrigel plugs compared with plating on matrigel alone or on uncoated wells; an effect that was inhibited by rSPARC (Fig. 2e). These results further sup- port the paracrine effect of adipocyte-SPARC and rSPARC inhibiting adipocyte-induced OvCa cell proliferation, hom- ing, adhesion, and invasiveness. in which OvCa cells were separated from adipocytes by 0.4 µm trans-well inserts placed in direct contact with the differentiated adipocytes for 72 h. Adipocytes and OvCa cells were collected, trypsinized, and counted by Trypan blue exclusion at the same time points. Consistent results were observed with OvCa counting and measuring ﬂuor- escent intensity of GFP, whereas the number of viable adipocyte did not exhibit signiﬁcant change during the experiment (data not shown). To further conﬁrm the role of adipocyte-SPARC on ovarian tumour growth in vivo, we injected ID8 cells with SP+/+ and SP–/– omental adipocytes (1:2, cancer cell:adipocyte ratio) subcutaneously in 6-week- old female athymic nude mice. Effect of SPARC on co-culture induced inﬂammatory chemokines in OvCa cells and adipocytes To further determine the effect of adipocyte-SPARC on the expression of cytokines in both tumours cells and adipo- cytes, we determined the mRNA expression of cytokines and chemokines in ID8 and SP–/– and SP+/+ adipocytes in mono- and co-cultures. We found that the association of Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC Fig. 3 Adipocyte-SPARC suppresses the reciprocal transcriptional activity of pro-inﬂammatory/adipogenic factors. a Schematic illustra- tion of the experimental design and the trans-well assays for the single and co-cultures. b The levels of adipokines in SP+/+ and SP–/– omental adipocytes and ID8 cells in single and in co-cultures for 24 h was determined by qRT-PCR. c Transcriptional activity of cEBP, NFκB, and AP-1 in primary SP+/+ and SP–/– omental adipocytes and ID8 cells in single and in co-cultures was determined by measuring the luciferase reporter activity in each cell type. Results were normalized to fold change of DNA content of each cell type measured before and after the experiment as determined by CyQuant assay. Bars represent mean ± SEM from one of three experiments, performed in triplicate. p < 0.05 Student’s t-test comparing SP+/+ and SP–/– adipocytes; #p < 0.05 Student’s t-test comparing adipocytes in single to co-cultures with ID8 cells; and p < 0.05 Student’s t-test comparing ID8 cells in single to co-culture with SP+/+ and SP–/– adipocytes Regulation of the bi directional cross talk between ovarian cancer cells and adipocytes by SPARC luciferase reporter activity in each cell type. Results were normalized to fold change of DNA content of each cell type measured before and after the experiment as determined by CyQuant assay. Bars represent mean ± SEM from one of three experiments, performed in triplicate. p < 0.05 Student’s t-test comparing SP+/+ and SP–/– adipocytes; #p < 0.05 Student’s t-test comparing adipocytes in single to co-cultures with ID8 cells; and p < 0.05 Student’s t-test comparing ID8 cells in single to co-culture with SP+/+ and SP–/– adipocytes rase reporter activity in each cell type. Results were normalized d change of DNA content of each cell type measured before and he experiment as determined by CyQuant assay. Bars represent ± SEM from one of three experiments, performed in triplicate. Effect of SPARC on co-culture induced inﬂammatory chemokines in OvCa cells and adipocytes .05 Student’s t-test comparing SP+/+ and SP–/– adipocytes; #p < tudent’s t-test comparing adipocytes in single to co-cultures with ells; and p < 0.05 Student’s t-test comparing ID8 cells in single culture with SP+/+ and SP–/– adipocytes Fig. 3 Adipocyte-SPARC suppresses the reciprocal transcriptional activity of pro-inﬂammatory/adipogenic factors. a Schematic illustra- tion of the experimental design and the trans-well assays for the single and co-cultures. b The levels of adipokines in SP+/+ and SP–/– omental adipocytes and ID8 cells in single and in co-cultures for 24 h was determined by qRT-PCR. c Transcriptional activity of cEBP, NFκB, and AP-1 in primary SP+/+ and SP–/– omental adipocytes and ID8 cells in single and in co-cultures was determined by measuring the luciferase reporter activity in each cell type. Results were normalized to fold change of DNA content of each cell type measured before and after the experiment as determined by CyQuant assay. Bars represent mean ± SEM from one of three experiments, performed in triplicate. p < 0.05 Student’s t-test comparing SP+/+ and SP–/– adipocytes; #p < 0.05 Student’s t-test comparing adipocytes in single to co-cultures with ID8 cells; and p < 0.05 Student’s t-test comparing ID8 cells in single to co-culture with SP+/+ and SP–/– adipocytes and AP-1. These TFs are not only implicated in adipocyte differentiation but are also recognized as oncogenes and markers of inﬂammation and aggressiveness of many can- cers including OvCa [21, 30–34]. To monitor the con- comitant changes in OvCa cells and adipocytes in co- culture, we co-cultured ID8 cells on 0.4 µm inserts on top of 3D SP–/– and SP+/+ omental adipocytes and determined the changes in NFkB, AP-1, and cEBPβ promoter activity in both cell types using luciferase reporters [35, 36]. Co- culture of SP–/– and SP+/+ adipocytes with ID8 cells elicited profound signiﬁcant increase in the activity of these TFs over single-cell culture, with SP–/– adipocytes exhibiting signiﬁcantly higher levels than the SP+/+. ID8 co-cultured with SP–/– adipocytes exhibited signiﬁcantly higher lucifer- ase reporter activation than those co-cultured with SP+/+ adipocytes (Fig. 3c). Consistently, human OvCa cell lines co- ID8 cells with adipocytes signiﬁcantly upregulated the expression of IL-6, CCL2, TNFα, VEGF, and leptin tran- scripts in both cell types compared with mono-cultures (Fig. 3a, b). Effect of SPARC on co-culture induced inﬂammatory chemokines in OvCa cells and adipocytes The expression of the transcripts of the aforemen- tioned adipokines were signiﬁcantly higher in SP–/– adipo- cytes than in SP+/+ (1.9-, 1.8-, 2-, 2.5-, and 1.7-fold for IL-6, CCL2, VEGF, TNFα, and leptin, respectively). Co-culture with ID8 cells signiﬁcantly upregulated the expression of each factor in SP–/– adipocytes over SP+/+ by 2.6- to 3-folds. Concomitantly, ID8 cells co-cultured with SP–/– adipocytes exhibited ~2- to 2.7-fold induction of these transcripts, compared with co-culture with SP+/+ adipocytes (Fig. 3a, b). In silico analysis of the common transcriptional regula- tion of the aforementioned chemokines using (http:// opossum.cisreg.ca/cgi-bin/oPOSSUM3/), predicted com- mon transcription machinery regulated by cEBPβ, NFκB, ID8 cells with adipocytes signiﬁcantly upregulated the expression of IL-6, CCL2, TNFα, VEGF, and leptin tran- scripts in both cell types compared with mono-cultures (Fig. 3a, b). The expression of the transcripts of the aforemen- tioned adipokines were signiﬁcantly higher in SP–/– adipo- cytes than in SP+/+ (1.9-, 1.8-, 2-, 2.5-, and 1.7-fold for IL-6, CCL2, VEGF, TNFα, and leptin, respectively). Co-culture with ID8 cells signiﬁcantly upregulated the expression of each factor in SP–/– adipocytes over SP+/+ by 2.6- to 3-folds. Concomitantly, ID8 cells co-cultured with SP–/– adipocytes exhibited ~2- to 2.7-fold induction of these transcripts, compared with co-culture with SP+/+ adipocytes (Fig. 3a, b). In silico analysis of the common transcriptional regula- tion of the aforementioned chemokines using (http:// opossum.cisreg.ca/cgi-bin/oPOSSUM3/), predicted com- mon transcription machinery regulated by cEBPβ, NFκB, B. John et al. Immunostaining of cEBPβ, NFκB, and cJun in tumours dissected from SP+/+ and SP–/– (magniﬁcation, ×200). c Box plots of the expression scores of the nuclear transcription factors in tumour cells and adipo- cytes of both genotypes. p < 0.05, Mann–Whitney test Fig. 4 SPARC suppresses pro-inﬂammatory/adipogenic factors in ID8 intraperitoneal tumour. a Immunoblots of total and phosphorylated cJun, p65NFκB, in lysates from ID8 intraperitoneal tumours growing in SP–/– and SP+/+ mice. Tubulin was used as a loading control. b Immunostaining of cEBPβ, NFκB, and cJun in tumours dissected from SP+/+ and SP–/– (magniﬁcation, ×200). c Box plots of the expression scores of the nuclear transcription factors in tumour cells and adipo- cytes of both genotypes. p < 0.05, Mann–Whitney test Fig. 4 SPARC suppresses pro-inﬂammatory/adipogenic factors in ID8 intraperitoneal tumour. a Immunoblots of total and phosphorylated cJun, p65NFκB, in lysates from ID8 intraperitoneal tumours growing in SP–/– and SP+/+ mice. Tubulin was used as a loading control. Effect of SPARC on co-culture induced inﬂammatory chemokines in OvCa cells and adipocytes Conversely, overexpression of SPARC in adipocytes, inhibited the activation of the three TFs in both OvCa cells and adipocytes (Supplement Figure 3B). Of note is that genetic manipulation of SPARC in primary hAdi by overexpression and knockdown signiﬁcantly inhibited/ increased the luciferase activity of NFκB and AP-1 but not cEBP prompter reporters in mono-cultures, respectively. Importantly, treating OvCa cells and adipocytes in single and co-culture with rSPARC phenocopied the effect of SPARC overexpression in adipocytes and signiﬁcantly inhibited the promoter activity of the three TFs in OvCa cells and adipo- cytes (Supplement Figure 3B). It is noteworthy that the sig- niﬁcantly reciprocated increase in promoter activity of the three TFs reported above is due to increase in activation and not due to increased cell proliferation as we determined changes cell proliferation of in mono- and co-cultures before (0 h) and after 24 h (end of the assay), and normalized the luciferase reporter activity in each cell type to the fold change in cell proliferation during the experiment. Differentiated human and murine adipocytes did not exhibit changes in proliferation as determined by CyQuant assay (data not shown). However, monoculture of ID8 cells exhibited 1.5-fold increase in proliferation, whereas, in co-culture with SP+/+ and SP–/– adipocytes, ID8 exhibited 2- and 3.8-fold change in proliferation (Supplement Figure 4A). Similarly, mono- cultures of SKOV3, OVCAR3, and CAOV3 exhibited 1.3-, Consistently, treatment of OvCa cells and primary omental adipocytes in mono-and co-cultures with rSPARC downregulated the transcript levels of IL-6, CCL2, VEGFA, TNFα, and LEP, all are downstream target genes of the three TFs in both OvCa cells and hAdi (Supplement Figure 5A, B). Effect of SPARC on co-culture induced inﬂammatory chemokines in OvCa cells and adipocytes b Immunostaining of cEBPβ, NFκB, and cJun in tumours dissected from SP+/+ and SP–/– (magniﬁcation, ×200). c Box plots of the expression scores of the nuclear transcription factors in tumour cells and adipo- cytes of both genotypes. p < 0.05, Mann–Whitney test 1.2-, and 1.7-fold change in proliferation in 24 h, respectively; whereas, in co-cultures with hAdi depleted, the three cell lines exhibited ~2- to 2.5-fold increase proliferation (Supplement Figure 4B, C). OvCa cells in co-cultures, with adipocytes overexpressing SPARC exhibited 1.3- to 1.4-fold increase in their numbers after 24 h; whereas co-cultures with adipocytes depleted of SPARC exhibited 3- to 4-fold increase in cell proliferation (Supplement Figure 4B, C). cultured with omental adipocytes depleted of SPARC (Sup- plement Figure 3A) exhibited signiﬁcant increase in the acti- vation of the three TFs in co-cultures compared with those in mono-cultures. Conversely, overexpression of SPARC in adipocytes, inhibited the activation of the three TFs in both OvCa cells and adipocytes (Supplement Figure 3B). Of note is that genetic manipulation of SPARC in primary hAdi by overexpression and knockdown signiﬁcantly inhibited/ increased the luciferase activity of NFκB and AP-1 but not cEBP prompter reporters in mono-cultures, respectively. Importantly, treating OvCa cells and adipocytes in single and co-culture with rSPARC phenocopied the effect of SPARC overexpression in adipocytes and signiﬁcantly inhibited the promoter activity of the three TFs in OvCa cells and adipo- cytes (Supplement Figure 3B). It is noteworthy that the sig- niﬁcantly reciprocated increase in promoter activity of the three TFs reported above is due to increase in activation and not due to increased cell proliferation as we determined changes cell proliferation of in mono- and co-cultures before (0 h) and after 24 h (end of the assay), and normalized the luciferase reporter activity in each cell type to the fold change in cell proliferation during the experiment. Differentiated human and murine adipocytes did not exhibit changes in proliferation as determined by CyQuant assay (data not shown). However, monoculture of ID8 cells exhibited 1.5-fold increase in proliferation, whereas, in co-culture with SP+/+ and SP–/– adipocytes, ID8 exhibited 2- and 3.8-fold change in proliferation (Supplement Figure 4A). Similarly, mono- cultures of SKOV3, OVCAR3, and CAOV3 exhibited 1.3-, cultured with omental adipocytes depleted of SPARC (Sup- plement Figure 3A) exhibited signiﬁcant increase in the acti- vation of the three TFs in co-cultures compared with those in mono-cultures. Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC compared with the SP+/+ tumours (Fig. 4b, c). Importantly, immunostaining of human OvCa specimens from OvCa tissue microarray (TMA, Fig. 5a, b) revealed signiﬁcant increase in the expression of nuclear TFs in advanced stage tumours, T3 and T4 (referred to thereafter as T3+), com- pared with early stage tumors, T1 and T2 (referred to Fig. 5 Correlation of tumour SPARC and cEBPβ, NFkB, and AP-1. a Photomicrograph of stage III HGSC specimens (CHTN) showing distinctive compartmentalization of SPARC in the cancerous vs stromal compartments and the expression of cEBPβ, NFkB, and cJun. b Scatter plots of the expression scores of SPARC, cEBPβ, NFkB, and cJun. p < 0.05, Mann–Whitney test. c Spearman’s correlation of the expression scores of tumour- SPARC and nuclear transcription factors. d Immunoﬂuorescence staining of the expression of omental metastases of HGSC specimens, showing the expression of cEBPβ, RelA, cJun, and SPARC in the tumour (T)–adipocyte (A) interface. e Spearman’s correlation of the nuclear expression of tumour and adipocyte cEBPβ, RelA, and cJun with tumour and adipocyte SPARC. Scale bars 100 µm Fig. 5 Correlation of tumour SPARC and cEBPβ, NFkB, and AP-1. a Photomicrograph of stage III HGSC specimens (CHTN) showing distinctive compartmentalization of SPARC in the cancerous vs stromal compartments and the expression of cEBPβ, NFkB, and cJun. b Scatter plots of the expression scores of SPARC, cEBPβ, NFkB, and cJun. p < 0.05, Mann–Whitney test. c Spearman’s correlation of the expression scores of tumour- SPARC and nuclear transcription factors. d Immunoﬂuorescence staining of the expression of omental metastases of HGSC specimens, showing the expression of cEBPβ, RelA, cJun, and SPARC in the tumour (T)–adipocyte (A) interface. e Spearman’s correlation of the nuclear expression of tumour and adipocyte cEBPβ, RelA, and cJun with tumour and adipocyte SPARC. Scale bars 100 µm ours (Fig. 4b, c). Importantly, vCa specimens from OvCa . 5a, b) revealed signiﬁcant increase in the expression of nuclear TFs in advanced stage tumours, T3 and T4 (referred to thereafter as T3+), com- pared with early stage tumors, T1 and T2 (referred to compared with the SP+/+ tumours (Fig. 4b, c). Importantly, immunostaining of human OvCa specimens from OvCa tissue microarray (TMA, Fig. Effect of SPARC on the activation of NFkB, AP-1, and cEBPβ in OvCa cells and adipocytes in vivo To determine the effect of SPARC on the three TFs in OvCa-adipocytes interactions in vivo, we ﬁrst determined the levels of total and phosphorylated proteins in lysates of syngeneic ID8 omental tumours that developed after ip injection in SP–/– and SP+/+ mice. We found marked increase in phosphorylation of cJun (S73), p65RelA (S276), as well as cEBPβ (T235) in SP–/– tumour lysates compared with SP+/+ tumours (Fig. 4a). Immunostaining of ID8 omental tumours revealed signiﬁcant increase in frequency of nuclear p65RelA, cJun and cEBPβ in both tumour cells and the juxtra-tumoral adipocytes in SP–/– tumours Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC To further conﬁrm whether these observations were due to a direct effect of SPARC, we treated human OvCa cells SKOV3 and OVCAR3 with increasing concentrations of rSPARC and found that SPARC exhibited concentration- dependent inhibition of FA uptake in both cell lines (Fig. 6f). Moreover, treating SKOV3 and OVCAR3 with rSPARC, signiﬁcantly decreased the expression of FA transporters FABP4 and CD36 as revealed by immuno- ﬂuorescence staining and quantiﬁcation of the ﬂuorescence intensity (Fig. 6g, h). To determine whether the increased FA uptake in ID8 cells was associated with changes in FA metabolism, we found that the transcript levels of enzymes involved in β-oxidation of FA as carnitine palmitoyl- transferase I (Cpt1a, b), acetyl-CoA acetyltransferase 2 (Acat2) a cholesterol acyltransferase, hydroxyacyl-CoA dehydrogenase (Hadh), medium and short chain FA dehy- drogenase, carnitine O-octanoyl transferase (Crot), acetyl- CoA acyltransferase 1 (Acaa1), ketoacyl-CoA thiolase 2 (Acaa2), and solute carrier family 25 (carnitine/acylcarnitine translocase member 20, Slc25a20) are increased in ID8 cells co-cultured with SP–/– adipocytes compared with either ID8 cultured alone or with SP+/+ adipocytes (Fig. 6i). Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC 5a, b) revealed signiﬁcant increase in the expression of nuclear TFs in advanced stage tumours, T3 and T4 (referred to thereafter as T3+), com- pared with early stage tumors, T1 and T2 (referred to correlation of the nuclear expression of tumour and adipocyte cEBPβ, RelA, and cJun with tumour and adipocyte SPARC. Scale bars 100 µm e compared with the SP+/+ tumours (Fig. 4b, c). Importantly, mmunostaining of human OvCa specimens from OvCa issue microarray (TMA, Fig. 5a, b) revealed signiﬁcant incre tumo pare adipocyte cEBPβ, RelA, and Jun with tumour and adipocyte SPARC. Scale bars 100 µm increase in the expression of nuclear TFs in advanced stage tumours, T3 and T4 (referred to thereafter as T3+), com- pared with early stage tumors, T1 and T2 (referred to compared with the SP+/+ tumours (Fig. 4b, c). Importantly, immunostaining of human OvCa specimens from OvCa tissue microarray (TMA, Fig. 5a, b) revealed signiﬁcant B. John et al. thereafter as T1+T2, Fig. 5a, b). Importantly, we found signiﬁcant negative correlation between tumour SPARC with nuclear expression of the p65RelA, cJun, and cEBPβ in advanced stage (T3+) OvCa specimens (Fig. 5c) in TMAs where only tumour cores are present. In an inde- pendent cohort of stage T3+ human OvCa specimens with adjacent omental tissue, we found signiﬁcant decrease of SPARC expression in the cancerous compartment with distinctive expression in the stroma (Fig. 5d). In this cohort, SPARC expression in tumour cells negatively correlated with the nuclear expression of the three TFs. In addition, SPARC expression in the juxtra-tumoral adipocytes nega- tively correlated with nuclear expression of the TFs in the adipocytes (Fig. 5d, e). These data further conﬁrm the negative correlation between SPARC protein expression and the activation of these TFs in tumour cells and adipo- cytes. It is noteworthy that in the human tissues, adipocytes exhibit detectable expression levels of SPARC protein with increasing intensity of expression as the juxta-tumoral adi- pocyte become smaller in size (Supplement Figure 6). Together with our earlier reports that SPARC is required for stromal cell differentiation and acquisition of cancer- associated phenotype [36], we sought to determine the effect of SPARC on adipocyte differentiation and acquisi- tion of CAA phenotype. Bodipy staining and electron microscopy (Fig. 6c–e). Effects of SPARC on reciprocal cross-talk between adipocytes and OvCa cells The enhanced growth and metabolic adaptation of OvCa that grow in adipose-rich peritoneal TME have been attributed to unconventional metabolism characterized by increased rates of the oxidation (β-oxidation) of FA released from adipocytes’ lipolysis [3]. FA and glycerol released from adipocytes are taken up by tumours cells, where FAs are used for β-oxidation, whereas glycerol may either be converted to glucose through gluconeogenesis or directly feeds into the glycolytic pathway providing energy for cellular metabolism [3, 39]. Therefore, we determined the effect of host-SPARC on the metabolic adaptation of ID8 omental tumour nodules developing in SP–/– and SP+/+ mice. We found signiﬁcant upregulation in the above mentioned enzymes involved in β-oxidation with exception of Cpt1 in ID8 tumours growing in SP–/– mice compared with those in the SP+/+ (Fig. 6j). Concomitantly, tumours growing in SP–/– mice exhibited low levels of acylcarnitine conjugated lipids as palmitoyl carnitine and stearoyl carni- tine suggesting reduced substrate availability due to enhanced lipid oxidation by the aforementioned enzymes. Consistently, signiﬁcantly increased levels of 3-dehy- drocarnitine, an intermediate of carnitine degradation were observed in in SP–/– tumours consistent with a tumour metabolic phenotype. In addition, multiple monoacyl gly- cerols including 1-oleoylglycerol accumulated in SP–/– Earlier reports demonstrated that tumour-adjacent adipo- cytes undergo phenotypic changes into CAAs to support cancer cells growth and survival. In addition to expression of pro-inﬂammatory cytokines and TFs [37], CAA release their lipids through lipolysis [3, 4]. Using the heterotypic cultures of ID8 and adipocytes, we found increased free fatty acids (FFA) production in conditioned media (CM) of co-cultures compared with single-cell cultures with higher FFA of in co-cultures including SP–/– adipocytes (Fig. 6a). This effect was inhibited by treating co-cultures of ID8 cells with SP+/+ and SP–/– adipocytes with inhibitors of NFkB, AP-1, and cEBPβ (Supplement Figure 7) inhibited co- culture-induced fatty acid (FA) release from adipocytes with modest effect on the adipocytes in monoculture (Supple- ment Figure 8). Consistently, SP–/– adipocytes expressed higher levels of adipose triglyceride lipase (ATGL) and total and phospho-hormone-sensitive lipase (HSL), the rate- limiting enzymes in the breakdown lipids and mobilization of FFA from adipocytes [38], compared with SP+/+ adi- pocytes (Fig. 6b). Effects of SPARC on reciprocal cross-talk between adipocytes and OvCa cells The number and size of lipid droplets (lower) was quantiﬁed in 10 cells/condition. Plots represent means ± SEM of the ﬂuorescent intensity quantiﬁed in 10 ﬁelds/ experimental condition (n = 3 experiments), and unpaired P < 0.05, t- test. f Effect of SPARC on FA uptake by human OvCa cells. p < 0.0 one-way ANOVA. g Immunoﬂuorescence showing the effect SPARC (5 µg/ml) on FABP4 and CD36 expression in SKOV3 an OVCAR3 cells. Scale bars, 10 µm. h Bars report means ± SEM ﬂuorescence intensity of FABP4 and CD36 quantiﬁed in 10 ﬁeld experimental condition, Student’s t-test. i mRNA expression enzymes involved in FA oxidation of ID8 in mono- and co-cultu with SP+/+ and SP–/– adipocytes. Bars report means ± SEM of representative of three independent experiments each in triplicate. < 0.05. Student’s t-test. j Bars depict mean ± SEM of the changes the transcript levels (n = 3/genotype). p-values are determined b unpaired Student’s t-test Fig. 6 Effect of SPARC on metabolic programming of adipocytes and OvCa cells. a Schema of the co-culture (upper), bars represent means ± SEM of FFA release in media of co-cultures for 48h. p < 0.05, Student’s t-test. b WB showing increased lipases in adipocytes in mono- and co-cultures with ID8 cells. c Schema showing co-cultures of ID8 and adipocytes. d Photomicrographs of FA uptake by ID8 cells visualized by Bodipy staining of in co-cultures (upper, 40×; scale bar 50 µm) and electron microscopy (lower, 6800×; scale bar, 500 nm). e Quantiﬁcation of Bodipy ﬂuorescence intensity in the in ID8 cells in mono- and co-cultures with adipocytes. The number and size of lipid droplets (lower) was quantiﬁed in 10 cells/condition. Plots represent means ± SEM of the ﬂuorescent intensity quantiﬁed in 10 ﬁelds/ experimental condition (n = 3 experiments), and unpaired P < 0 05, t- test. f Effect of SPARC on FA uptake by human OvCa cells. p < 0. one-way ANOVA. g Immunoﬂuorescence showing the effect SPARC (5 µg/ml) on FABP4 and CD36 expression in SKOV3 a OVCAR3 cells. Scale bars, 10 µm. h Bars report means ± SEM ﬂuorescence intensity of FABP4 and CD36 quantiﬁed in 10 ﬁel experimental condition, Student’s t-test. i mRNA expression enzymes involved in FA oxidation of ID8 in mono- and co-cult with SP+/+ and SP–/– adipocytes. Bars report means ± SEM of representative of three independent experiments each in triplicate. < 0.05. Student’s t-test. Effects of SPARC on reciprocal cross-talk between adipocytes and OvCa cells ID8 cells exposed to the same co-culture conditions with adipocytes exhibited higher levels of intracellular FA and larger lipid droplets in co-cultures with SP–/– compared with SP+/+ adipocytes as determined by Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC Fig. 6 Effect of SPARC on metabolic programming of adipocytes and OvCa cells. a Schema of the co-culture (upper), bars represent means ± SEM of FFA release in media of co-cultures for 48h. p < 0.05, Student’s t-test. b WB showing increased lipases in adipocytes in mono- and co-cultures with ID8 cells. c Schema showing co-cultures of ID8 and adipocytes. d Photomicrographs of FA uptake by ID8 cells visualized by Bodipy staining of in co-cultures (upper, 40×; scale bar 50 µm) and electron microscopy (lower, 6800×; scale bar, 500 nm). e Quantiﬁcation of Bodipy ﬂuorescence intensity in the in ID8 cells in mono- and co-cultures with adipocytes. The number and size of lipid droplets (lower) was quantiﬁed in 10 cells/condition. Plots represent test. f Effect of SPARC on FA uptake by human OvCa cells. p < 0.05, one-way ANOVA. g Immunoﬂuorescence showing the effect of SPARC (5 µg/ml) on FABP4 and CD36 expression in SKOV3 and OVCAR3 cells. Scale bars, 10 µm. h Bars report means ± SEM of ﬂuorescence intensity of FABP4 and CD36 quantiﬁed in 10 ﬁelds/ experimental condition, Student’s t-test. i mRNA expression of enzymes involved in FA oxidation of ID8 in mono- and co-culture with SP+/+ and SP–/– adipocytes. Bars report means ± SEM of a representative of three independent experiments each in triplicate. p < 0.05. Student’s t-test. j Bars depict mean ± SEM of the changes in the transcript levels (n = 3/genotype). p-values are determined by Fig. 6 Effect of SPARC on metabolic programming of adipocytes and OvCa cells. a Schema of the co-culture (upper), bars represent means ± SEM of FFA release in media of co-cultures for 48h. p < 0.05, Student’s t-test. b WB showing increased lipases in adipocytes in mono- and co-cultures with ID8 cells. c Schema showing co-cultures of ID8 and adipocytes. d Photomicrographs of FA uptake by ID8 cells visualized by Bodipy staining of in co-cultures (upper, 40×; scale bar 50 µm) and electron microscopy (lower, 6800×; scale bar, 500 nm). e Quantiﬁcation of Bodipy ﬂuorescence intensity in the in ID8 cells in mono- and co-cultures with adipocytes. Effects of SPARC on reciprocal cross-talk between adipocytes and OvCa cells j Bars depict mean ± SEM of the changes the transcript levels (n = 3/genotype). p-values are determined unpaired Student’s t-test Fig. 6 Effect of SPARC on metabolic programming of adipocytes and OvCa cells. a Schema of the co-culture (upper), bars represent means ± SEM of FFA release in media of co-cultures for 48h. p < 0.05, Student’s t-test. b WB showing increased lipases in adipocytes in mono- and co-cultures with ID8 cells. c Schema showing co-cultures of ID8 and adipocytes. d Photomicrographs of FA uptake by ID8 cells visualized by Bodipy staining of in co-cultures (upper, 40×; scale bar 50 µm) and electron microscopy (lower, 6800×; scale bar, 500 nm). e Quantiﬁcation of Bodipy ﬂuorescence intensity in the in ID8 cells in mono- and co-cultures with adipocytes. The number and size of lipid droplets (lower) was quantiﬁed in 10 cells/condition. Plots represent means ± SEM of the ﬂuorescent intensity quantiﬁed in 10 ﬁelds/ experimental condition (n = 3 experiments), and unpaired P < 0.05, t- test. f Effect of SPARC on FA uptake by human OvCa cells. p < 0.05, one-way ANOVA. g Immunoﬂuorescence showing the effect of SPARC (5 µg/ml) on FABP4 and CD36 expression in SKOV3 and OVCAR3 cells. Scale bars, 10 µm. h Bars report means ± SEM of ﬂuorescence intensity of FABP4 and CD36 quantiﬁed in 10 ﬁelds/ experimental condition, Student’s t-test. i mRNA expression of enzymes involved in FA oxidation of ID8 in mono- and co-culture with SP+/+ and SP–/– adipocytes. Bars report means ± SEM of a representative of three independent experiments each in triplicate. p < 0.05. Student’s t-test. j Bars depict mean ± SEM of the changes in the transcript levels (n = 3/genotype). p-values are determined by unpaired Student’s t-test B. John et al. tissues indicating a change in complex lipid hydrolysis evidenced by elevated levels of glycerol (Supplement Fig- ure 9). These data suggest a novel signiﬁcant role of adi- pocyte SPARC on metabolic reprogramming of OvCa cells in the unique peritoneal milieu. Inhibitory effect of SPARC on adipogenic differentiation The above described data prompted us to investigate the effect of SPARC on omental adipocytes’ differentiation and tissues indicating a change in complex lipid hydrolysis evidenced by elevated levels of glycerol (Supplement Fig- ure 9). These data suggest a novel signiﬁcant role of adi- pocyte SPARC on metabolic reprogramming of OvCa cells in the unique peritoneal milieu. Effects of SPARC on reciprocal cross-talk between adipocytes and OvCa cells Inhibitory effect of SPARC on adipogenic differentiation The above described data prompted us to investigate the effect of SPARC on omental adipocytes’ differentiation and Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC (Fig. 7h). Consistently, SPARC inhibited the expression of adipogenic TFs PPARγ, cEBPα, and cEBPβ, as well as markers of adipogentic differentiation HSL, ATGL, and FABP4 (Fig. 7i). Interestingly, consistent with our obser- vation with SP+/+ adipocytes, SPARC protein expression increased in differentiating 3T3L adipocytes by D3 then decreased to basal levels by D7 and D10 (Fig. 7j). In addition, treatment with rSPARC decreased the mRNA levels of the master regulator of differentiation Pparg and Cebp isoforms, as well as markers of early and late differ- entiation as Fasn, Adipoq, lep, Cd36, Acly, Scd1, Acaca, Pnpla2/Atgl1, Lipe/Hsl, and Screbp-1c (Supplement Figure 10). These data further support the effect of SPARC inhi- biting omental adipocyte differentiation and in accord with earlier reports of the anti-adipogenic effect of SPARC on different adipocyte and mesenchymal stem cell niches. Importantly, when these adipocytes are challenged by OvCa cells, SPARC suppresses their interaction with cancer cells and consequently inhibits the acquisition of CAA pheno- type that fosters OvCa omental dissemination and colonization. Fig. 7 SPARC exerts anti-adipogenic effect on omental adipocytes and 3T3-L1 cells. a Oil Red O (ORO) staining of differentiating SP+/+ and SP–/– pre-adipocytes (×10 magniﬁcation). b SP–/– adipocytes exhibit increased size and number of fat droplets day 10 (D10) post- differentiation as shown by ORO (×20 magniﬁcation) and electron microscopy (×6800). c Plots indicate mean ± SEM of the number (upper) and size (lower) of lipid droplets quantiﬁed in EM images (10 cells/experimental condition). P values are determined by Student's t- test. d Western blots showing the kinetics of expression of adipogenic transcription factors during differentiation of SP+/+ and SP–/– pre- adipocytes. e The expression of SPARC protein during differentiation of SP+/+ adipocytes. f Effect of SPARC (5 µg/ml in PBS–0.4% BSA) on the expression of the adipogenic transcription factors during dif- ferentiation of SP+/+ adipocytes. Tubulin was used as loading control. g Conﬂuent 3T3-L1 pre-adipocytes (D0) were differentiated either in the presence or absence of 5 μg/ml rSPARC up to day 10. The cells were harvested at days 0, 3, 7, and 10. Intracellular lipids were stained with ORO. Effects of SPARC on reciprocal cross-talk between adipocytes and OvCa cells h Bars (right) represent means ± SEM of the quantiﬁcation of ORO-stained cells solubilized with 100% isopropanol for 5min and measuring the absorbance A492 nm. *p < 0.0001, two-way ANOVA with Sidak post-hoc test. h Images of Bodipy ﬂuorescent staining of intracellular lipids in differentiating 3T3-L1 pre-adipocytes in the presence or absence of 5 μg/ml rSPARC. i Western blots showing the expression of SPARC and the adipogenic differentiation markers in differentiating 3T3-L1 cells in the presence or absence of SPARC plasticity especially that the anti-adipogenic function of SPARC was earlier reported [25, 29, 40, 41]. Omental tis- sues isolated from age-matched SP–/– and SP+/+ female mice did not exhibit discernible macroscopic or microscopic differences. Omental adipocytes were isolated from the buoyant layer after digestion and centrifugation of omental tissues (Supplement Figure 1A). SP–/– adipocytes exhibited increased lipid droplets compared with SP+/+ (Supplement Figure 1A). Pelleted stromal cells were composed mainly of adipocytes, pre-adipocytes, and vascular smooth muscle cells [29] and exhibited ﬁbrobalstoid phenotype in standard culture as stained with α-smooth muscle actin (Supplement Figure 1C). When conﬂuent monolayers of SP+/+ and SP–/– omental stromal cells were exposed to adipogenic growth medium (ADM) for 7 days D7, SP–/– exhibited faster adi- pogenic differentiation, with increased accumulation of intracellular lipids as determined by Oil Red O (ORO, Fig. 7a), and electron microscopy with signiﬁcantly larger numbers and size of lipid droplets (Fig. 7b, c). Fully dif- ferentiated SP–/– adipocytes exhibited signiﬁcantly higher levels of adipogenic TFs PPARγ, and c/EBPβ than SP+/+ adipocytes (Fig. 7d). SPARC protein exhibited differential expression in differentiating SP+/+ adipocytes being highest after 3 days (D3), in adipogenic medium and declined to basal levels in fully differentiated adipocytes at D7 (Fig. 7e). Exogenous SPARC suppressed the expression of adi- pogenic factors when added to differentiating SP–/– adipo- cytes (Fig. 7f). Independently, treating 3T3-L1 pre- adipocytes with rSPARC (5 μg/ml) for 10 days signiﬁcantly inhibited the accumulation of intracellular lipids as deter- mined by ORO (Fig. 7g) and Bodipy ﬂuorescent staining Discussion In the present study, we expand our knowledge on the biological roles of SPARC in OvCa by investigating its role in regulating the interactions between OvCa cells and omental adipocytes. Our ﬁndings in the present study indicate that SPARC exerts a tumour-suppressor effect on OvCa cells in part through inhibiting their interactions with omental adipocytes; the main site of metastasis of OvCa [3, 42, 43], and the most common cause of mortality in OvCa patients [43]. We used multipronged approach employing human and murine OvCa cell lines, as well as primary human and murine omental adipocytes in single and co-culture. We conﬁrmed the inhibitory effect of SPARC by using primary adipocytes from SP–/– and SP+/+ mice in single and co- cultures with murine ID8 cell line, a model system that is extensively used in syngeneic models of OvCa in normal and transgenic mice C57B6 background and is well characterized as has been reported in ~145 publications. However, ID8 cell line may not be considered a faithful representation of HGSC because it was originally derived by spontaneous transfor- mation of high passage murine ovarian surface epithelium [44]. To circumvent this limitation of the mouse model, and to ascertain that our ﬁndings are not limited to the phenotype of one cell line or model system, we complemented our mechanistic and phenotypic studies with three human OvCa cell lines in mono- and co-cultures with human omental (pre) adipocytes, as well as human tumour specimens, as well as genetic manipulation of SPARC in human primary omental B. John et al. correlated with the expression of SPARC protein in the cancerous compartment, as well as in the juxta-tumoral adipocytes in two independent patients’ cohorts. Survival data curated from The Cancer Genome Atlas (TCGA) revealed that the expression of cEBPβ, cJun, cFos, and NFkB1 were associated with poor patients’ survival (Sup- plement Figure 11). SPARC protein expression in the OvCa cells negatively correlates with nuclear localization of these TFs in patients’ specimens and in syngeneic tumours growing in Sparc-deﬁcient mice. In tumour specimens with adjacent omental tissues, we show that the negative corre- lation between both tumour and adipocyte SPARC with the nuclear localization of these factors in tumour cells and juxta-tumoral adipocytes. SPARC transcript was not cor- related with patients’ survival, most probably due to the distinctive compartmentalization of SPARC protein expression in patients’ tumours. Discussion In addition, our unpub- lished data indicated that genetically manipulating SPARC in SPARC-proﬁcient CAOV3 cell line by overexpression or knockdown inversely correlated with malignant phenotype in vitro and peritoneal dissemination and mouse survival when they were injected ip in nude mice. Furthermore, we provide evidence that SPARC exerts a tumour-suppressor effect in OvCa linking inﬂammation to metabolic pro- gramming, a process termed “metaﬂammation” that has been implicated in diseases associated with inﬂammation and perturbed bioenergetics as diabetes, obesity and aging [52]. Together with our recent report that SPARC inhibits metabolic plasticity and mitochondrial bioenergetics in OvCa [53], our data underscore the multi-faceted sup- pressor role of SPARC in limiting peritoneal dissemination of OvCa. (pre)adipocytes and human OvCa cells. We further demon- strated the role of SPARC in omental adipocyte differentia- tion as SP–/– adipocytes exhibited accelerated adipogenic differentiation and signiﬁcantly accumulated more lipids than SP+/+ adipocytes. This ﬁnding is consistent with earlier report of increased tendency of SP–/– bone marrow cells for adipogenic differentiation [25, 29, 40]. Indeed, the use of the 3D co-cultures of OvCa cells and adipocytes provided a useful tool for mechanistic and functional studies of tumour cells and adipocytes with monitoring concomitant changes in both cell types in response to genetic manipulations and pharmacologic inhibitors. We conﬁrmed the speciﬁcity of the inhibitory effect of adipocyte-SPARC on the functional cross-talk between OvCa cells and adipocytes independent of con- tribution of other cells in the TME and the complex host background. This approach ﬁrst conﬁrmed the direct auto- crine and paracrine effects of SPARC on tumour cell–adipocyte interactions. Adipocyte-SPARC exerted a suppressor effect at multiple levels. First, adipocyte-SPARC decreased homing, adhesion, proliferation of OvCa cells to omental adipocytes and inhibited their invasiveness. We showed that these effects were due to the effects of SPARC mitigating inﬂammatory secretome as evidenced by func- tional blocking antibodies. These are in accord with our earlier reports of the anti-inﬂammatory role of SPARC in OvCa [6–8]. Second, adipocyte-SPARC, as well as rSPARC suppressed the phenotypic switch of omental adipocytes and their acquisition of CAA inﬂammatory phenotype through suppression of OvCa cell-induced acti- vation of cEBPβ, NFκB, and AP-1 TFs and their down- stream inﬂammatory and metabolic effects. Discussion Pharmacologic inhibitors of these TFs not only phenocopied the effect of rSPARC on FFA release from adipocytes in mono-and co- cultures with OvCa cells, but they also mitigated the increase FFA release from SP–/– adipocytes in co-cultures with ID8 cells. Inﬂammatory chemokines as TNFα, IL-2, and IL-6, were reported to increase lipolysis and negatively affect metabolic homeostasis [15, 45–48]. Moreover, the reciprocated transactivation between cEBPβ, NFkB, and AP-1 in myriad physiological and pathological contexts including lipid homeostasis, differentiation, inﬂammation, and cancer has been established and involves the tran- scription of multiple common factors including the enzymes, and chemokines studied herein. Our data using pharmacologic inhibitors of NFkB further supported the transactivation loop between NFkB and cEBPβ that has been earlier reported and conﬁrmed [16, 21]. The associa- tion of these TFs with aggressiveness of many cancers, including OvCa, have been reported [28, 49–51]. Our data using patients’ specimens indicated the progressive increase and nuclear localization of these TFs as a function of OvCa progression Importantly their expression inversely Using OvCa cell-adipocytes co-cultures, we found that SPARC inhibited the increased lipolysis and FFA release from adipocytes concomitant with inhibition of increased FA uptake by OvCa cells. We show that the effect of SPARC is due to differential effects on adipocytes and OvCa cells; not only inhibiting inﬂammation-induced lipolysis and FFA release in adipocytes, but also down- regulating the surface expression of FA transporters CD36 and FABP4, both have been associated with poor prognosis in patients with OvCa [54–56]. FABP4 has been recently reported as hypoxia-regulated gene [56], and its expression in tumour endothelial cells is associated with increased angiogenesis, especially in low-grade stroma-rich tumours [57]. In addition, FABP4 has been reported as predictor of residual disease in HGSC [55] and its overexpression in patients’ tumours is associated with increased metastatic burden and poor survival [56]. As a consequence of increase FFA uptake, OvCa cells have been shown to rely on FA oxidation for energy pro- duction [3]. Recent reports [58] demonstrated the link between FA oxidation and cancer cell proliferation and Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC survival through activation of salt-inducible kinase-2 (SIK2) in tumour cells co-cultured with omental adipocytes. Activated SIK2 phosphorylated acetyl-CoA carboxylase (ACC) and phosphatidyl inositol 3 kinase (PI3K) and, thus, simultaneously regulates both fatty acid oxidation and cancer cell proliferation and survival. Discussion Consistently, our recent report [53] showed that SPARC inhibits mitochon- drial bioenergetics and ATP, glycolysis and ATP produc- tion, “metabolic plasticity” through inhibition of activation of adenosine monophosphate kinase, ACC, as well as mammalian target of rapamycin and its downstream targets; thus linking metabolic programming and mitochondrial dysfunction to cancer cell proliferation and survival. Together with our results herein, it is plausible that the effect of SPARC on cancer cell-adipocyte metabolic pro- gramming through an effect on SIK2, or through an effect on enzymatic processes that occur in the mitochondria including FA oxidation, leading to signiﬁcant reactive oxygen species (ROS) generation and activation of the transcriptional inﬂammatory oncogenic machinery orche- strated by NFkB, cJun, and cEBPβ. differentiation medium. Transient and stable overexpression of SPARC was done using adenoviral and retroviral vectors as previously described [7, 36]. Transduction efﬁciency was optimized to overcome negative selection due to the anti- proliferative effect of SPARC. SPARC depletion was done by transduction using lentiviral vectors with short-hairpin RNA targeting SPARC (shSPARC) with control shRNA targeting non-human target [36]. All were purchased from Sigma, TRC (TRCN000008710, TRCN000008709, and TRCN000008711). Culture media, supplements, anti- biotics, and growth factor-reduced matrigel were from Invitrogen (Grand Island, NY) and BD Biosciences (Franklin Lakes, NJ). Recombinant human and murine SPARC were purchased from Peprotech (Rocky Hill, NJ, catalog# 120-36) and R&D Systems Inc. (Minneapolis, MN, catalog# 942-SP). Stock solutions of rSPARC from the same lot numbers were reconstituted in Dulbecco's phos- phate buffered saline (DPBS)–0.1% bovine serum albumin (BSA) (1 mg/ml), aliquoted and stored at –08 oC till used. The purity of SPARC was validated by SDS-PAGE under reducing and non-reducing conditions, and Commassie blue staining showing the abundance of the protein at the appropriate molecular weight along with the BSA (Sup- plement Figure 1). Unless otherwise stated, all reagents were purchased from Sigma Aldrich (St. Louis, MO) and ThermoFisher (Pittsburgh, PA). In summary, this is the ﬁrst investigation of the role of adipocyte-SPARC in regulation of omental adipocytes and its impact on OvCa progression and omental metastasis. In vivo syngeneic model SP+/+ and SP–/– mice are maintained on C57BL/6 back- ground for at least 10 backcrosses. Mice were housed in a speciﬁc pathogen-free (SPF) facility. All animal experi- ments were approved by IACUC of the University of Vir- ginia (IACUC# 3879) and Wake Forest University Schools of Medicine (IACUC# A16-165). ID8 (4×106/100 µl sterile PBS) ip injections in SP+/+ and SP–/– mice was previously described [5, 7]. Dissected tumour tissues were either snap frozen in liquid nitrogen then stored at –80oC till used or ﬁxed in 10% neutral zinc formalin and embedded in par- afﬁn. For in vivo homing experiments, ID8-GFP cells (4×106) were injected ip in 5-week-old female SP+/+ and SP–/– mice [5]. Mice were euthanized at the time points indicated in ﬁgure legends, omenta were dissected, placed in wells of six-well plates, and ID8-GFP cell adhesion was determined using a ﬂuorescent inverted microscope as earlier described [36]. GFP ﬂuorescence intensity was quantiﬁed in ﬁve images/mouse omentum using Image J software. Background ﬂuorescence was normalized to the ﬂuorescence of images of omenta isolated from sham (PBS)-injected mice. For functional blocking experiments, mouse IL-6 (MAB406; 1 µg/ml), mouse TNFα (MAB4101, 1 µg/ml), CCL2/MCP-1 (AB-479-NA; 30 µg/ml), mouse Discussion Our study provides novel information on the tumour- suppressor role of the SPARC in the regulation of OvCa- omental cross-talk and highlights the role of over- expression, as well as exogenous recombinant SPARC not only on mitigating the effect of loss of SPARC in adipo- cytes but on suppressing pro-tumorigenic and metabolic programming, thus making the omental adipocyte niche unfavorable for seeding and colonization of tumour cell. We show the effect of SPARC on activation of the main TFs orchestrating inﬂammation, adipocyte dysfunction, and cancer progression. Our data highlight the importance of SPARC protein as a promising therapeutic target in OvCa. Antibodies, reagents, and western blots Monoclonal and polyclonal antibodies against total and phosphorylated cEBPβ, PPAR-γ, HSL, p-HSL (ser660), cJun, p65NFκB, ATGL, human and murine SPARC, α- smooth muscle actin (α-sma), and β-tubulin antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA), Abcam (Cambridge, MA), and Santa Cruz (Santa Cruz, CA). Cells were lysed and protein concentration were determined as earlier described [7, 36]. Cellular proteins (20 µg) were resolved by 4–20% SDS-PAGE, transferred to polyvinylidene diﬂuoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA), and probed with primary and the appropriate ﬂuorescent- and horseradish peroxidase (HRP)- conjugated secondary antibodies (Licor, NE and Abcam). Blots were visualized using Odyssey 3v and Amersham Imagers. Isolation of primary murine omental adipocytes Omenta were isolated from euthanized 6- to 8-week-old female mice and were digested by collagenase/dispase for 2 h at 37 oC with gentle agitation [25]. Slurries were allowed to separate into ﬂoating mature adipocytes (top layer) (Supplement Figure 1) and pellets mainly consisting of ﬁbroblasts, smooth muscle cells, and pre-adipocytes [25, 41]. Mature adipocytes were immediately used for the assays involving murine cells. Adipocytes were maintained in adipocytes maintenance medium (ADMM, Zen Bio, Raleigh, NC) supplemented with 10% fetal bovine serum (FBS), antibiotic–antimycotic solution. Omental stromal cells were maintained in Dulbecco’s modiﬁed Eagle’s medium/F12 with 10% FBS and antibiotic–antimycotic solution. Adipogenic differentiation was initiated in con- ﬂuent ﬁbroblastoid omental stromal cells, by adipogenic medium (ADM, Zen Bio) for 3 days, after which they were switched to ADMM for further 10–14 days. The differ- entiation of adipocytes was compared by microscopic examination and stained with ORO staining and Bodipy ﬂuorescent staining (Molecular Probes, ThermoFisher) [3]. For in vitro homing experiments, ID8-GFP cells (1 × 105) were added in the top chamber of 8 µm-pore trans-well inserts and were allowed to migrate towards SP–/– and SP +/+ primary adipocytes or SP–/– and SP+/+ adipocytes in the bottom chambers for 6 h at 37 °C. For invasion assays, 1×105 OvCa cells/100 µl SFM were added on top of matrigel-coated 8 µm-pore trans-well inserts, and incubated at 37 oC for 6 h with SPARC-deﬁcient and SPARC- proﬁcient omental adipocytes or their CM in the bottom chambers of the trans-wells [3, 59, 60]. 3D omental cultures and in vitro proliferation, adhesion, and invasion assays The 3D omental culture was assembled by plating 5 × 105 fresh omental adipocytes in 500 µl of reduced growth factor–matrigel mixed with growth media (1:3) into a 24- well culture plate. For proliferation assays, ID8-GFP cells (5000 cells/100 µl serum-free media (SFM)–2% FBS) were added on top of adipocytes embedded in matrigel. Green ﬂuorescence at A488 was determined daily after plating for 72 h. As controls, parallel experiments were performed using ID8-GFP cells grown under similar conditions in absence of adipocytes and/or matrigel. The number of adi- pocytes was determined by parallel experiments counting the number of adipocytes after sorting out GFP-labeled cancer cells before, during, and at the end of corresponding experiments. For proliferation experiments (~72 h), parallel Cell culture, plasmid transfections, and viral transductions Murine (ID8) were earlier described [5, 7]. Human OvCa (SKOV3, OVCAR3, CAOV3) cell lines were originally from ATCC and were maintained at low passage and in complete growth media as earlier described [5, 7]. Cells were tested for mycoplasma once/month at Wake Forest Baptist Cell and Viral Vector Core Lab (CVVL). Primary human omental pre-adipocytes were obtained from Zen Bio, Inc. (Raleigh, NC, USA) and were maintained in omental pre-adipocytes medium and differentiated using adipocyte B. John et al. anti-leptin (AF 498, 0.3 µg/ml), mouse CCL3/MIP1 (AF450, 0.3 µg/ml), mouse IL-2 (MAB702, 1 µg/ml), and VEGF (AF-493-NA; 1 µg/ml), were injected ip in SP+/+ and SP–/– mice 30 min before ID8 injection. Normal isotype controls included rat anti-mouse IgG1 (MAB005), goat anti-mouse IgG (AB-108-C) injected in the same dose. All neutralizing antibodies were from R&D Systems. In some experiments, mice, athymic nude mice (Jackson Labs, Bar Harbor, ME) were injected subcutaneously (SC) with murine ID8 cells mixed with SP+/+ or SP–/– adipocytes in a ratio of 1:2 (ID8:adipocytes). SC tumours were measured twice weekly by caliper for 6 weeks [5, 7, 36]. experiments were performed as follows: for SP–/– and SP+/+ adipocytes and ID8-GFP cells, another parallel experiment in which adipocytes in matrigel were separated from ID8- GFP cells by 0.4 µm trans-well inserts (ID8 cells added in the top chambers and the inserts were touching adipocytes). ID8 proliferation was determined by measuring A488 ﬂuorescence and cell counting by Trypan blue exclusion. Adipocytes in the bottom chambers were trypsinized and counted. Similarly, human omental pre-adipocytes embed- ded in matrigel were allowed to differentiate and OvCa- GFP expressing cells were added on the top chamber of trans-well inserts (0.4 µm) directly placed on the adipocytes plugs, only separated by the membranes. At the time points indicated in the ﬁgures and ﬁgure legends, OvCa cells were collected and their proliferation were determined by cell counting and Trypan blue exclusion, as well as measuring the GFP ﬂuorescence. Adipocytes in the bottom were trypsinized and counted by Trypan blue exclusion. We did not ﬁnd changes in the adipocytes numbers between the experimental conditions up to 72 h. Immunohistochemistry Monoclonal and polyclonal antibodies against SPARC, p65NFκB, cEBPα, cJun, CD36 and FABP4 and horse raddish peroxidase (HRP)- and ﬂuorescent-labeled second- ary antibodies were purchased from sources described in Supplement Table 2. For immunohistochemistry parafﬁn, HRP secondary antibodies were used and signal was developed with ImmPACT DAB Peroxidase (HRP) Sub- strate (Vector Labs, Burlingame, CA) and counterstained with hematoxylin. For immunoﬂuorescence staining, appropriate Alexa-ﬂuor 488 and 594 secondary antibodies were used and nuclei were counterstained with prolong anti- fade mounting media (Invitrogen). Nuclear and cytoplasmic expression of the aforementioned proteins in cancer cells and adipocytes were determined as earlier described [35, 36, 60]. Images were acquired using Aperio Scanscope (Leica Microsystems, Buffalo Grove, IL), and Olympus VS-110 Virtual Imaging System (Life Science Solutions, Center Valley, Pennsylvania). Digital image analysis of protein intensity and frequency was performed using Halo software (Indica Lab, Corrales, NM) and VisioPharm (Broomﬁeld, CO) to segment tumour vs stroma and nuclear vs cytoplasmic and tissue alignment of sections stained with hematoxylin and eosin (H&E) with those stained colori- metric and ﬂuorescent stains. We speciﬁcally focused on the areas that include tumour–adipocyte interface and classiﬁed the regions containing mainly adipocytes as “stroma” as guided by aligned matching H&E slides. Staining frequency and intensity were assigned arbitrary scores as earlier described [35, 36, 60]. Composite expression score (H- score) was calculated by multiplying the frequency and intensity scores [35, 36, 60]. For CD36 and FABP4 immunoﬂuorescence staining, cells were seeded in dupli- cate wells LabTek eight-well slide chambers and were treated with SPARC (5 µg/ml) for 24 h. Immunostaining was carried out using rabbit anti-CD36 and FABP4 after ﬁxing cells with 4% paraformaldehyde and permeabiliza- tion with Triton X-100. Alexa-ﬂuor 594 secondary anti- bodies (Invitrogen) were used. Image acquisition was done using Leica AF6000 Modular System confocal microscope (Leica). Image acquisition, deconvolution, and maximum projection analysis were performed with the program LAS AF (Leica). Morphometric analysis was performed by Image J [53] in 10 random ﬁelds/experimental condition. Each experiment is performed three times. In some experiments, the following inhibitors were included in the co-cultures of OvCa cells and adipocytes: NFkB inhibitory peptides for RelA/NFkB p65 (p Ser276, NBP2-26505, 50 µM), which functions as a p65 decoy inhibiting Ser276 phosphorylation of RelA, and NFkB p50 (NLS, NBP2-29323, 50 µM) inhibitory peptide that blocks p50 nuclear translocation, as well as their control peptide (NBP2-29334, 50 µM). Cytokine, chemokine, and metabolic assays The levels of cytokines/chemokines in the CM of different experimental conditions were determined using the appro- priate species-speciﬁc commercial kits from R&D Systems, and RayBiotech Inc., as per the manufacturer’s recom- mendations. Intracellular fatty acids were determined by Bodipy ﬂuorescent staining (Molecular Probes) and oil red staining. Fatty Acid Fluorometric Assay and uptake assay kits were from Cayman Chemicals (Ann Arbor, MI) and Abcam. Immunohistochemistry NFkB inhibitory peptides were purchased from R&D Systems. JNK inhibitor SP600125 was from SelleckChem, Inc. (Houston, TX) supplied as10 µM in dimethylsulfoxide (DMSO). Of note is that most of the commercially available cEBPβ inhibitors function by virtue of their anti-inﬂammatory effect [61] and were also reported to inhibit p65RelA subunit of NFkB [62]. Thus, we used NFkB inhibitors and conﬁrmed their inhibitory effects on cEBPβ activity. Luciferase reporter assays NFκB and AP-1 luciferase reporters were described pre- viously [35, 36]. The cEBPβ responsive luciferase vector Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC (pGL2–5xcEBPβ-TK-Luc) and matching empty vector control [21] were kindly provided by Dr. Xianjun Fang at Medical College of Virginia, Richmond, VA. Differential luciferase reporter assays in co-cultures of OvCa cells and adipocytes were performed as earlier described [35, 36]. For investigation of the TF reporter activity, ID8 and omental adipocytes were transfected with the luciferase reporter plasmids using Fugene 6 (Promega, Madison WI) 24 h prior to co-culture. As an internal control for transfection efﬁ- ciency, cells were co-transfected with TK-Reinella-lucifer- ase plasmid (at a ratio of 20:1 ﬁreﬂy:TK-Reinella). Cells were co-cultured for 24 h and the luciferase activity was measured using Dual Luciferase Reporter Assay kit (Pro- mega) as per manufacturer’s instruction. Results were fur- ther normalized to DNA content of the cells determined by CyQuant assay to correct for changes in cell proliferation/ experimental condition [35, 36, 59]. human OvCa tumour tissues with the adjacent omental tissues were obtained from Wake Forest Tumor Tissue and Pathol- ogy Shared Resources (WF-TTPSR, IRB#IRB00036014). The clinical data of the samples are summarized in Supple- ment Table 1. Compliance with ethical standards Conﬂict of interest The authors declare that they have no conﬂict of interest. 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/. RNA isolation and qRT-PCR expression scores in stained tissues were performed using Spearman’s correlation. Differences were deemed sig- niﬁcant at p < 0.05. GraphPad Prism 7.0 (San Diego, CA). expression scores in stained tissues were performed using Spearman’s correlation. Differences were deemed sig- niﬁcant at p < 0.05. GraphPad Prism 7.0 (San Diego, CA). Total RNA was extracted from cultured cells by RNeasy kits (Qiagen, Valencia, CA) and qRT-PCR was carried out using iScript cDNA Synthesis Kit and iQSYbr Green Supermix and Bio-Rad CFX thermal cycler (Bio-Rad, Hercules, CA). The primer sequences for mouse and human genes are summarized in Supplement Tables 3-4. Each experiment was performed in triplicate and repeated three times. Acknowledgements The authors wish to acknowledge the support of the Wake Forest Baptist Comprehensive Cancer Center Tumor Tissue and Pathology Shared Resources (TTPSR), Cellular Imaging Shared Resources, Virtual microscopy core, and Cell and Viral Vector Laboratory Shared Resource. The authors also wish to thank members of the University of Virginia Biorepository Tumor Research Facility (BTRF) for providing TMAs and their technical help with immunos- taining, and Mr. Michael White for help with immunoﬂuorescence and confocal microscopy. References 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018. CA Cancer J Clin. 2018;68:7–30. 2. Lengyel E, Burdette JE, Kenny HA, Matei D, Pilrose J, Haluska P, et al. Epithelial ovarian cancer experimental models. Oncogene. 2014;33:3619–33. 3. Nieman KM, Kenny HA, Penicka CV, Ladanyi A, Buell-Gutbrod R, Zillhardt MR, et al. Adipocytes promote ovarian cancer metastasis and provide energy for rapid tumor growth. Nat Med. 2011;17:1498–503. 4. Yeung TL, Leung CS, Yip KP, Au Yeung CL, Wong ST, Mok SC. Cellular and molecular processes in ovarian cancer metastasis. A review in the theme: cell and molecular processes in cancer metastasis. Am J Physiol Cell Physiol. 2015;309:C444–456. Metabolomic proﬁling Funding This work is supported by Marsha Rivkin Pilot Award, WFBMC Department of Pathology Research Pilot Funds and R01 CA193437 (NS) and P30CA012197 to the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC). Snap-frozen dissected omental tumour nodules that devel- oped in SP+/+ and SP–/– mice (n = 6/cohort) were prepared as previously described [63]. Brieﬂy, samples were de- proteinized and protein-associated metabolites were extracted. The resulting extracts were analyzed by ultra- performance liquid chromatography and mass spectroscopy (UPLC-MS/MS) with positive and negative ion mode electrospray ionization, as well as gas chromatography and mass spectroscopy (GC-MS). Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadru- pole mass spectrometer using electron impact ionization (EI) and operated at unit mass resolving power. Raw data were extracted, peak identiﬁed, QC processed, and nor- malized using proprietary Metabolon’s hardware and soft- ware. Compounds were identiﬁed by comparison with library entries of puriﬁed standards or recurrent unknown entities [63]. Human ovarian tumour tissues Human OvCa TMA was obtained from the University of Virginia Cooperative Human Tumor Network (CHTN). We included results from serous papillary and poorly differ- entiated subtypes form CHTN TMA. Thirty-ﬁve de-identiﬁed B. John et al. Transmission electron microscopy (TEM) Tumour tissue sections were processed for TEM at Wake Forest Baptist Medical Center (WFBMC) Imaging Core Facility according to standard protocols [64]. Sections were viewed with an FEI Tecnai Spirit TEM operating at 80 kV and images were acquired with an AMT 2Vu CCD camera. Image analysis was performed by counting the number of lipid droplets/cells in 10 cells/experimental condition. Lipid droplet size was measured in images ×1800 magniﬁcation by ImageJ. The averages of the area of lipid droplets/cell were calculated in 10 cells/experimental condition and was presented as arbitrary units. Statistical analysis 2012;18:25–34. 31. Guo L, Li X, Huang JX, Huang HY, Zhang YY, Qian SW, et al. Histone demethylase Kdm4b functions as a co-factor of C/EBPβ to promote mitotic clonal expansion during differentiation of 3T3- L1 preadipocytes. Cell Death Differ. 2012;19:1917–27. 13. Howe LR, Subbaramaiah K, Hudis CA, Dannenberg AJ. Mole- cular pathways: adipose inﬂammation as a mediator of obesity- associated cancer. Clin Cancer Res. 2013;19:6074–83. 32. Guo L, Li X, Tang Q-Q. Transcriptional regulation of adipocyte differentiation: a central role for CCAAT/enhancer-binding pro- tein (C/EBP) β. J Biol Chem. 2015;290:755–61. 14. Ji C, Chen X, Gao C, Jiao L, Wang J, Xu G, et al. IL-6 induces lipolysis and mitochondrial dysfunction, but does not affect insulin-mediated glucose transport in 3T3-L1 adipocytes. J Bioenerg Biomembr. 2011;43:367–75. 33. Hernandez L, Hsu S, Davidson B, Birrer MJ, Kohn EC, Annun- ziata CM. Activation of NF-κB signaling by IKKβ increases aggressiveness of ovarian cancer. Cancer Res. 2010;70:4005–14. 15. Zhang W, Mottillo EP, Zhao J, Gartung A, VanHecke GC, Lee JF, et al. Adipocyte lipolysis-stimulated interleukin-6 production requires sphingosine kinase 1 activity. J Biol Chem. 2014;289:32178–85. 34. House CD, Jordan E, Hernandez L, Ozaki M, James JM, Kim M. et al. NF-kB promotes ovarian tumorigenesis via classical path- ways supporting proliferative cancer cells and alternative path- ways supporting ALDH+ cancer stem-like cells. Cancer Res. 2017;77:6927–40. 16. Zhang Z, Bryan JL, DeLassus E, Chang L-W, Liao W, Sandell LJ. CCAAT/enhancer-binding protein β and NF-κB mediate high level expression of chemokine genes CCL3 and CCL4 by human chondrocytes in response to IL-1β. J Biol Chem. 2010;285:33092–103. 35. Al-Hassan NN, Behzadian A, Caldwell R, Ivanova VS, Syed V, Motamed K, et al. Differential roles of uPAR in peritoneal ovarian carcinomatosis. Neoplasia. 2012;14:259–70. 17. Esteves CL, Kelly V, Breton A, Taylor AI, West CC, Donadeu FX, et al. Proinﬂammatory cytokine induction of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in human adipocytes is mediated by MEK, C/EBPβ, and NF-κB/RelA. J Clin Endocrinol Metab. 2014;99:E160–E168. 36. Said N, Frierson HF, Sanchez-Carbayo M, Brekken RA, Theo- dorescu D. Loss of SPARC in bladder cancer enhances carcino- genesis and progression. J Clin Invest. 2013;123:751–66. 37. Iyengar P, Combs TP, Shah SJ, Gouon-Evans V, Pollard JW, Albanese C, et al. Adipocyte-secreted factors synergistically promote mammary tumorigenesis through induction of anti- apoptotic transcriptional programs and proto-oncogene stabiliza- tion. Oncogene. 2003;22:6408–23. 18. Statistical analysis All other data were analyzed by two-tailed unpaired Stu- dent's t-test and one- and two-way analysis of variance (ANOVA) with Sidak or Tukey post-hoc tests. The asso- ciation of the expression of different proteins were eval- uated using the nonparametric Wilcoxon–Mann–Whitney and Kruskal–Wallis tests. Correlation between the 5. Said N, Motamed K. Absence of host-secreted protein acidic and rich in cysteine (SPARC) augments peritoneal ovarian carcino- matosis. Am J Pathol. 2005;167:1739–52. 6. Said N, Najwer I, Motamed K. Secreted protein acidic and rich in cysteine (SPARC) inhibits integrin-mediated adhesion and growth factor-dependent survival signaling in ovarian cancer. Am J Pathol. 2007;170:1054–63. Regulation of the bi-directional cross-talk between ovarian cancer cells and adipocytes by SPARC 7. Said N, Socha MJ, Olearczyk JJ, Elmarakby AA, Imig JD, Motamed K. Normalization of the ovarian cancer microenviron- ment by SPARC. Mol Cancer Res. 2007;5:1015–30. 26. Piconese S, Costanza M, Tripodo C, Sangaletti S, Musio S, Pittoni P, et al. The matricellular protein SPARC supports follicular dendritic cell networking toward Th17 responses. J Autoimmun. 2011;37:300–10. 8. Said NA, Najwer I, Socha MJ, Fulton DJ, Mok SC, Motamed K. SPARC inhibits LPA-mediated mesothelial-ovarian cancer cell crosstalk. Neoplasia. 2007;9:23–35. 27. Said NA, Elmarakby AA, Imig JD, Fulton DJ, Motamed K. SPARC ameliorates ovarian cancer-associated inﬂammation. Neoplasia. 2008;10:1092–104. 9. Dirat B, Bochet L, Dabek M, Daviaud D, Dauvillier S, Majed B, et al. Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion. Cancer Res. 2011;71:2455–65. 28. Kulbe H, Chakravarty P, Leinster DA, Charles KA, Kwong J, Thompson RG, et al. A dynamic inﬂammatory cytokine network in the human ovarian cancer microenvironment. Cancer Res. 2012;72:66–75. 10. Das SK, Eder S, Schauer S, Diwoky C, Temmel H, Guertl B, et al. Adipose triglyceride lipase contributes to cancer-associated cachexia. Science. 2011;333:233–8. 29. Bradshaw AD, Graves DC, Motamed K, Sage EH. SPARC-null mice exhibit increased adiposity without signiﬁcant differences in overall body weight. Proc Natl Acad Sci USA. 2003;100:6045–50. 11. Ebadi M, Mazurak VC. Evidence and mechanisms of fat depletion in cancer. Nutrients. 2014;6:5280–97. 30. Ewing SJ, Zhu S, Zhu F, House JS, Smart RC. C/EBPβ represses p53 to promote cell survival downstream of DNA damage inde- pendent of oncogenic Ras and p19(Arf). Cell Death Differ. 2008;15:1734–44. 12. Grisouard J, Bouillet E, Timper K, Radimerski T, Dembinski K, Frey DM, et al. Both inﬂammatory and classical lipolytic path- ways are involved in lipopolysaccharide-induced lipolysis in human adipocytes. Innate Immun. Statistical analysis surgical debulking of high-grade serous ovarian cancer. Clin Cancer Res. 2014;20:3280–8. 45. Vaysse C, Lomo J, Garred O, Fjeldheim F, Lofteroed T, Schlichting E, et al. Inﬂammation of mammary adipose tissue occurs in overweight and obese patients exhibiting early-stage breast cancer. NPJ Breast Cancer. 2017;3:19. 56. Gharpure KM, Pradeep S, Sans M, Rupaimoole R, Ivan C, Wu SY, et al. FABP4 as a key determinant of metastatic potential of ovarian cancer. Nat Commun. 2018;9:2923. 46. De Pergola G, Silvestris F. Obesity as a major risk factor for cancer. J Obes. 2013;2013:291546. 57. Harjes U, Bridges E, Gharpure KM, Roxanis I, Sheldon H, Miranda F, et al. Antiangiogenic and tumour inhibitory effects of down- regulating tumour endothelial FABP4. Oncogene. 2017;36:912–21. 47. Fontana L, Eagon JC, Trujillo ME, Scherer PE, Klein S. Visceral fat adipokine secretion is associated with systemic inﬂammation in obese humans. Diabetes. 2007;56:1010–3. 48. Doerstling SS, O’Flanagan CH, Hursting SD. Obesity and cancer metabolism: a perspective on interacting tumor–intrinsic and extrinsic factors. Front Oncol. 2017;7:216. 58. Miranda F, Mannion D, Liu S, Zheng Y, Mangala LS, Redondo C, et al. Salt-inducible kinase 2 couples ovarian cancer cell metabolism with survival at the adipocyte-rich metastatic niche. Cancer Cell. 2016;30:273–89. 49. Barbolina MV, Moss NM, Westfall SD, Liu Y, Burkhalter RJ, Marga F, et al. Microenvironmental regulation of ovarian cancer metastasis. Cancer Treat Res. 2009;149:319–34. 59. Said N, Sanchez-Carbayo M, Smith SC, Theodorescu D. RhoGDI2 suppresses lung metastasis in mice by reducing tumor versican expression and macrophage inﬁltration. J Clin Invest. 2012;122:1503–18. 50. Hein S, Mahner S, Kanowski C, Loning T, Janicke F, Milde- Langosch K. Expression of Jun and Fos proteins in ovarian tumors of different malignant potential and in ovarian cancer cell lines. Oncol Rep. 2009;22:177–83. 60. Said N, Smith S, Sanchez-Carbayo M, Theodorescu D. Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer. J Clin Invest. 2011;121:132–47. 51. Liu D, Zhang X-X, Li M-C, Cao C-H, Wan D-Y, Xi B-X, et al. C/ EBPβ enhances platinum resistance of ovarian cancer cells by reprogramming H3K79 methylation. Nat Commun. 2018;9:1739. 61. Jakobs A, Steinmann S, Henrich SM, Schmidt TJ, Klempnauer K- H. Helenalin acetate, a natural sesquiterpene lactone with anti- inﬂammatory and anti-cancer activity, disrupts the cooperation of CCAAT box/enhancer-binding protein β (C/EBPβ) and co- activator p300. J Biol Chem. 2016;291:26098–108. p g g y 52. Hotamisligil GS. Inﬂammation, metaﬂammation and immuno- metabolic disorders. Statistical analysis Rahman SM, Janssen RC, Choudhury M, Baquero KC, Aikens RM, de la Houssaye BA, et al. CCAAT/enhancer-binding protein β (C/EBPβ) expression regulates dietary-induced inﬂammation in macrophages and adipose tissue in mice. J Biol Chem. 2012;287:34349–60. 38. Schweiger M, Schreiber R, Haemmerle G, Lass A, Fledelius C, Jacobsen P, et al. Adipose triglyceride lipase and hormone- sensitive lipase are the major enzymes in adipose tissue tria- cylglycerol catabolism. J Biol Chem. 2006;281:40236–41. 19. Ramji DP, Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation. Biochem J. 2002;365:561–75. 20. Zhang Z, Zhang Z, Kang Y, Hou C, Duan X, Sheng P, et al. Resistin stimulates expression of chemokine genes in chon- drocytes via combinatorial regulation of C/EBPβ and NF-κB. Int J Mol Sci. 2014;15:17242–55. 39. Currie E, Schulze A, Zechner R, Walther Tobias C, Farese Robert V. Cellular fatty acid metabolism and cancer. Cell Metab. 2013;18:153–61. 40. Delany AM, Kalajzic I, Bradshaw AD, Sage EH, Canalis E. Osteonectin-null mutation compromises osteoblast formation, maturation, and survival. Endocrinology. 2003;144:2588–96. 21. Oyesanya RA, Lee ZP, Wu J, Chen J, Song Y, Mukherjee A, et al. Transcriptional and post-transcriptional mechanisms for lysopho- sphatidic acid-induced cyclooxygenase-2 expression in ovarian cancer cells. FASEB J. 2008;22:2639–51. 41. Nie J, Bradshaw AD, Delany AM, Sage EH. Inactivation of SPARC enhances high-fat diet-induced obesity in mice. Connect Tissue Res. 2011;52:99–108. 22. Rozenberg JM, Bhattacharya P, Chatterjee R, Glass K, Vinson C. Combinatorial recruitment of CREB, C/EBPβ and c-Jun deter- mines activation of promoters upon keratinocyte differentiation. PLoS ONE. 2013;8:e78179. 42. Pradeep S, Kim SW, Wu SY, Nishimura M, Chaluvally-Raghavan P, Miyake T, et al. Hematogenous metastasis of ovarian cancer: rethinking mode of spread. Cancer Cell. 2014;26:77–91. 23. Said N, Theodorescu D. Secreted protein acidic and rich in cysteine (Sparc) in cancer. J Carcinog Mutagen. 2013;4:151. 43. Bowtell DD, Bohm S, Ahmed AA, Aspuria PJ, Bast RC Jr., Beral V, et al. Rethinking ovarian cancer II: reducing mortality from high- grade serous ovarian cancer. Nat Rev Cancer. 2015;15:668–79. 24. Delany AM, Hankenson KD. Thrombospondin-2 and SPARC/ osteonectin are critical regulators of bone remodeling. J Cell Commun Signal. 2009;3:227–38. 44. Roby KF, Taylor CC, Sweetwood JP, Cheng Y, Pace JL, Tawﬁk O, et al. Development of a syngeneic mouse model for events related to ovarian cancer. Carcinogenesis. 2000;21:585–91. 25. Nie J, Sage EH. SPARC functions as an inhibitor of adipogenesis. J Cell Commun Signal. 2009;3:247–54. B. John et al. Statistical analysis Nature. 2017;542:177. 53. Naczki C, John B, Patel C, Lafferty A, Ghoneum A, Aﬁfy H. et al. SPARC inhibits metabolic plasticity in ovarian cancer. Cancers. 2018;10:385. 62. Huber R, Pietsch D, Panterodt T, Brand K. Regulation of C/EBPβ and resulting functions in cells of the monocytic lineage. Cell Signal. 2012;24:1287–96. 54. Ladanyi A, Mukherjee A, Kenny HA, Johnson A, Mitra AK, Sundaresan S, et al. Adipocyte-induced CD36 expression drives ovarian cancer progression and metastasis. Oncogene. 2018;37:2285–301. 63. Chinnaiyan P, Kensicki E, Bloom G, Prabhu A, Sarcar B, Kahali S, et al. The metabolomic signature of malignant glioma reﬂects accelerated anabolic metabolism. Cancer Res. 2012;72:5878–88. 55. Tucker SL, Gharpure K, Herbrich SM, Unruh AK, Nick AM, Crane EK, et al. Molecular biomarkers of residual disease after 64. Cheville NF, Stasko J. Techniques in electron microscopy of animal tissue. Vet Pathol. 2014;51:28–41.
https://openalex.org/W3187771243	https://www.researchsquare.com/article/rs-709565/latest.pdf	English	null	Impact of Urbanization on Surface Energy Balance Components Over Metropolitan Cities of India During 2000–2018 Winter Seasons	Research Square (Research Square)	2,021	cc-by	19,271	Impact of Urbanization on Surface Energy Balance Components Over Metropolitan Cities of India During 2000–2018 Winter Seasons Sabiha Sultana Indian Institute of Technology Kharagpur Dr A.N.V. Satyanarayana (  achanta.satya@gmail.com ) Indian Institute of Technology Kharagpur https://orcid.org/0000-0002-2875-6403 DOI: https://doi.org/10.21203/rs.3.rs-709565/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Theoretical and Applied Climatology on February 12th, 2022. See the published version at https://doi.org/10.1007/s00704-022-03937-5. Impact of Urbanization on Surface Energy Balance Components over Metropolitan Cities of India during 2000–2018 winter seasons Sabiha Sultana and A.N.V. Satyanarayana* Centre for Oceans, Rivers, Atmosphere and Land Sciences Indian Institute of Technology Kharagpur, Kharagpur-721 302 India Corresponding Author: Dr A N V Satyanarayana Associate Professor Centre for Oceans, Rivers, Atmosphere and Land Sciences Indian Institute of Technology Kharagpur Kharagpur-721 302 India E-mail: anvsatya@coral.iitkgp.ac.in , achanta.satya@gmail.com Tel: +91 3222 281820 / 281821 Impact of Urbanization on Surface Energy Balance Components over Metropolitan Cities of India during 2000–2018 winter seasons India E-mail: anvsatya@coral.iitkgp.ac.in , achanta.satya@gmail.com 1 1. Introduction The explosive population rise and fleeting industrialization in the developing countries of tropical and subtropical climate has lead to brisk and abrupt expansion of the artificial surfaces and in turn warmer environment over the cities, the phenomena of urban heat island (UHI) (Oke, 1982; Voogt and Oke, 2003; Sobrino et al., 2012; Roth, 2013). The impact of expanding cities is not just limited to the city scale, but influences the regional and the global climate through radiation balance and greenhouse gas emission (Roth, 2007; Grimmond, 2007; Seto et al., 2012; Kuang et al., 2014). The higher temperature experienced due to UHI effect over the cities is mostly the result of the declining vegetation cover, replacement of natural surfaces by artificial materials, anthropogenic heating and the increasing pollution in the atmosphere (Grimmond, 2007; Grimmond, Ward and Kotthaus, 2016). The artificial materials (such as asphalt, concrete, marbles, tiles etc) of high heat capacities and conductivities store more heat and increase the surface temperature (Yang et al., 2019) whereas reduction in the surface temperature through evapotranspiration is declined due to the loss of the vegetation cover (Kato and Yamaguchi, 2005, 2007; Kuang et al., 2014). The artificial impervious surface has significant contribution in altering the surface energy fluxes and hence impacts the regional climate dynamics. It is essential to analyze the surface heat balance over urban and rural domain to understand the boundary layer dynamics over the fast developing cities (Grimmond, 1992; Hanna et al., 2011; Kotthaus and Grimmond, 2014) and quantitatively investigate the influence of heat fluxes on the temperature rise associated with UHI (Kato et al., 2008). The explosive population rise and fleeting industrialization in the developing countries of tropical and subtropical climate has lead to brisk and abrupt expansion of the artificial surfaces and in turn warmer environment over the cities, the phenomena of urban heat island (UHI) (Oke, 1982; Voogt and Oke, 2003; Sobrino et al., 2012; Roth, 2013). The impact of expanding cities is not just limited to the city scale, but influences the regional and the global climate through radiation balance and greenhouse gas emission (Roth, 2007; Grimmond, 2007; Seto et al., 2012; Kuang et al., 2014). Abstract The present study attempts to evaluate the urban energy balance components with respect to increasing urbanization over Indian metropolitan cities during 2000 – 2018 winter seasons by using Landsat 7 and 8 satellite imageries. The impact of increasing artificial surfaces on different energy fluxes is analyzed for the study period over these cities. The results indicate that the estimated ranges of the energy fluxes are in the range of typical values that are reported in the earlier literatures over cities. The Sensible Heat Flux (SHF) is observed to be increased considerably whereas the Latent Heat Flux (LHF) is noticed to slightly decreasing. The mean SHF over the buildup areas and the dry lands of Delhi noticed to record maximum increase of 28.2 Wm-2and 39.7 Wm-2 respectively during the study period. The SHF (LHF) shows positive (negative) correlation with the land surface temperature. The SHF (LHF) is observed to be about 19% – 33% (1.9% – 15%) of the net radiation flux and the residual heat flux is found to be about 60% to 80% of the net radiation flux. The study advocates the substantiate changes of the surface energy balance parameters would have profound influence on the energy exchange mechanism and which in turn affects regional climatic change. Keywords: Surface Energy Balance, Net Radiation Flux, Sensible Heat Flux, Latent Heat Flux 2 1. Introduction The higher temperature experienced due to UHI effect over the cities is mostly the result of the declining vegetation cover, replacement of natural surfaces by artificial materials, anthropogenic heating and the increasing pollution in the atmosphere (Grimmond, 2007; Grimmond, Ward and Kotthaus, 2016). The artificial materials (such as asphalt, concrete, marbles, tiles etc) of high heat capacities and conductivities store more heat and increase the surface temperature (Yang et al., 2019) whereas reduction in the surface temperature through evapotranspiration is declined due to the loss of the vegetation cover (Kato and Yamaguchi, 2005, 2007; Kuang et al., 2014). The artificial impervious surface has significant contribution in altering the surface energy fluxes and hence impacts the regional climate dynamics. It is essential to analyze the surface heat balance over urban and rural domain to understand the boundary layer dynamics over the fast developing cities (Grimmond, 1992; Hanna et al., 2011; Kotthaus and Grimmond, 2014) and quantitatively investigate the influence of heat fluxes on the temperature rise associated with UHI (Kato et al., 2008). The concept of surface energy balance was utilized in studying the surface dynamics as early as The concept of surface energy balance was utilized in studying the surface dynamics as early as in the end of the nineteenth century and many significant work has been spotted regarding urban climatology in terms of the heat balance involving observation data and numerical modelling 3 during the second half of the twentieth century (Sundborg, 1951; Munn 1966; Tag, 1968; Myrup, 1969; Probald, 1971; Oke et al., 1972) indicating that the observational results were coarse and incomplete and the model results were rather simple. The first study over any tropical or subtropical city related to energy balance was executed for Mexico City (Oke et al., 1992) and for next couple of decades very few and selective studies related urban energy balance (UEB) are noticed (Goldreich, 1992; Salmond, 1999, 2005; Roth, 2007). 1. Introduction At the beginning of the twenty- first century many scientific researchers focused on deducing the energy balance components over the urban regions using the ground observations and the numerical modeling (Pearlmutter et al., 1999; Crawford and Bluestein, 2000; Moriwaki and Kanda, 2004; Offerle et al., 2005; Pearlmutter et al., 2005, 2006; Hanna et al., 2011; Kotthaus and Grimmond, 2014; Templeton et al., 2018) The urban geometry, structure and the spread plays important role in quantifying the changes in the fluxes into and out of the surface and the storage changes as well. The high-resolution remote sensing techniques are found to be extremely useful in the recent years for monitoring the changes in the land use land cover (LULC) patterns and map the abrupt and rapid changes in the urban areas that influencing the corresponding surface heat fluxes (Kato et al., 2008). Kato and Yamaguchi (2005) separated the artificial increase in the sensible heat fluxes using the surface heat balance assumption over an urban area of Nagoya, Japan using ASTER and Landsat data. In 2007, Kato and Yamaguchi proposed to consider the storage heat as the heat flux between the surface and the canopy layer and the results reasonably agreed to earlier reported studies (Oke et al., 1999; Spronken-Smith, 2002; Grimmond et al., 2004; Moriwaki and Kanda, 2004; Christen and Vogt, 2004; Offerle et al., 2005). Roupioz et al., (2016) attempted to generate a time series of radiation fluxes of better spatial and temporal resolution using remote sensing data over the 4 4 heterogeneous topography of Tibetan Plateau. Yang, Wong and Menetti (2016) estimated the land surface temperature (LST) from remotely sensed data with and without considering the urban geometry to evaluate the turbulent heat flux. Consideration of urban geometry appears to lower the values of sensible heat. Brenner et al. (2017) used a high resolution thermal imager mounted on an unmanned aircraft to estimate LST and turbulent energy fluxes. In 2018, Chrysoulakis et al. investigated the potential of the satellite observations data (MODIS and Sentinel 3) downscaled to 100 m × 100 m to estimate UEB fluxes at a local scale along with the suitable ground observations. In 2019 Yang et al. employed a numerical microclimate model – TUF-3D (Temperatures of Urban Facets in 3-D) to obtain radiometric and complete surface temperatures for better estimation of SHF. 1. Introduction Hrisko et al., (2021) utilized multispectral satellite radiance for quantifying the storage heat through gradient-boosted regression trees method. Wetherley et al., (2021) investigated the variability of urban energy fluxes for land cover and climate gradient over Los Angeles, USA using remote sensing and energy balance model. Many remote sensing modeling methodologies are adapted in the recent studies to estimate the influence of urbanization over the surface heat fluxes (Kuang et al., 2014; Eswar et al., 2017) and address the energy-balance-closure problem (Nelli et al., 2019; Mauder et al., 2020). Both earth observation and the ground measurements can be utilized in the estimating the surface energy balance over the urban areas but, the ground observation method requires sufficient number of weather stations, vast amount of data, lots of manpower, huge cost within a small observational area for a productive study (Hanna et al., 2011; Kotthaus and Grimmond, 2014; Templeton et al., 2018) whereas the earth observation data is easily accessible and covers larger area. Recent studies indicated the potential of earth observation in accounting the changes in the fluxes is high 5 5 (Weng et al., 2014; Chen and Hu, 2017; Eswar et al., 2017; Rahman and Zhang, 2019; Liang et al., 2019). The phenomena of UHI have been studied widely using the earth observations and the remote sensing tool but the corresponding heat fluxes studies appears to be limited to only few selective studies (Chrysoulakis et al, 2018). Considering the review of literature, it is noticed that the study corresponding to surface energy fluxes are mostly emphasized on western tropical or subtropical cities. India being the seventh-largest country by area and the second-most populated country in the world, it has many highly populated metropolitan cities with rapidly spreading infrastructure. The phenomena of UHI have been studied widely using the earth observations and the remote sensing tool but the corresponding heat fluxes studies appears to be limited to only few selective studies (Chrysoulakis et al, 2018). Considering the review of literature, it is noticed that the study corresponding to surface energy fluxes are mostly emphasized on western tropical or subtropical cities. India being the seventh-largest country by area and the second-most populated country in the world, it has many highly populated metropolitan cities with rapidly spreading infrastructure. 1. Introduction The smaller cities are developing widely for recent couple of decades leading to modifications in the regional climate and hence summons a thorough investigation of energy fluxes over Indian metropolitan cities. In our earlier studies the pattern of land surface temperature (LST) and UHI intensities over the different LULC class changes are analyzed over 11 (7) Indian metropolitan cities for winter (summer) seasons during 2000 – 2018 (Sultana and Satyanarayana, 2018, 2019, 2020). Considering the fast development of the cities in India, it is essential to quantify the surface energy balance and investigate its influence on the temperature rise associated with UHI over these cities. The present study attempts to quantify the various surface energy balance components over the different Indian metropolitan cities and investigate the influence of rapid urbanization on variation in the UEB, specifically on sensible and latent heat fluxes, using Landsat-7 and 8 satellite data. the world, it has many highly populated metropolitan cities with rapidly spreading infrastructure. The smaller cities are developing widely for recent couple of decades leading to modifications in the regional climate and hence summons a thorough investigation of energy fluxes over Indian metropolitan cities. In our earlier studies the pattern of land surface temperature (LST) and UHI intensities over the different LULC class changes are analyzed over 11 (7) Indian metropolitan cities for winter (summer) seasons during 2000 – 2018 (Sultana and Satyanarayana, 2018, 2019, 2020). Considering the fast development of the cities in India, it is essential to quantify the surface energy balance and investigate its influence on the temperature rise associated with UHI over these cities. The present study attempts to quantify the various surface energy balance components over the different Indian metropolitan cities and investigate the influence of rapid urbanization on variation in the UEB, specifically on sensible and latent heat fluxes, using Landsat-7 and 8 satellite data. 2. Study area and Data 2.1. Study area India is a fast developing tropical country with around 400 cities of population one lakh or more and about 53 urban agglomerations with one million or above residents (Census of India, 2011). 6 Urban population in India is increasing in a tremendous rate of around 34% as estimated in 2017 (World Bank, 2017). The urbanization rate over the developing cities (viz.Lucknow, Chandigarh, Jaipur, Nagpur) is much higher than the developed ones with close-packed high density urban areas (viz. Mumbai, Delhi, Kolkata, Bengaluru). It is essential to monitor the urban expansion over the developing cities along with the developed cities, in order to assess the variation in the urban climate dynamics. Present study includes 11 Indian metropolitan cities of different climatic zones, such as Kolkata (22°34’21’’N, 88°21’50’’E) and Visakhapatnam (17°41’12’’N, 83°13’07’’E) in the east coast, Mumbai (19°04’34’’N, 72°52’40’’E) and Kochi(9°55’52’’N, 76°16’02’’E) in the west coast, the northern inland cities Delhi (28°42’15’’N, 77°06’09’’E), Chandigarh (30°43’59’’N, 76°46’46’’E), Jaipur (26°54’45’’N, 75°47’14’’E) and Lucknow (26°50’48’’N, 80°56’46’’E),Nagpur (21°08’45’’N, 79°05’18’’E) the city in the central India and southern peninsular cities Hyderabad (17°23’06’’N, 78°29’12’’E) and Bengaluru (12°58’18’’N, 77°35’41’’E) (Fig 1).The particulars of the cities such as locations, area, population, climatic conditions can be referred in Sultana and Satyanarayana 2018 (Table 1) and Sultana and Satyanarayana 2020. 2.2. Data Landsat 7 and Landsat 8 imageries (cloud-free conditions) of the representative day of the winter seasons (December – February) in 3 to 6 years’ intervals during 2000–2018 are utilized for the present study over each study regions. The Landsat images are obtained from the USGS Earth Explorer user interface (https://earthexplorer.usgs.gov/).The details of the data used for the present study are given in Table 1. For the purpose of the present study, the imageries are pre- processed through spatial, spectral and radiometric corrections using ERDAS IMAGINE 2014. 7 7 The LULC classification of the study areas for the study periods is retrieved using the multi- spectral bands of the Landsat (Sultana and Satyanarayana, 2018, 2019, 2020). The reflectance and the spectral radiance are also estimated for the multi-spectral bands (Band 1 – 5 and 7 of Landsat 7 and Band 2 – 7 of Landsat 8) which are used for estimating the albedo (Allen et al., 2002) and the Normalized Difference Vegetation Index (NDVI) (Carlson and Ripley, 1997; Sultana and Satyanarayana, 2018).The at-satellite brightness temperature can be calculated using the infrared (IR) thermal bands (Band 6 of Landsat 7 and Band 10 and 11 of Landsat 8). The brightness temperature along with the NDVI is employed in estimating the emissivity-corrected LST over the study regions for the study period. The estimated albedo, LST and LULC classification along with the meteorological data are utilized in evaluating the components of the surface energy balance as discussed in the section 3. The relative digital elevation model (DEM) data from the Advanced Space-borne Thermal Emission and Reflection radiometer (ASTER) is used for estimating the obstacle height (https://mirador.gsfc.nasa.gov/). The LULC classification of the study areas for the study periods is retrieved using the multi- spectral bands of the Landsat (Sultana and Satyanarayana, 2018, 2019, 2020). The reflectance and the spectral radiance are also estimated for the multi-spectral bands (Band 1 – 5 and 7 of Landsat 7 and Band 2 – 7 of Landsat 8) which are used for estimating the albedo (Allen et al., 2002) and the Normalized Difference Vegetation Index (NDVI) (Carlson and Ripley, 1997; Sultana and Satyanarayana, 2018).The at-satellite brightness temperature can be calculated using the infrared (IR) thermal bands (Band 6 of Landsat 7 and Band 10 and 11 of Landsat 8). 2.2. Data The brightness temperature along with the NDVI is employed in estimating the emissivity-corrected LST over the study regions for the study period. The estimated albedo, LST and LULC classification along with the meteorological data are utilized in evaluating the components of the surface energy balance as discussed in the section 3. The relative digital elevation model (DEM) data from the Advanced Space-borne Thermal Emission and Reflection radiometer (ASTER) is used for estimating the obstacle height (https://mirador.gsfc.nasa.gov/). The meteorological data parameters such as the near surface air temperature (T near surface air) data are collected from NCEP/DOE 2 Reanalysis data products, Physical Science Division (PSD), NOAA Earth System Research Laboratory. The available data are 4 times daily averaged with a spatial coverage of 2.5 x2.5 degree grids, where the temperature is collected at 2 m height. (https://psl.noaa.gov/data/gridded/data.ncep.reanalysis2.html/). Precipitable water (w) data has been obtained from University of Wyoming (http://weather.uwyo.edu/upperair/sounding.html/), the upper air soundings data. Other Meteorological data (Wind speed, Atmospheric temperature, Relative humidity, Dew point temperature, etc.) has been obtained from local weather stations present approximately at center of our chosen study region from Weather Underground atmospheric soundings (https://www.wunderground.com/). 8 8 3. Methodology Investigating the influence of rapid urbanization on the surface energy balance requires different methodologies to estimate the energy components. The expression for the surface balance can be presented as (Oke, 1982, 1988; Masson et al., 2002), 𝑸∗+ 𝑸𝑭= 𝑸𝑯+ 𝑸𝑬+ ∆𝑸𝑺+ ∆𝑸𝑨 (1) (1) where, Q is the net all-wave radiation flux (Wm-2), QH and QE are the turbulent sensible and latent heat flux (Wm-2) and QF is the anthropogenic heat flux (Wm-2). The ΔQS is the total change in heat storage in the surface (Wm-2) and ΔQA is the total change in advected heat flux (Wm-2), which is considered to be small in comparison to other components and could be neglected. The net radiation flux can be calculated using the Surface Energy Balance Algorithms for Land - SEBAL algorithm (Allen et al., 2002). The turbulent heat fluxes are estimated by the use of Aerodynamic Resistance Method- ARM (Nishida et al., 2003; Kato and Yamaguchi, 2005, 2007; Kato et al., 2008). The heat storage and anthropogenic heat flux over urban surfaces cannot be estimated separately by remote sensing data analysis as they depend on surface characteristics and anthropogenic activities. Hence, the QF and ΔQS are combined as the residual heat flux, QR and estimated from the energy balance equation as follows (Kato and Yamaguchi, 2005, 2007). 𝑸𝑹= ∆𝑸𝑺− 𝑸𝑭= 𝑸∗−(𝑸𝑯+ 𝑸𝑬) (2) (2) The net radiation, also called as net flux at the surface, is the actual radiant energy available to earth at its surface. It can be calculated by subtracting the total outgoing fluxes from the total incoming fluxes (Allen et al., 2002). It can be expressed as, The net radiation, also called as net flux at the surface, is the actual radiant energy available to earth at its surface. It can be calculated by subtracting the total outgoing fluxes from the total incoming fluxes (Allen et al., 2002). It can be expressed as, 𝑸∗= (𝑹𝑰𝑺𝑾+ 𝑹𝑰𝑳𝑾) −(𝑹𝑶𝑺𝑾+ 𝑹𝑶𝑳𝑾) (3) (3) 9 9 where, RISWis the incoming short wave (SW) radiation reaching the earth surface (Wm-2), RILW is the incoming long wave (LW) radiation emitted by the atmosphere (Wm-2), ROSW is the SW radiation reflected back to the atmosphere (Wm-2), ROLW is the outgoing LW radiation that emitted by earth surface into the atmosphere (Wm-2). 3. Methodology The equation (3) can be rewritten as, where, RISWis the incoming short wave (SW) radiation reaching the earth surface (Wm-2), RILW is the incoming long wave (LW) radiation emitted by the atmosphere (Wm-2), ROSW is the SW radiation reflected back to the atmosphere (Wm-2), ROLW is the outgoing LW radiation that emitted by earth surface into the atmosphere (Wm-2). The equation (3) can be rewritten as, 𝑸∗= (𝟏−𝜶) × 𝑹𝑰𝑺𝑾+ (𝑹𝑰𝑳𝑾−𝑹𝑶𝑳𝑾) −(𝟏−𝝐𝟎) × 𝑹𝑰𝑳𝑾 (4) (4) where, α is the albedo of earth surface and ϵ0 is the planetary surface emissivity. The additional final term in the Eq. (2), (1 – ε0) RILW, represents the fraction of incoming LW radiation that is reflected back (Allen et al., 2002). where, α is the albedo of earth surface and ϵ0 is the planetary surface emissivity. The additional final term in the Eq. (2), (1 – ε0) RILW, represents the fraction of incoming LW radiation that is reflected back (Allen et al., 2002). R ISW is estimated using the relation given in the following (Allen et al., 2002), R ISW is estimated using the relation given in the following (Allen et al., 2002), 𝑹𝑰𝑺𝑾= 𝑮𝒔𝒄× 𝝉× 𝑫𝒓× 𝐜𝐨𝐬𝜽 (5) (5) where, Gsc is the solar constant (1367 Wm-2), τ is the atmospheric transmissivity, Dr is the Earth- Sun distance corrected for eccentricity of planet orbit and θ is the Zenith angle. The Dr and θ can be obtained using Meta data available with the Landsat-8 imageries. The atmospheric transmissivity can be estimated by the equation as follows (Allen et al., 2007), −(𝟎.𝟎𝟎𝟏𝟒𝟔×𝑷 𝑲𝒕×𝐜𝐨𝐬𝜽)−𝟎.𝟎𝟕𝟓×( 𝒘 𝐜𝐨𝐬𝜽) 𝟎.𝟒 ] (6) 𝝉= 𝟎. 𝟑𝟓+ 𝟎. 𝟔𝟐𝟕× 𝒆 [−(𝟎.𝟎𝟎𝟏𝟒𝟔×𝑷 𝑲𝒕×𝐜𝐨𝐬𝜽)−𝟎.𝟎𝟕𝟓×( 𝒘 𝐜𝐨𝐬𝜽) 𝟎.𝟒 ] (6) (6) (6) where, P is the local atmospheric pressure (K Pa), w is the precipitable water (mm) and Kt is the unit less air turbidity coefficient (0 <Kt≤ 1; for clear air, 0.5 for polluted air). where, P is the local atmospheric pressure (K Pa), w is the precipitable water (mm) and Kt is the unit less air turbidity coefficient (0 <Kt≤ 1; for clear air, 0.5 for polluted air). Surface albedo can be defined as the ratio of reflected solar radiance to the amount of irradiance received. The albedo (α) is estimated using the following expression used by Allen et al. 3. Methodology (2002), 𝜶= (𝜶𝑻𝑶𝑨−𝜶𝒂𝒕𝒎) 𝝉𝟐 (7) (7) (7) 10 where, α is the Planetary albedo corrected for atmosphere effects, αTOA is the Planetary albedo without correction for corresponding atmosphere effects and α atm is the Atmospheric albedo (a typical constant value of 0.03 is taken based on Bastiaanssen, 2000). The αTOAcan be estimated using the method given by (Allen et al., 2002; Silva et al., 2015), 𝜶𝑻𝑶𝑨= ∑(𝝎𝝀× 𝝆𝝀) (8) (8) (8) where, ρλ is the reflectivity of the multispectral bands and ωλ is the weighting coefficient for eachband, are computed using methods mentioned in the Landsat User Handbooks (Landsat 7 Data Users Handbook, 2019; Landsat 8 Data user Handbook, 2019). where, ρλ is the reflectivity of the multispectral bands and ωλ is the weighting coefficient for eachband, are computed using methods mentioned in the Landsat User Handbooks (Landsat 7 Data Users Handbook, 2019; Landsat 8 Data user Handbook, 2019). Both the incoming and the outgoing LW radiation are estimated using the Stefan-Boltzmann equation given as follows (Allen et al., 2002; An, Hemmati and Cui, 2017), 𝑹𝑰𝑳𝑾= 𝝐𝒂𝒊𝒓× 𝝈× 𝑻𝒏𝒆𝒂𝒓 𝒔𝒖𝒓𝒇𝒂𝒄𝒆 𝒂𝒊𝒓 (9) 𝑹𝑶𝑳𝑾= 𝝐𝟎× 𝝈× 𝑳𝑺𝑻 (10) 𝑹𝑰𝑳𝑾= 𝝐𝒂𝒊𝒓× 𝝈× 𝑻𝒏𝒆𝒂𝒓 𝒔𝒖𝒓𝒇𝒂𝒄𝒆 𝒂𝒊𝒓 (9) (9) 𝑹𝑶𝑳𝑾= 𝝐𝟎× 𝝈× 𝑳𝑺𝑻 (10) 𝑹𝑶𝑳𝑾= 𝝐𝟎× 𝝈× 𝑳𝑺𝑻 (10) (10) where, σ is Stefan-Boltzmann constant (5.67 × 10-8 in W m-2K4), ϵairis the atmospheric emissivity computed using the relation which is a function of atmospheric transmissivity (Bastiaanssen, 1995; Allen et al., 2002), 𝝐𝒂𝒊𝒓= 𝟎. 𝟖𝟓× (−𝐥𝐧𝝉)𝟎.𝟗 (11) 𝝐𝒂𝒊𝒓= 𝟎. 𝟖𝟓× (−𝐥𝐧𝝉)𝟎.𝟗 (11) (11) The planetary surface emissivity, ϵ0 is calculated using the NDVI (Carlson and Ripley, 1997) and LST is obtained using the thermal bands of Landsat data. The methodology to calculate LST and ϵ0from Landsat imageries is discussed in detail by Sultana and Satyanarayana (2018, 2019 and 2020). Sensible heat flux is the conductive transfer of heat flux from the Earth's surface to the atmosphere. 3. Methodology 12 Latent heat flux is the flux of energy associated with evaporation or transpiration of water from the Earth's surface to the atmosphere and vice versa. Latent heat flux can be estimated from the following equation (Kato and Yamaguchi, 2005, 2007; Kato et al., 2008), Latent heat flux is the flux of energy associated with evaporation or transpiration of water from the Earth's surface to the atmosphere and vice versa. Latent heat flux can be estimated from the following equation (Kato and Yamaguchi, 2005, 2007; Kato et al., 2008), 𝑸𝑬= 𝝆× 𝑪𝑷× (𝒆𝒔−𝒆𝒂) 𝜸× (𝑹𝒂+ 𝑹𝑺) (15) (15) where,  is the psychrometric constant (0.67 hPa K-1), es is the saturated water vapor pressure (hpa) at surface temperature, ea is the Atmospheric water vapor pressure (hpa) and RSis the stomatal resistance.Stomatal resistance can be computedusing the proposed method as follows (Nishida et al., 2003, Kato et al., 2008), where,  is the psychrometric constant (0.67 hPa K-1), es is the saturated water vapor pressure (hpa) at surface temperature, ea is the Atmospheric water vapor pressure (hpa) and RSis the stomatal resistance.Stomatal resistance can be computedusing the proposed method as follows (Nishida et al., 2003, Kato et al., 2008), 𝟏 𝑹𝑺 = 𝒇𝟏(𝑷𝑨𝑹) × 𝒇𝟐(𝑷𝑨𝑹) 𝑹𝑺𝒎𝒊𝒏 + 𝟏 𝑹𝒄𝒖𝒕𝒊𝒄𝒍𝒆 (16) 𝟏 𝑹𝑺 = 𝒇𝟏(𝑷𝑨𝑹) × 𝒇𝟐(𝑷𝑨𝑹) 𝑹𝑺𝒎𝒊𝒏 + 𝟏 𝑹𝒄𝒖𝒕𝒊𝒄𝒍𝒆 (16) (16) where, Rsmin is the minimum stomatal resistance and Rcuticleis the canopy resistance related to the diffusion through the cuticle layer of leaves (105s m-1). The typical values of Rsmin over different LULC classes are considered as proposed by Kato et al., (2008); Kato and Yamaguchi, (2005, 2007), are summarized in Table 3. The f1(PAR) and f2(PAR) are given by (Nishida et al., 2003), 𝒇𝟏(𝑷𝑨𝑹) = (𝑻𝒂−𝑻𝒏 𝑻𝟎−𝑻𝒏 ) × (𝑻𝒙−𝑻𝒂 𝑻𝒙−𝑻𝟎 ) (𝑻𝒙−𝑻𝟎 𝑻𝟎−𝑻𝒏) (17) (17) 𝒇𝟐(𝑷𝑨𝑹) = 𝑷𝑨𝑹 (𝑷𝑨𝑹+ 𝑨) (18) (18) where, Tn, Tx and T0 and are the minimum, maximum and the optimal temperature for stomatal activity, and the typical values for them as considered are 2.7 °C, 45.3 °C and 31.1 °C respectively. Ais the parameterconcerning photon absorption efficiency at low light intensity (152 µ mol m-2 s-1), PAR is the Photosynthetic active radiation (W m-2), can be calculated as f * R ISW (where, f is 2.05 µmol m-2 s-1). 3. Methodology Sensible heat flux can be estimated using the equation, (Kato and Yamaguchi, 2005, 2007; Kato et al., 2008), 11 11 𝑸𝑯= 𝝆× 𝑪𝑷× (𝑳𝑺𝑻−𝑻𝒂𝒕𝒎) 𝑹𝒂 𝑸𝑯= 𝝆× 𝑪𝑷× (𝑳𝑺𝑻−𝑻𝒂𝒕𝒎) 𝑹𝒂 (12) 𝑹𝒂 where, ρ is the air density (1.225 kg m-3), CP is the Specific heat of air at constant pressure (1005 KJ Kg-1K-1), Tatmis the Atmospheric air temperature (K) andRa is the Aerodynamic resistance (sm-1)for heat and momentum exchange and can be estimated by using the equation as given by Brutsaert (1982), where, ρ is the air density (1.225 kg m-3), CP is the Specific heat of air at constant pressure (1005 KJ Kg-1K-1), Tatmis the Atmospheric air temperature (K) andRa is the Aerodynamic resistance (sm-1)for heat and momentum exchange and can be estimated by using the equation as given by Brutsaert (1982), 𝑹𝒂= [ 𝐥𝐧(𝒁𝒓𝒆𝒇−𝑫𝟎) 𝒁𝒐𝒎 −𝜳𝒎] × [ 𝐥𝐧(𝒁𝒓𝒆𝒇−𝑫𝟎) 𝒁𝒐𝒉 −𝜳𝒉] 𝑲𝟐𝑼 (13) 𝑹𝒂= [ 𝐥𝐧(𝒁𝒓𝒆𝒇−𝑫𝟎) 𝒁𝒐𝒎 −𝜳𝒎] × [ 𝐥𝐧(𝒁𝒓𝒆𝒇−𝑫𝟎) 𝒁𝒐𝒉 −𝜳𝒉] 𝑲𝟐𝑼 (13) (13) where, Zref is the reference height (m) at which wind speed and temperature are measured, U is the wind speed (m s-1) at the reference height, D0 is the displacement height (m), Zoh and Zom is the roughness length(m) for heat and momentum exchange, ψmand ψhare the stability correction functions for heat and momentum exchange depending on Monin–Obukhov length and K is the von Karman constant (0.4). For the present study, typical values of Zoh and Zomwere considered for different LULC classes (Table 2) as proposed by Kato and Yamaguchi, (2005, 2007); Kato et al., (2008) as it is quite difficult to acquire specific required data to calculate the Zoh and Zomvalues by the methods proposed earlier (Macdonald et al., 1998; Grimmondand Oke, 1999; Duijm, 1999). The displacement height, D0 is estimated by the expression given by Macdonald et al. (1988) and Kato and Yamaguchi (2005), 𝑫𝟎= 𝒛𝒉[𝟏+ 𝑨−𝝀𝑷(𝝀𝑷−𝟏)] (14) (14) where, zh is the obstacleheight (m), A is a constant (=4.43 for staggered arrays), and λP is the plan areadensity of obstacles, i.e., the ratio of the build-up area tothe total area. The obstacle height is being estimated from ASTER DEM data. Stability correction function calculated as a function of Monin–Obukhov length by the proposed methodology (Paulson, 1970; Webb, 1970; Allen et al., 2002; Prueger and Kustas, 2005). 3. Methodology where, Tn, Tx and T0 and are the minimum, maximum and the optimal temperature for stomatal activity, and the typical values for them as considered are 2.7 °C, 45.3 °C and 31.1 °C respectively. Ais the parameterconcerning photon absorption efficiency at low light intensity (152 µ mol m-2 s-1), PAR is the Photosynthetic active radiation (W m-2), can be calculated as f * R ISW (where, f is 2.05 µmol m-2 s-1). 13 4. Results and Discussion In this section the energy components as estimated over the study region are analysed with respect to the changes in the LULC classes, specifically over BA and DL possessing the higher LST. Additionally, the mean of the energy balance components during the study period are analysed over the DL, BA and the vegetated areas. The proportional sharing of the net radiation flux in heat fluxes are estimated for each case and are compared with the earlier studies. In our earlier studies (Sultana and Satyanarayana, 2018, 2020) the cities under consideration are classified in to 5 LULC classes; water bodies (WB), built up areas (BA), dry lands (DL), crop/grass land (CGL) and dense vegetation (DV). The methodology for the classification, variation in the LULC classes and the accuracy assessment of these classes are also discussed in detail. The LST pattern and the UHI intensities in relation to the variation in LULC classes over the cities considered for the present study are also analysed. 4.1. Estimated surface energy components During the study it is noticed that the net radiation flux and the latent heat flux (LHF) over the BA and the DL are comparatively lower than the vegetated areas and the water bodies have the highest values. On the other hand, the sensible heat flux (SHF) is observed to be higher over the BA and DL in comparison to the vegetation and the water bodies possess lowest SHF values (small positive or negative). As the present study focuses on the high LST possessing BA and DL, in this section the analysis is conducted over these LULC classes only and hence the Figures 2 – 12 depict the energy balance components only over the DL and the BA and the portions over the vegetated areas and the water bodies are removed. 14 Kolkata Net Radiation Flux: The spatial distributions of the net radiation flux during the study period for BA and the DL are shown in Figure 2a. The figure indicates that the lower values of net radiation flux are observed over the DL and fallow lands in comparison to the BA. The ranges over the DL are 411.2 – 460 Wm-2, 407.4 – 465 Wm-2, 428.4 – 475 Wm-2 and 438.4 – 495 Wm-2for the years 2002, 2007, 2012 and 2017 respectively whereas the ranges for BA are 445.1 – 495 Wm-2, 450.1 – 530 Wm-2, 460.1 – 510 Wm-2 and 480.1 – 530 Wm-2 for the same years. It can be noticed in the Fig. 2a that more patches of higher values of net radiation flux appear over the outer regions of the BA due to appearance of DL in the later part of study period. Sensible heat flux: The spatial distribution for sensible heat flux over the BA and the DL are depicted in Fig. 2b for the study period. The SHF over the central BA is observed to be highest followed by the DL and the fallow lands in the outer regions. The ranges of SHF over DL are 75.1 – 105 Wm-2, 75.1 – 110 Wm-2, 85.1 – 120 Wm-2 and 85.1 – 120 Wm-2 during 2002, 2007, 2012 and 2017 respectively. The values of SHF over BA ranged 95.1 – 125.2 Wm-2, 100.1 – 138.4 Wm-2,110.1 – 166.3 Wm-2 and 110.1 – 154.2 Wm-2 respectively during the study period. Latent heat flux: The Fig. 2c shows the spatial distribution of the LHF over the BA and the DL for the study period. The central BA shows the lowest range of values (blue shades) and the DL noticed have comparatively higher values (brown shades). The value of LHF over BA is observed to be30Wm-2or less during the study period due to artificial surfaces lack moisture content. The ranges over the DL during the study period are around 20.1 – 45 Wm-2, 20.1 – 45 Wm-2, 25.1 – 45 Wm-2and 20.1 – 40 Wm-2. Residual heat flux: The Fig. 2d shows the spatial distribution of the residual heat flux over BA and the DL and the ranges over these regions are noticed to be lower in comparison to the other 15 LULC classes (vegetation and water bodies). Kolkata The values are noticed to be higher in the outer region of the BA during the later part of the study period due to presence of the DL. Visakhapatnam Net Radiation Flux: The Fig. 3a depicts spatial distributions of the net radiation flux during the study period for BA and the DL over Visakhapatnam. The lower values are mostly noticed over the DL, the ranges during the study period being 374.3 – 440 Wm-2, 382.3 – 450 Wm-2, 386.7 – 440 Wm-2and 391.6 – 450 Wm-2. The ranges over the BA are little higher in comparison to the DL, which are 425.1 – 470 Wm-2, 435.1 – 485 Wm-2, 425.1 – 475 Wm-2 and 440.1 – 490 Wm-2for year 2001, 2008, 2012 and 2017 respectively. Sensible heat flux: The spatial distribution for sensible heat flux over the BA and the DL are depicted in Fig. 3b for the study period. The highest values of SHF are noticed over the DL whereas the values over BA are comparatively lower. The ranges of SHF over DL are 90.1 – 135.4 Wm-2, 100.1 – 146.1 Wm-2, 105.1 – 164.5 Wm-2 and 110.1 – 156.2 Wm-2 during 2001, 2008, 2012 and 2017 respectively and the ranges of SHF over the BA are 40.1 – 100 Wm-2, 80.1 – 110 Wm-2, 85.1 – 115 Wm-2 and 90.1 – 115 Wm-2 respectively during the same years. Latent heat flux: The Figure 3c shows the spatial distribution of the LHF over the BA and the DL over Visakhapatnam. The LHF over the DL are observed to be low in comparison to the BA. The ranges of LHF over DL are 5.1 – 30 Wm-2, 3.4 – 25 Wm-2, 2.9 – 25 Wm-2 and 2.1 – 20 Wm-2 during 2001, 2008, 2012 and 2017 respectively. The ranges over the BA during the study period are 25.1 –55 Wm-2, 20.1 –50 Wm-2, 20.1 – 45 Wm-2and 15.1 – 45 Wm-2. Residual heat flux: The Figure 3d depicts the spatial distribution of the residual heat flux over BA and the DL. The ranges of residual heat over the DL are observed to be lower than that of BA and the ranges over water and vegetation are higher than both the BA and DL ranges. 16 Mumbai Net radiation flux: Figure 4a shows the spatial distribution of net radiation flux over BA and DL of Mumbai. The dry barren lands, sandy region and pavements are observed to possess lower range of values (blue shades). The ranges for DL are 437.3 – 470 Wm-2, 439.1 – 480 Wm-2, 446.3 – 485 Wm-2 and 472.5 – 520 Wm-2 for year 2002, 2008, 2012 and 2018 respectively. The ranges over BA are 460.1 – 500 Wm-2, 470.1 – 505 Wm-2, 475.1 – 510 Wm-2 and 510.1 – 545 Wm- 2(yellow and brown shades) during the study period and are little higher than the DL. Net radiation flux: Figure 4a shows the spatial distribution of net radiation flux over BA and DL of Mumbai. The dry barren lands, sandy region and pavements are observed to possess lower range of values (blue shades). The ranges for DL are 437.3 – 470 Wm-2, 439.1 – 480 Wm-2, 446.3 – 485 Wm-2 and 472.5 – 520 Wm-2 for year 2002, 2008, 2012 and 2018 respectively. The ranges over BA are 460.1 – 500 Wm-2, 470.1 – 505 Wm-2, 475.1 – 510 Wm-2 and 510.1 – 545 Wm- 2(yellow and brown shades) during the study period and are little higher than the DL. Sensible heat flux: The spatial distribution for the SHF over Mumbai for BA and DL are depicted in Fig. 4b. In the figure the DL, sandy areas, pavements and the dense BA are noticed to possess highest SHF values (yellow and brown shades), the ranges being 120.1 – 158.3 Wm-2, 120.1 – 163.7 Wm-2, 125.1 – 168.4 Wm-2 and 140.1 – 180.7 Wm-2 during the study period. The less dense BA, open lands with slight vegetation and regions close to water body (western boundary) possess lower ranges 80.1 – 130 Wm-2, 85.1 – 130 Wm-2, 90.1 – 135 Wm-2 and 100.1 – 150 Wm-2 during 2002, 2008, 2012 and 2018 respectively (blue shades). Sensible heat flux: The spatial distribution for the SHF over Mumbai for BA and DL are depicted in Fig. 4b. In the figure the DL, sandy areas, pavements and the dense BA are noticed to possess highest SHF values (yellow and brown shades), the ranges being 120.1 – 158.3 Wm-2, 120.1 – 163.7 Wm-2, 125.1 – 168.4 Wm-2 and 140.1 – 180.7 Wm-2 during the study period. Mumbai The less dense BA, open lands with slight vegetation and regions close to water body (western boundary) possess lower ranges 80.1 – 130 Wm-2, 85.1 – 130 Wm-2, 90.1 – 135 Wm-2 and 100.1 – 150 Wm-2 during 2002, 2008, 2012 and 2018 respectively (blue shades). Latent heat flux: The spatial distribution of LHF over BA and DL during the study period is depicted in Fig. 4c. The LHF over the city appears to be mostly uniform except for some exceptions. The dry lands and the pavements are noticed to have lowest range of values less than 60Wm-2 during the study period whereas BA has little higher values of ranges 55.1 – 95 Wm-2, 50.1 – 85 Wm-2, 40.1 – 80 Wm-2and 35.1 – 80 Wm-2 during 2002, 2008, 2012 and 2018 respectively. Residual heat flux: Figure 4d shows the spatial distribution of the residual heat flux over BA and the DL and the ranges over these regions are noticed to be lower, where the DL has lowest range of values followed by moderate values over the BA. 17 17 Delhi Net radiation flux: The spatial distribution of net radiation flux for Delhi over BA and DL is shown in Fig.6a. The DL have the lowest range of values (284.9 – 325 Wm-2, 297.4 – 335 Wm-2, 317.3 – 370 Wm-2 and 334.7 – 375 Wm-2) during the study period. The BA has the moderate ranges of values (yellow shades) with zones of little higher values (brown shades) close to the water bodies or the vegetated areas. The ranges of net radiation flux over BA during the study period are 310.1 – 350 Wm-2, 320.1 – 365 Wm-2, 355.1 – 395 Wm-2 and 365.1 – 405 Wm-2. shown in Fig.6a. The DL have the lowest range of values (284.9 – 325 Wm-2, 297.4 – 335 Wm-2, 317.3 – 370 Wm-2 and 334.7 – 375 Wm-2) during the study period. The BA has the moderate ranges of values (yellow shades) with zones of little higher values (brown shades) close to the water bodies or the vegetated areas. The ranges of net radiation flux over BA during the study period are 310.1 – 350 Wm-2, 320.1 – 365 Wm-2, 355.1 – 395 Wm-2 and 365.1 – 405 Wm-2. Sensible heat flux: The spatial variation in the SHF over BA and DL is depicted in the Fig. 6b. The highest range of SHF values is noticed over the DL and the dense BA with lower values (blue shades) over the build up regions close to the water body and/or vegetation. During the years 2002, 2007, 2013 and 2017 the ranges over the DL and dense BA are 95.1 – 138.3 Wm-2, 110.1 – 146.3 Wm-2, 125.1 -161.4 Wm-2and 130.1 – 179.3 Wm-2 respectively. The sparse BA noticed to have the ranges 65.1 – 105 Wm-2, 80.1 – 120 Wm-2, 85.1 – 135 Wm-2and 90.1 – 140 Wm-2respectively during the study period. Sensible heat flux: The spatial variation in the SHF over BA and DL is depicted in the Fig. 6b. The highest range of SHF values is noticed over the DL and the dense BA with lower values (blue shades) over the build up regions close to the water body and/or vegetation. During the years 2002, 2007, 2013 and 2017 the ranges over the DL and dense BA are 95.1 – 138.3 Wm-2, 110.1 – 146.3 Wm-2, 125.1 -161.4 Wm-2and 130.1 – 179.3 Wm-2 respectively. Kochi Net radiation flux: The spatial distribution of net radiation flux over BA and DL for Kochi is shown in Fig. 5a. The lowest range of net radiation is observed over the BA but the DL and fallow lands are noticed to have comparatively higher values. The ranges over the BA during 2002, 2007, 2013 and 2018 are 368.8 – 415 Wm-2, 358.4 – 415 Wm-2, 381.9 – 435 Wm-2 and 382.4 – 440 Wm-2respectively. The DL observed to have the net radiation flux ranges 405.1 – 445 Wm-2, 405.1 – 450 Wm-2, 425.1 – 465 Wm-2and 430.1 – 470 Wm-2for the years 2002, 2007, 2013 and 2018 respectively. The regions close to the water bodies are noticed to possess higher values. Sensible heat flux: The spatial variation in the SHF over BA and DL is shown in the Fig. 5b. The highest values of the SHF are observed over the dense BA followed by the DL. The higher range of values is noticed to be more prominent during the later part of the study period indicate the increase in impermeability. The ranges of SHF over BA are 90.1 – 132.9 Wm-2, 100.1 – 139.9 Wm-2, 105.1 – 145.4 Wm-2and 110.1 – 154.6 Wm-2for the years 2002, 2007, 2013 and 2018 respectively and the ranges over DL are 70.1 – 100 Wm-2, 75.1 – 110 Wm-2, 80.1 – 115 Wm-2 and 80.1 – 120 Wm-2 respectively for the same years. Latent heat flux: Figure 5c depicts spatial distribution of LHF over BA and DL during the study period for Kochi. The BA observed to possess lowest values of LHF ranging 3.4 – 30 Wm-2, 1.6 – 30Wm-2, 2.7 – 25 Wm-2and 5.7 – 20 Wm-2 for the year 2002, 2007, 2013 and 2018 respectively. The LHF over the DL and the open lands close to the water bodies have comparatively higher values ranging 25.1 – 60Wm-2, 25.1 – 55 Wm-2, 20.1 –50 Wm-2 and 15.1 – 45 Wm-2 respectively for the study period. 18 Residual heat flux: Figure 5d shows the spatial distribution of the residual heat flux over BA and the DL for Kochi during the study period. The BA noticed to possess the lowest values of residual flux followed by the DL with moderate values. Delhi The sparse BA noticed to have the ranges 65.1 – 105 Wm-2, 80.1 – 120 Wm-2, 85.1 – 135 Wm-2and 90.1 – 140 Wm-2respectively during the study period. Latent heat flux: The spatial distribution of LHF over the BA and DL of Delhi is shown in Fig. 6c. The LHF values over the BA are ranged as 25.1 – 50 Wm-2, 20.1 – 40 Wm-2, 25.1 – 40 Wm- 2and 20.1 – 40 Wm-2 during the study period. The zones of higher ranges are noticed increasing during the study period. The DL and the fallow lands over the outer edge of the BA are noticed to possess the lowest values (≤ 35 Wm-2) during the study period. 19 Residual heat flux: The DL and BA are noticed to store lesser residual heat in comparison to vegetated areas and water bodies during the study period. The residual heat flux over the DL is noticed to be lower than the BA in case of Delhi as shown in Fig. 6d. Chandigarh noticed to possess comparatively higher values in the ranges of 25.1 – 55 Wm-2, 20.1 –50 Wm-2, 20.1 – 45 Wm-2, 15.1 – 45 Wm-2 and 15.1 – 40 Wm-2respectively during the study years. noticed to possess comparatively higher values in the ranges of 25.1 – 55 Wm-2, 20.1 –50 Wm-2, 20.1 – 45 Wm-2, 15.1 – 45 Wm-2 and 15.1 – 40 Wm-2respectively during the study years. Residual heat flux: The spatial distribution of the residual heat flux over the BA and the DL are depicted in the Fig.7d. The values of residual heat flux over the BA and the DL are noticed to be lower and the DL and the industrial areas have the lowest range of values. Chandigarh Net Radiation flux: The spatial distribution of net radiation flux for Chandigarh over BA and DL is depicted in Figure 7a. The figure indicates that the lowest range of values of net radiation flux is mostly over the DL, pavements and fallow lands (blue shades). The ranges over these areas are 229.5 – 275 Wm-2, 236.4 – 280 Wm-2, 247.6 – 285 Wm-2, 261.1 – 295 Wm-2and 276.1 – 305 Wm-2 during 2000, 2005, 2010, 2013 and 2018 respectively. The BA noticed to possess relatively higher ranges 265.1 – 300 Wm-2, 270.1 – 310 Wm-2, 285.1 – 325 Wm-2, 285.1 – 325 Wm-2and 295.1 – 335 Wm-2 during the study period. Sensible heat flux: Figure 7b shows the spatial distribution of the SHF over the BA and the DL of Chandigarh city. The highest range of values is observed over the dense BA, pavements and DL. The ranges of SHF over these regions are 110.1 – 148.9 Wm-2, 115.1 – 155.5 Wm-2, 120.1 – 159.2 Wm-2, 125.1 – 164.3 Wm-2 and 125.1 – 165.5 Wm-2 for the years 2000, 2005, 2010, 2013 and 2018 respectively. The build-up regions possess moderate values with patches of lower values (blue shades) and the ranges are80.1 – 120 Wm-2, 85.1 – 125 Wm-2, 90.1 – 130 Wm-2, 90.1 – 135 Wm-2 and 95.1 – 135 Wm-2 during the years 2000, 2005, 2010, 2013 and 2018 respectively. Latent heat flux: The spatial distribution of LHF over the BA and DL is shown in Fig. 7c. The figure indicates that the DL, industrial areas and the pavements possess very low range of LHF values. For the years 2000, 2005, 2010, 2013 and 2018 the ranges over these areas are 4.3 – 30 Wm-2, 3.1 – 25 Wm-2, 1.4 – 25 Wm-2, 2.4 – 20 Wm-2 and 2.2 – 20 Wm-2 respectively. The BA 20 noticed to possess comparatively higher values in the ranges of 25.1 – 55 Wm-2, 20.1 –50 Wm-2, 20.1 – 45 Wm-2, 15.1 – 45 Wm-2 and 15.1 – 40 Wm-2respectively during the study years. Residual heat flux: The spatial distribution of the residual heat flux over the BA and the DL are depicted in the Fig.7d. The values of residual heat flux over the BA and the DL are noticed to be lower and the DL and the industrial areas have the lowest range of values. Jaipur Net radiation flux: Figure 8a shows the spatial distribution of net radiation flux over BA and DL of Jaipur. The dry barren lands, sandy-rocky region and fallow lands in the outer region are noticed to possess lower range of values. The ranges of net radiation over these regions are 323.4 – 385 Wm-2, 332.2 – 390 Wm-2, 353.8 – 400 Wm-2 and 363.7 – 410 Wm-2 for year 2002, 2007, 2012 and 2017 respectively. The ranges over the central BA are 370.1 – 405 Wm-2, 380.1 – 415 Wm-2, 385.1 – 420 Wm-2 and 395.1 – 445 Wm-2 during the study period, are comparatively higher than the ranges over the DL. Sensible heat flux: The spatial distribution for the SHF over BA and DL are depicted in Fig.8b. The DL, sandy-rocky areas and fallow lands are noticed to possess highest SHF values, the ranges being 110.1 – 176.6 Wm-2, 120.1 – 183.1 Wm-2, 120.1 – 186.3 Wm-2 and 125.1 – 197.2 Wm-2 during the study period. The ranges of SHF over the BA are 85.1 – 120 Wm-2, 90.1 – 130 Wm-2, 95.1 – 130 Wm-2 and 100.1 – 135 Wm-2, are little lower than that of the DL. Latent heat flux: The spatial distribution of LHF over BA and DL during the study period is depicted in Fig. 8c. The DL and the fallow lands have lowest range of values 8.1 – 30Wm-2, 4.3 – 25 Wm-2, 3.4 – 20 Wm-2 and 5.4 – 20 Wm-2 during the study period. The BA in the central part of the city possess little higher values of ranges 25.1 – 55 Wm-2, 20.1 – 55 Wm-2, 15.1 – 50 Wm-2 and 15.1 – 45 Wm-2 during 2002, 2007, 2012 and 2017 respectively. 21 21 Residual heat flux: Figure 8d shows the spatial distribution of the residual heat flux over BA and the DL for Jaipur and the ranges over these regions are noticed to be lower with respect to vegetation and water bodies. The DL and the fallow lands possess lowest range of values followed by moderate to higher values over the BA. Lucknow Net Radiation flux: The spatial distribution of net radiation flux for Lucknow over BA and DL is depicted in Fig. 9a. The figure indicates that the lowest range of values of net radiation flux is mostly over the DL, pavements and fallow lands (blue shades). The net radiation flux ranges during the study period are 327.7 – 370 Wm-2, 354.6 – 405 Wm-2, 362.4 – 425 Wm-2 and 389.8 – 430 Wm-2. The central BA noticed to possess relatively higher values of range 370.1 - 415Wm-2, 405.1 – 440 Wm-2, 425.1 – 465 Wm-2 and 430.1 – 470 Wm-2 during 2002, 2007, 2012 and 2017 respectively. Sensible heat flux: Figure 9b shows the spatial distribution of the SHF over the BA and the DL of Lucknow. The highest range of values is observed over the dense BA, fallow lands and DL in the outer part of the city. The ranges of SHF over these areas are 115.1 – 167.2 Wm-2, 120.1 – 178.1 Wm-2, 125.1 – 169.2 Wm-2and 130.1 – 184.9 Wm-2for the years 2002, 2007, 2012 and 2017 respectively. The sparse build up regions are observed to have comparatively lower values of the range 85.1 – 125 Wm-2, 90.1 – 130 Wm-2, 95.1 – 135 Wm-2 and 95.1 – 140 Wm-2for the years 2002, 2007, 2012 and 2017 respectively. Latent heat flux: The spatial distribution of LHF over the BA and DL is shown in Fig. 9c. The figure shows that the DL and the fallow lands possess very low range of LHF values (blue shades). For the years 2002, 2007, 2012, and 2017 the ranges over these areas are 1.2 – 30 Wm-2, 1.7 – 25 Wm-2, 2.3 – 20 Wm-2and 4.9 – 15 Wm-2 respectively. The BA noticed to possess 22 moderate values with comparatively higher values over sparse BA close to vegetation and water bodies. The ranges of LHF over BA are observed to be 30.1 – 60 Wm-2, 25.1 – 60 Wm-2, 20.1 – 55 Wm-2and 15.1 – 55 Wm-2during the years 2002, 2007, 2012 and 2017 respectively. Residual heat flux: The spatial distribution of the residual heat flux over the BA and the DL are depicted in the Fig. 9d. The values of residual heat flux over the BA noticed to be moderate and the DL and the fallow lands have the lowest range of values. Lucknow The higher values are noticed over the vegetated areas and the water bodies have the highest values. Nagpur Net Radiation flux: The spatial distribution of net radiation flux over the BA and the DL of Nagpur are shown in Fig. 10a. The DL in the outer region of the city are noticed to have lowest ranges of values, 325.1 – 380 Wm-2, 334.6 – 385 Wm-2, 330.2 – 390 Wm-2 and 351.7 – 405 Wm-2 during the 2002, 2007, 2013 and 2018 respectively. The central BA have comparatively higher values of 370.1 – 405 Wm-2, 375.1 – 410 Wm-2, 380.1 – 415 Wm-2 and 395.1 – 435 Wm-2 for the duration of the study period. The higher value zones are noticed to be increasing indicating spread of impervious surfaces. Sensible heat flux: Figure 10b shows the spatial distribution of the SHF over the BA and the DL of Nagpur city. The DL possess the highest range of values, 95.1 – 148.2 Wm-2, 100.1 – 147.3 Wm-2, 105.1 – 155.9 Wm-2 and 110.1 – 166.2 Wm-2 for the years 2002, 2007, 2013 and 2018 respectively. The values of SHF over BA are of comparatively lower range, 65.1 – 105 Wm-2, 65.1 – 110 Wm-2, 75.1 – 115 Wm-2 and 80.1 – 120 Wm-2 for the study period. The low values (blue shades) over the BA are observed to be replaced by high range zones (yellow and brown) during the study period indicate increase in imperviousness. 23 Latent heat flux: The spatial distribution for the LHF over the BA and the DL of Nagpur are shown in the Fig. 10c. The DL and the fallow lands are observed to have lowest range of LHF values less than 25 Wm-2 during the study period whereas for BA the ranges are 20.1 – 45 Wm-2, 20.1 – 40 Wm-2, 20.1 – 35 Wm-2 and 15.1 – 35 Wm-2 for the years 2002, 2007, 2013 and 2018 respectively. The high-value zones over the BA are noticed to be disappearing during the study period indicates gradual loss of moisture content in the surface. Residual heat flux: The spatial distribution of the residual heat flux over BA and the DL of Nagpur are depicted in the Fig. 10d. The values of residual heat flux over the BA and the DL are lower in comparison to that of the vegetation and the water bodies with DL having the lowest range of values. Hyderabad Net radiation flux: The spatial distribution of net radiation flux over BA and DL for Hyderabad is shown in Fig. 11a. The lowest range of net radiation is observed over the DL and fallow lands and the BA noticed to have comparatively higher values. The ranges over the DL during 2002, 2007, 2012 and 2017 are 371.3 – 425 Wm-2, 365.8 – 420 Wm-2, 367.2 – 420 Wm-2 and 384.3 – 430 Wm-2respectively. The ranges over BA observed to be 410.1 – 450 Wm-2, 405.1 – 445 Wm-2, 410.1 – 455 Wm-2 and 420.1 – 455 Wm-2 for the years 2002, 2007, 2012 and 2017 respectively. Net radiation flux: The spatial distribution of net radiation flux over BA and DL for Hyderabad is shown in Fig. 11a. The lowest range of net radiation is observed over the DL and fallow lands and the BA noticed to have comparatively higher values. The ranges over the DL during 2002, 2007, 2012 and 2017 are 371.3 – 425 Wm-2, 365.8 – 420 Wm-2, 367.2 – 420 Wm-2 and 384.3 – 430 Wm-2respectively. The ranges over BA observed to be 410.1 – 450 Wm-2, 405.1 – 445 Wm-2, 410.1 – 455 Wm-2 and 420.1 – 455 Wm-2 for the years 2002, 2007, 2012 and 2017 respectively. Sensible heat flux: The spatial variation in the SHF over the BA and the DL are shown in the Fig. 11b for Hyderabad. The highest values of the SHF are observed over the dense BA followed by the DL. The ranges of SHF over BA are 115.1 – 183.3 Wm-2, 120.1 – 190.5 Wm-2, 125.1 – 187.7 Wm-2 and 130.1 – 192.7 Wm-2 for the years 2002, 2007, 2012 and 2017 respectively and the ranges over the DL are 85.1 – 125 Wm-2, 90.1 – 130 Wm-2, 95.1 – 135 Wm-2 and 100.1 – 140 24 Wm-2 respectively for the same years. The patches of higher values over the BA are noticed to be increased due to increase and spread in imperviousness. Latent heat flux: The Figure 11c depicts spatial distribution of LHF over BA and DL during the study period. The BA observed to possessing lowest values of LHF ranging 5.5 – 40 Wm-2, 6.3 – 35 Wm-2, 2.8 – 30 Wm-2 and 3.6 – 25 Wm-2 for the year 2002, 2007, 2012 and 2017 respectively. Hyderabad The LHF over DL have comparatively higher values ranging 30.1 – 70 Wm-2, 25.1 – 70 Wm-2, 25.1 – 65 Wm-2 and 20.1 – 60 Wm-2 respectively for the same years. The lower valuezones over the BA are noticed be spreading and can be attributed to the continuous loss of moisture content natural surfaces. Residual heat flux: The Figure 11 (d) shows the spatial distribution of the residual heat flux over BA and the DL for Hyderabad during the study period. The DL noticed to possess the lowest values of residual flux followed by the BA with moderate values which are much lower than that of the vegetated and the water bodies. Bengaluru Net radiation flux: The spatial distribution of the net radiation flux over the BA and DL are shown in the Fig.12a. The DL observed to possess lowest values of the ranges 418.3 – 460 Wm-2, 408.6 – 470 Wm-2, 423.1 – 465 Wm-2and 428.4 – 475 Wm-2whereas the central BA possess comparatively higher range of values, 450.1 – 490 Wm-2, 460.1 – 495 Wm-2, 455.1 – 510 Wm-2 and 465.1 – 505 Wm-2 during the years 2002, 2008, 2013 and 2018 respectively. Sensible heat flux: The spatial distribution of SHF over the BA and the DL of Bengaluru are shown in Fig. 12b. The DL and the fallow lands are noticed to possess highest ranges of values, 120.1 – 177.5 Wm-2, 130.1 – 184.7 Wm-2, 140.1 – 192.5 Wm-2 and 140.1 – 185.2 Wm-2 during the 25 study period. The central BA observed to have slightly lower range of values 95.1 – 130 Wm-2, 100.1 – 140 Wm-2, 110.1 – 150 Wm-2 and 115.1 – 150 Wm-2 during this period. Latent heat flux: The spatial distribution for LHF over BA and DL are depicted in the Fig. 12c. The LHF values over the DL and the fallow land during the study period are noticed to be as low as 25 Wm-2or less. The ranges over the BA are observed to be slightly higher than DL. During the study period the ranges over the BA are 20.1 – 55 Wm-2, 15.1 –50 Wm-2, 15.1 – 45 Wm-2 and 15.1 – 40 Wm-2. Residual heat flux: The values of residual heat flux over the DL patches are lowest and the central BA have slightly high values as shown in Fig. 12d which are much lower than the values over the vegetated and the water bodies. The energy components estimated for the study regions indicate that the ranges of these components are in agreement with the results of the earlier studies. The Net radiation flux during the study period over the cities for BA and DL are ranged around 200 – 550 Wm-2 agrees with values obtained by Offerle et al., (2005); Hanna et al. (2011), Templeton et al. (2018) and Chrysoulakis et al. (2018). Bengaluru The ranges of the SHF and LHF in the present study are around 50 – 200 Wm-2 and 0 – 100 Wm-2 respectively for BA and/or DL, these ranges are in well agreement with the results obtained by Offerle et al., (2005); Kato and Yamaguchi (2005 and 2007) and Chrysoulakis et al. (2018). 4.2. Variation in surface energy component over different LULC classes During the study period the artificial surface (build up areas) over the study regions are noticed to be increased significantly (Sultana and Satyanarayana, 2018, 2020). The surface heat fluxes are therefore noticed to vary considerably during this period. In this section the mean net radiation flux (Q), the mean SHF (QH), the mean LHF (QE) and mean residual heat flux (QR) 26 with respect to different LULC classes such as BA, DL and vegetation over the cities under consideration are discussed. The variations in the mean values of the energy components; ∆Q, ∆QH and ∆QE during the study period for these cities are tabulated in Table 3. The ∆Qand ∆QH are notice to be positive whereas the ∆QE is found to be negative for all the study regions. Kolkata Figure 13a depicts the variation in the mean of the energy components during the study period with respect to different LULC classes (BA, DL and vegetation) of Kolkata. The Q over BA (DL) is observed to be 459.1 Wm-2 (426.5 Wm-2) in 2002 which increased by 57.7 Wm-2(47.1 Wm-2) to 516.8 Wm-2(473.6 Wm-2) in 2017. For vegetation the value increases from 509.5 Wm-2in 2002 to 554.0 Wm-2 in 2017 and the increase in Q* is found to be 44.5 Wm-2during this period. In 2002 the QH over BA (DL) was 112.3Wm-2 (89.3Wm-2) and is increased to 136.7 Wm-2 (103.4 Wm-2) in 2017. The QH is noticed to be increased by 24.4 Wm-2(14.1 Wm-2) for BA (DL) during the study period. In the vegetated area the values are increased from 46.5 Wm-2 to 50.8 Wm- 2during 2002 – 2017; hence the ∆QH is 4.3Wm-2for this LULC class. The QE over BA (DL) was 13.7 Wm-2 (34.6 Wm-2) in 2002, and decreased to 9.7 Wm-2(31.4 Wm-2) in 2017. Over vegetated area the QE noticed to slightly decreasing from 74.9 Wm-2to 73.3 Wm-2 during the study period. The ∆QE over BA, DL and vegetation are – 3.2 Wm-2, – 4.0 Wm-2 and – 1.6 Wm-2respectively for the study period. Visakhapatnam The mean of the energy components with respect to different LULC classes (BA, DL and vegetation) over Visakhapatnam is depicted in Fig. 13b for the study period. The Q* over BA (DL) in 2001 is 452.8 Wm-2(406.5 Wm-2) and in 2017 it increased to 474.8 Wm-2(426.2 Wm-2). Over vegetated area the mean net radiation flux increased from 505.1 Wm-2 to 526.6 Wm-2during 27 27 2001 – 2017. The overall increase for the duration of 2001 – 2017 is 22.0 Wm-2, 19.7 Wm-2 and 21.5 for BA, DL and vegetation respectively. The QH during 2001 – 2017 increased from 86.1 Wm-2 (117.6 Wm-2) to 104.9 Wm-2 (136.2 Wm-2) over BA (DL). In vegetated area the values increased from 41.9 Wm-2 to 58.2 Wm-2 during 2001 – 2017. The ∆QH over BA, DL and vegetation are 18.8 Wm-2, 18.6 Wm-2 and 16.3 Wm-2respectively for the study period. During the study period the QE observed to be decreasing from 39.4 Wm-2 (17.3 Wm-2) to 29.1 Wm-2(9.6 Wm- 2) over BA (DL). In the vegetated area the QE decreases from 87.9 Wm-2 to 74.2 Wm-2. The variations in mean LHF, ∆QE during 2001–2017 are noticed to be – 10.3 Wm-2, – 7.7 Wm-2 and – 13.7 Wm-2 for BA, DL and vegetation respectively. The QR does not appear to be varied much during the study period. 2001 – 2017. The overall increase for the duration of 2001 – 2017 is 22.0 Wm-2, 19.7 Wm-2 and 21.5 for BA, DL and vegetation respectively. The QH during 2001 – 2017 increased from 86.1 Wm-2 (117.6 Wm-2) to 104.9 Wm-2 (136.2 Wm-2) over BA (DL). In vegetated area the values increased from 41.9 Wm-2 to 58.2 Wm-2 during 2001 – 2017. The ∆QH over BA, DL and vegetation are 18.8 Wm-2, 18.6 Wm-2 and 16.3 Wm-2respectively for the study period. During the study period the QE observed to be decreasing from 39.4 Wm-2 (17.3 Wm-2) to 29.1 Wm-2(9.6 Wm- 2) over BA (DL). In the vegetated area the QE decreases from 87.9 Wm-2 to 74.2 Wm-2. The variations in mean LHF, ∆QE during 2001–2017 are noticed to be – 10.3 Wm-2, – 7.7 Wm-2 and – 13.7 Wm-2 for BA, DL and vegetation respectively. The QR does not appear to be varied much during the study period. Mumbai From Fig.13c the mean net radiation flux over BA (DL) in 2002 is observed to be 483.7 Wm- 2(455.8 Wm-2) and in 2018 it increased to 529.8 Wm-2(497.3 Wm-2) whereas over the vegetation it increased from 524.1 Wm-2 to 573.4 Wm-2 during the same duration. The ∆Q* over BA, DL and vegetated areas of Mumbai for the duration of the study, 2002 – 2018 are around 46.1 Wm-2, 41.5 Wm-2 and 49.3 Wm-2 respectively. The QH during 2002 – 2018 noticed to increase from 140.9 Wm-2(104.4 Wm-2) to 163.9 Wm-2(124.3 Wm-2) over BA (DL); hence, the ∆QH over the BA and DL are 23.0 Wm-2and 19.9 Wm-2 respectively. In vegetated area the values increased from 48.2 Wm-2to 64.8 Wm-2, the ∆QH being 16.6 Wm-2during 2002 – 2018. During the study period the QEover Mumbai observed to be decreasing from 77.2 Wm-2 (39.4 Wm-2) to 63.7 Wm-2(25.7 Wm-2) for BA (DL). In the vegetated area the QEdecreased from 130.9 Wm-2 to 121.2 Wm-2.The ∆QE over the BA, DL and the vegetation are – 13.5Wm-2, – 13.7 Wm-2and – 9.7 Wm-2respectively. Kochi Kochi The ∆QE during the study period is observed to be – 5.2 Wm-2, – 10.8 Wm-2and – 12.0 Wm-2over the BA, DL and the vegetation respectively. Kochi 28 28 Figure 13d depicts the variation in the mean of the energy components during the study period with respect to LULC classes, BA, DL and vegetation of Kochi. The Q* over BA (DL) is observed to be 390.8 Wm-2 (429.7 Wm-2) in 2002 which is increased by 15.9 Wm-2 (19.5 Wm-2) to 406.7Wm-2(449.2 Wm-2) in 2018. For vegetation the value increases from 484.8 Wm-2in 2002 to 505.6 Wm-2 in 2018. The ∆Qfor the vegetated areas is 20.8 Wm-2 for the study period. In 2002 the QH over BA (DL) was 113.6 Wm-2 (83.1 Wm-2) and is increased to 133.1 Wm-2 (97.2 Wm-2) in 2018. In the vegetated area the values are increased from 42.4 Wm-2 to 51.9 Wm-2 during 2002 – 2018. The ∆QH over the BA, DL and vegetation are observed to be 19.5 Wm-2, 14.1 Wm-2 and 9.5 Wm-2 respectively during the study period. The QEover BA (DL) was 16.6 Wm-2 (44.2 Wm-2) in 2002, and decreased to 11.4 Wm-2 (33.4 Wm-2) in 2017. Over vegetated area the QEdecreased from 91.2 Wm-2to 79.2 Wm-2 during the study period. The ∆QE during the study period is observed to be – 5.2 Wm-2, – 10.8 Wm-2and – 12.0 Wm-2over the BA, DL and the vegetation respectively. Figure 13d depicts the variation in the mean of the energy components during the study period with respect to LULC classes, BA, DL and vegetation of Kochi. The Q over BA (DL) is observed to be 390.8 Wm-2 (429.7 Wm-2) in 2002 which is increased by 15.9 Wm-2 (19.5 Wm-2) to 406.7Wm-2(449.2 Wm-2) in 2018. For vegetation the value increases from 484.8 Wm-2in 2002 to 505.6 Wm-2 in 2018. The ∆Qfor the vegetated areas is 20.8 Wm-2 for the study period. In 2002 the QH over BA (DL) was 113.6 Wm-2 (83.1 Wm-2) and is increased to 133.1 Wm-2 (97.2 Wm-2) in 2018. In the vegetated area the values are increased from 42.4 Wm-2 to 51.9 Wm-2 during 2002 – 2018. The ∆QH over the BA, DL and vegetation are observed to be 19.5 Wm-2, 14.1 Wm-2 and 9.5 Wm-2 respectively during the study period. The QEover BA (DL) was 16.6 Wm-2 (44.2 Wm-2) in 2002, and decreased to 11.4 Wm-2 (33.4 Wm-2) in 2017. Over vegetated area the QEdecreased from 91.2 Wm-2to 79.2 Wm-2 during the study period. Delhi Figure 14a depicts the variation in the mean values of the energy components during the study period with respect to different LULC classes (BA, DL and vegetation) of Delhi. The Q shows an increasing trend during 2002 – 2017 for all the classes. For 2002 over BA (DL) the Q* value is 334.2 Wm-2 (304.7 Wm-2) which increased to 388.4 Wm-2(350.7 Wm-2) in 2017. For vegetation the value increases from 385.9 Wm-2in 2002 to 440.5 Wm-2 in 2017. The ∆Q* over the BA, DL and the vegetation for the study period are 54.2 Wm-2, 46.0 Wm-2and 54.6 Wm-2respectively. In 2002 the QHover BA (DL) was 84.6 Wm-2 (117.5 Wm-2) and is increased to 112.8 Wm-2 (157.2 Wm-2) in 2017. Over the vegetation the values are increased from 36.2 Wm-2 to 51.4 Wm-2 during 2002 – 2017. The ∆QH over the BA, DL and vegetation are observed to be 28.2 Wm-2, 39.7 Wm-2 29 and 15.2 Wm-2respectively for the study period. The QEover the BA (DL) was 38.2 Wm-2 (23.2 Wm-2) in 2002, and slightly decreased to 32.6 Wm-2 (14.9 Wm-2) in 2017. Over the vegetated area the QE showed small decrease from 79.5 Wm-2to 70.5 Wm-2 during the study period. During the study period the ∆QE over the BA, DL and the vegetated area found to be – 5.6 Wm-2, – 8.3 Wm-2 and – 9.0 Wm-2 respectively. The QR observed to be increasing for all the LULC classes over Delhi. Chandigarh For Chandigarh, Fig. 14b depicts the variation in the mean values of the energy components during the study period with respect to different LULC classes (BA, DL and vegetation). The Q* is noticed increased during 2000 – 2018 for all the classes. For 2000 over BA (DL) the value is 287.3 Wm-2 (252.6 Wm-2) which increased to 319.2 Wm-2(290.4 Wm-2) in 2018. For vegetation the value increases from 331.5 Wm-2in 2000 to 366.5 Wm-2 in 2018.The ∆Q* during the study period over the BA, DL and the vegetation are observed to be 31.9 Wm-2, 37.8 Wm-2and 35.0 Wm-2respectively. In 2000, the QH over BA (DL) was 98.2 Wm-2 (130.7 Wm-2) and is increased to 108.6 Wm-2 (145.2 Wm-2) in 2018 and over the vegetated area the value increased from 52.2 Wm- 2 to 62.1 Wm-2 during this period. The ∆QH found to be 10.4 Wm-2, 14.5 Wm-2and 9.9 Wm-2 over BA, DL and vegetation respectively during the study period. The QE over BA (DL) was 43.7 Wm-2 (16.7 Wm-2) in 2000, and decreased to 28.1 Wm-2 (7.4 Wm-2) in 2018. Over vegetated area the QE decreased from 83.9 Wm-2to 72.1 Wm-2 during the study period. The ∆QE during the study period over the BA, DL and vegetation are observed to be – 15.6 Wm-2, – 9.3 Wm-2and – 11.8 Wm-2respectively. The QR does not appear to be increasing during the study period. Jaipur 30 The mean of the energy components respect to different LULC classes (BA, DL and vegetation) is depicted in Fig. 14c for Jaipur for the study period. The Q* over BA (DL) in 2002 is 392.4 Wm-2(352.7 Wm-2) and in 2017 it increased to 427.6 Wm-2(386.7 Wm-2). Over vegetated area the Q* increased from 438.9 Wm-2 to 480.2 Wm-2during 2002 – 2017. The ∆Q* over the BA, DL and vegetation are found to be 35.2 Wm-2, 34.0 Wm-2and 41.3 Wm-2respectively. The QH during 2002 – 2017 increased from 101.1 Wm-2(145.4 Wm-2) to 114.6 Wm-2(161.5 Wm-2) over BA (DL). In vegetated area the values increased from 53.1 Wm-2to 63.3 Wm-2during the study period. The ∆QH during the study period are found to be 13.5 Wm-2, 16.1 Wm-2and 10.2 Wm-2over the BA, DL and the vegetation respectively. During the study period the QE observed to be decreasing from 42.8 Wm-2 (18.7 Wm-2) to 32.4 Wm-2(9.8 Wm-2) over BA (DL). In the vegetated area the QE decreased from 90.9 Wm-2 to 77.9 Wm-2. The ∆QE during the study period over the BA, DL and the vegetated areas are observed to be – 10.4 Wm-2, – 8.9 Wm-2and – 13.0 Wm-2respectively. Nagpur Figure 15a depicts the variation in the mean values of the energy components with respect to different LULC classes over Nagpur during the study period. The Q* over BA (DL) in 2002 is 388.1 Wm-2(349.6 Wm-2) and in 2018 it increased to 419.6 Wm-2(378.6 Wm-2). The Q* over the vegetation is noticed to increase from 431.8 Wm-2 to 471.7 Wm-2during 2002 – 2018. The ∆Q* as estimated over the BA, DL and vegetation are 31.5 Wm-2, 29.0 Wm-2and 39.9 Wm-2respectively. The QHduring 2002 – 2018 increased from 85.2 Wm-2(122.8 Wm-2) to 99.1 Wm-2(142.9 Wm-2) over BA (DL). For the vegetated area the values increased from 38.6 Wm-2to 53.9 Wm-2during 2002 – 2018. The ∆QH over the BA, DL and the vegetated areas are found to be 13.9 Wm-2, 20.1 Wm-2and 15.3 Wm-2respectively. During the study period the QEover Nagpur observed to be decreasing from 33.2 Wm-2 (14.7 Wm-2) to 26.2 Wm-2(8.5 Wm-2) over BA (DL). In the vegetated area the QE decreased from 67.8 Wm-2 to 54.7 Wm-2. The ∆QE over the BA, DL and vegetation as estimated for the study period are – 7.0 Wm-2, – 6.2 Wm-2 and – 13.1 Wm-2 respectively. Lucknow Figure 14d shows the variation in the mean values of the energy components during the study period with respect to different LULC classes (BA, DL and vegetation) for Lucknow. For 2002 over BA (DL) the Q* value observed to be 398.4 Wm-2 (354.4 Wm-2) which increased to 454.8 Wm-2(414.6 Wm-2) in 2017 and for vegetation the value increases from 454.1 Wm-2in 2002 to 501.7 Wm-2 in 2017. The ∆Q* over the BA, DL and vegetation as observed for the study period are 56.4 Wm-2, 60.2 Wm-2 and 47.6 Wm-2 respectively. In 2002 the QH over BA (DL) was 101.4 Wm-2 (142.3 Wm-2) and is increased to 116.7 Wm-2 (157.3 Wm-2) in 2017. In the vegetated area the values are increased from 53.1 Wm-2 to 66.7 Wm-2 during 2002 – 2017. The ∆QH is observed to be 15.3 Wm-2, 15.0 Wm-2and 13.6 Wm-2over the BA, DL and vegetation respectively during the study period. The QE over BA (DL) was 44.8 Wm-2 (15.7 Wm-2) in 2002, and noticed to be 31 31 decreased to 37.8 Wm-2 (10.6 Wm-2) in 2017. Over vegetated area the QE decreased from 94.1 Wm-2to 85.8 Wm-2 during the study period. The ∆QE during the study period over the BA, DL and vegetation are found to be – 7.0 Wm-2, – 5.1 Wm-2 and – 8.3 Wm-2 respectively. Hyderabad For Hyderabad, Fig. 15b depicts the variation in the mean of the energy components during the study period with respect to different LULC classes (BA, DL and vegetation). The Q* over BA (DL) is observed to be 424.1 Wm-2 (386.4 Wm-2) in 2002 which is slightly increased to 439.1 Wm-2(405.2 Wm-2) in 2017 and for vegetation the value increases from 487.2 Wm-2in 2002 to 498.8 Wm-2 in 2017. The ∆Q* during the study period over the BA, DL and vegetation as estimated are 15.0 Wm-2, 18.8 Wm-2and 11.6 Wm-2respectively. In 2002, the QHover BA (DL) 32 was 152.3 Wm-2 (103.8 Wm-2) and is increased to 163.9 Wm-2 (116.4 Wm-2) in 2017. For the vegetated area the values are increased from 52.1 Wm-2 to 63.9 Wm-2 during 2002 – 2017. The ∆QH during the study period over the BA, DL and vegetated areas are 11.6 Wm-2, 12.6 Wm-2and 11.8 Wm-2respectively. The QE over BA (DL) was 21.5 Wm-2 (54.2 Wm-2) in 2002, and noticed to decreased to 14.3 Wm-2 (42.5 Wm-2) in 2017. Over vegetated area the QE decreased from 101.9 Wm-2 to 93.4Wm-2 during the study period. The ∆QE during the study period found to be – 7.2 Wm-2, – 11.7 Wm-2 and – 8.5 Wm-2over the BA, DL and vegetation respectively. was 152.3 Wm-2 (103.8 Wm-2) and is increased to 163.9 Wm-2 (116.4 Wm-2) in 2017. For the vegetated area the values are increased from 52.1 Wm-2 to 63.9 Wm-2 during 2002 – 2017. The ∆QH during the study period over the BA, DL and vegetated areas are 11.6 Wm-2, 12.6 Wm-2and 11.8 Wm-2respectively. The QE over BA (DL) was 21.5 Wm-2 (54.2 Wm-2) in 2002, and noticed to decreased to 14.3 Wm-2 (42.5 Wm-2) in 2017. Over vegetated area the QE decreased from 101.9 Wm-2 to 93.4Wm-2 during the study period. The ∆QE during the study period found to be – 7.2 Wm-2, – 11.7 Wm-2 and – 8.5 Wm-2over the BA, DL and vegetation respectively. Bengaluru Figure 15c depicts the variation in the mean values of the energy components during the study period with respect to different LULC classes (BA, DL and vegetation) of Bengaluru. For 2002 over BA (DL) the value is 474.5 Wm-2 (436.7 Wm-2) which is noticed to increased to 486.2 Wm- 2(451.2 Wm-2) in 2018. For vegetation the value increases from 529.9 Wm-2in 2002 to 538.3 Wm-2 in 2018. The∆Q* during the study period over the BA, DL and vegetation are 11.7 Wm-2, 14.5 Wm-2and 8.4 Wm-2respectively. In 2002, the QH over BA (DL) observed to be 113.5 Wm-2 (151.9 Wm-2) and is increased to 131.8 Wm-2 (168.9 Wm-2) in 2018. In the vegetated area the values are increased from 58.2 Wm-2 to 76.9 Wm-2 during 2002 – 2018. The ∆QH as estimated for the study duration over the BA, DL and vegetation are 18.3 Wm-2, 17.0 Wm-2and 18.7 Wm-2respectively. The QE over BA (DL) was 38.7 Wm-2 (13.4 Wm-2) in 2002, and decreased to 28.4 Wm-2(9.7 Wm- 2) in 2018. Over vegetated areas the QE decreased from 88.9 Wm-2to 71.9 Wm-2 during the study period. The ∆QE during the study period over the BA, DL and vegetation are – 10.3 Wm-2, – 3.7 Wm-2and – 17.0 Wm-2respectively. The above analysis indicate that the mean SHF, QH is increasing significantly over the BA and the DL during the study period with maximum increase of 28.2 Wm-2 (39.7 Wm-2) over the BA 33 (DL) of Delhi. The ∆QH over the BA of Kolkata (24.4 Wm-2) and Mumbai (23.0 Wm-2) are also found to be higher. On the other hand the mean LHF, QE is noticed to slightly decrease (< 20 Wm-2) over all the LULC classes during the study period. The Q* is also observed to increase considerably during the study period. 4.3. SHF and LHF values with respect to LST over different LULC classes 4.3. SHF and LHF values with respect to LST over different LULC classes The above results indicate that the LHF values are small due to absence of moisture over the BA and the DL over the study regions and the SHF is dominating over these areas. The vegetated areas are noticed to possess the higher LHF values and smaller SHF values. In our earlier studies the estimated LST values over the BA and the DL are observed to be higher than the vegetations and the hotspots are identified mostly over the DL and dense BA for the study regions (Sultana and Satyanarayana, 2018, 2020). In this section the SHF and the LHF values are analyzed with respect to the LST ranges over Different LULC classes (Table 4 – 9). For Kolkata (Table 4), Kochi (Table 5) and Hyderabad (Table 9) the SHF (LHF) values are higher (lower) over the BA than the DL but the LST as noticed in the earlier studies are higher over the DL than that of BA (Sultana and Satyanarayana, 2018, 2020), whereas for the rest of the cites SHF (LHF) have higher (lower) ranges over the DL than that of the BA like the LST ranges. On the other hand, the vegetated areas are found to possess higher (lower) LHF (SHF) and thus LST ranges are noticed to be considerably lower than the BA and DL. It is also noticed that the SHF and LHF ranges over the BA and the DL are slightly overlapping which is also featured in LST values for all cities. 4.4. Estimation of proportion of the heat fluxes with respect to the net radiation flux In this section the ratio of the mean heat fluxes to the mean net radiation flux is analyzed and compared with earlier studies. The ratio of the mean SHF, QH, mean LHF, QE and mean residual 34 heat flux, QR to the mean net radiation flux, Q* is estimated for the urban regions of the cities under consideration for the duration of the study period (Table 10). The proportion of SHF is noticed to be about 19% (Visakhapatnam in 2001) to 33% (Kochi in 2018) of the net radiation flux and are found to increased slightly (1% – 4%) during the study period, with maximum increase for Delhi and Kochi (4% for each). Though Kochi is a coastal city rich in estuaries, the high SHF proportion can be attributed to the soil-type which is mostly loamy sand and poor in water holding capacity (http://www.keralasoils.gov.in/index.php/2016-04-27-09-26-39/soils-of- kerala). For Delhi and Mumbai where higher SHF proportion is noticed are densely-packed in built up areas with negligible moisture-content surfaces. On the other hand, the LHF are noticed to be 1.9% (Kolkata in 2017) to 15% (Chandigarh in 2000) of the net radiation flux and are found to be decreasing for all the cities during the study period. The residual heat flux is found to be about 60% to 80% of the net radiation flux. For Hyderabad the residual heat is found to be higher among the cities under consideration, which can be due to the dense BA and the rocky terrain of granites over the suburban area. Delhi and Kochi also possess higher proportion of residual heat which can be attributed to the dense BA and the soil types of the cities. The proportion of the heat flux with respect to net radiation flux found in the present study is compared with the earlier studies for winter season in the Table 11. It indicates that the present study agrees well with Oke et al., 1999; Moriwaki and Kanda, 2004; Kato and Yamaguchi, 2005, 2007; Kato et al., 2008; Hanna et al., 2011. The residual flux as estimated by Kato and Yamaguchi, 2005 appeared to be over-estimated whereas the SHF and LHF over commercial area by Kato and Yamaguchi, 2007 appear to be underestimated and the residual heat flux is found to be overestimated. 4.4. Estimation of proportion of the heat fluxes with respect to the net radiation flux Comparison of present results with the published ground observation might appear not suitable as they are mean daytime values over different cities, but the 35 comparison could be considered significant as the surface heat fluxes are mostly subjected to surface characteristics. comparison could be considered significant as the surface heat fluxes are mostly subjected to surface characteristics. 5. Summary and Conclusion Though the present study concentrated on the BA and DL, the net all wave radiation flux as estimated over the vegetation and the water bodies are observed to be higher due to lower albedo in comparison to the BA and the DL. The DLs are notices to possess lowest range of net radiation flux values followed by BA for all the cities under consideration except Kochi where BA possess lowest range of values. The SHF is observed to be higher over the BA and DL in comparison to vegetation and water bodies, whereas the LHF is found to be lower over BA and DL due to presence of artificial impervious surfaces lower in moisture contents. The SHF values are noticed to be higher over BA than that of the DLfor the cities like Kolkata, Mumbai, Kochi and Hyderabad whereas for rest of the cities under study the highest SHF values are observed over DL. Similarly, the LHF over the BA are noticed to be lowest for Kolkata, Kochi and Hyderabad and for the rest of the cities the LHF is lowest over DL. The residual heat flux over all the cities under consideration noticed to be higher for the water bodies and vegetation due to higher heat capacity property of water whereas the BA possess moderate values and DL possess the lowest values. For all the cities under consideration the net radiation flux and the SHF are observed to be increased over all the LULC classes during the study period of 2000 – 2018. The SHF is noticed to be increased significantly during the study period over the BA and the DL of the cities, but the LHF is noticed to be slightly decreased over these regions. The increase in SHF and the decreasing trend in LHF can be attributed to the increase of artificial materials and loss of moisture in the surface. 36 The analysis of the relation between SHF/LHF with the LST indicate that the SHF (LHF) values are higher (lower) over the BA than the DL for Kolkata, Kochi and Hyderabad whereas the LST ranges are higher over the DL than that of BA. For rest of the cities the SHF and LHF ranges are noticed to be in linear with the LST ranges. 5. Summary and Conclusion In the present study the estimated proportion of heat flux with respect to net radiation flux indicate that the SHF is about 19% – 33% with higher values over Mumbai, Kochi and Delhi. The LHF is found to be about 1.9% – 15% of the net radiation flux for the study regions. In addition to that the results also indicate that about 60% – 80% of net radiation flux is end up as residual heat flux. The proportion of SHF is noticed to be slightly increased (by 4% for both Delhi and Kochi) and the proportion of LHF is decreased during the study period. The comparison between present results with the earlier studies appears to be in good agreement. The surface energy balance components estimated in the present study are based on earth observation and are could not be validated with the ground observations due to unavailability of high resolution data. In addition to that the energy balance components estimated over the water bodies found to be containing error for few study areas. Hence, the satellite imageries and the ground observations of higher resolution are essential for better estimation of the energy balance components and ultimately the surface dynamics over the fast developing cities. Inferences from the results emanated from the present study has shown substantial variations of surface energy fluxes related to different LULC classes and would certainly influence the energy exchange mechanism from the BA and results in change in the regional climate on the city scale. 37 Acknowledgements The first author of the manuscript would gratefully acknowledge the Indian Institute of Technology, Kharagpur for providing fellowship and necessary facilities to conduct PhD work. Authors are thankful to USGS Earth Resources Observation Systems (EROS) data Centre for freely providing Landsat imageries used in the study. Authors are thankful to the Wyoming Weather Web – Atmospheric sounding (University of Wyoming) and Weather Underground for freely availing atmospheric soundings. Authors are also thankful to the NOAA data archive for freely availing NCEP-DOE Reanalysis 2 data sets. Author Contribution: SS and ANVS designed and framed the study. SS analyzed the data and wrote the manuscript. ANVS provided the intellectual advice and work directions along with review and editing of the manuscript. Funding: Not applicable. Funding: Not applicable. Data availability: The data utilized for the study are available freely for users in the corresponding websites as mentioned in the ‘data’ section of the manuscript. Code availability: Software: ArcGIS 10.3, ERDAS Imagine, Matlab Code availability: Software: ArcGIS 10.3, ERDAS Imagine, Matlab Declarations Ethics approval and consent to participate: The authors confirm that this article is an original research and is not been published in any journal earlier. Consent to publication: The authors agree to submit the manuscript in the current form for publication in the journal. Conflict of interest: The authors declare no conflict of interest. 38 References Allen R., Waters R., Tasumi M., Trezza R., and Bas-tiaanssen W. (2002) SEBAL, Surface energy balance algorithms for land, Idaho Implementation. Advanced Training and User’s manual, version 1.0. Allen, R. G., Tasumi, M., Trezza, R. (2007). Satellite-based energy balance for mapping evapotranspiration with internalized calibration (METRIC) - Model. Journal of Irrigation and Drainage Engineering, v.133, p.380-394. http://dx.doi.org/10.1061/(ASCE)0733- 9437133:4(380). Allen, R. G., Tasumi, M., Trezza, R. (2007). Satellite-based energy balance for mapping evapotranspiration with internalized calibration (METRIC) - Model. Journal of Irrigation and Drainage Engineering, v.133, p.380-394. http://dx.doi.org/10.1061/(ASCE)0733- 9437133:4(380). An N., Hemmati S., Cui Y., (2017). Assessment of the methods for determining net radiation at different time-scales of meteorological variables, Journal of Rock Mechanics and Geotechnical Engineering, 9 (2), 239 – 246, https://doi.org/10.1016/j.jrmge.2016.10.004 An N., Hemmati S., Cui Y., (2017). Assessment of the methods for determining net radiation at different time-scales of meteorological variables, Journal of Rock Mechanics and Geotechnical Engineering, 9 (2), 239 – 246, https://doi.org/10.1016/j.jrmge.2016.10.004 Bastiaanssen, W. G. M., (1995). “Regionalization of surface flux densities and moisture indicators in composite terrain: A remote sensing approach under clear skies in Mediterranean climates.” Ph.D. Dissertation, CIP Data KoninklijkeBibliotheek, Den Haag, The Netherlands. Bastiaanssen, W. G. M., (2000). “SEBAL-based sensible and latent heat fluxes in the irrigated Gediz Basin, Turkey.” J. Hydrol., 229, 87–100. Brenner C., Thiem C. E., Wizemann H., Bernhardt M., Schulz K., (2017). Estimating spatially distributed turbulent heat fluxes from high-resolution thermal imagery acquired with a UAV system, International Journal of Remote Sensing, 38, 8–10, 3003–3026. DOI: 10.1080/01431161.2017.1280202 Brutsaert, W. (1982). In Evaporation into the atmosphere -Theory, History and Applications-; Reidel, D., Ed.; publishing Company: Dordrecht. 39 Carlson, T. N., and D. A. Ripley. (1997). On the Relation between NDVI, Fractional Vegetation Cover, and Leaf Area Index. Remote Sensing of Environment 62 (3): 241–252. doi:10.1016/S0034-4257 (97)00104-1. Carlson, T. N., and D. A. Ripley. (1997). On the Relation between NDVI, Fractional Vegetation Cover, and Leaf Area Index. Remote Sensing of Environment 62 (3): 241–252. doi:10.1016/S0034-4257 (97)00104-1. Census 2011 of India. Accessed 29 June 2020.http://www.censusindia.gov.in Chen, S. and Hu, D., (2017). Parameterizing Anthropogenic Heat Flux with an Energy- Consumption Inventory and Multi-Source Remote Sensing Data. Remote Sens.9, 1165. Chen, S. and Hu, D., (2017). Parameterizing Anthropogenic Heat Flux with an Energy- Consumption Inventory and Multi-Source Remote Sensing Data. Remote Sens.9, 1165. Christen, A. and Vogt, R. (2004). Energy and radiation balance of a central European city. Int. J. Climatol.,24, 1395-1421. References Christen, A. and Vogt, R. (2004). Energy and radiation balance of a central European city. Int. J. Climatol.,24, 1395-1421. Chrysoulakis N., et al., (2018). Urban energy exchanges monitoring from space.Scientific Reports8:11498. DOI:10.1038/s41598-018-29873-x Chrysoulakis N., et al., (2018). Urban energy exchanges monitoring from space.Scientific Reports8:11498. DOI:10.1038/s41598-018-29873-x Crawford T.M., Bluestein H. B., (2000). An Operational, Diagnostic Surface Energy Budget Model, Journal of Applied Meteorology, 39, 1196 – 1217. Eswar, R.,Sekhar, M., Bhattacharya, B. K., (2017). Comparison of three remotesensing based models for the estimation of latent heat flux over India, Hydrological SciencesJournal, 62:16, 2705-2719, DOI: 10.1080/02626667.2017.1404067 Eswar, R.,Sekhar, M., Bhattacharya, B. K., (2017). Comparison of three remotesensing based models for the estimation of latent heat flux over India, Hydrological SciencesJournal, 62:16, 2705-2719, DOI: 10.1080/02626667.2017.1404067 Eswar, R.,Sekhar, M., Bhattacharya, B. K., (2017). Comparison of three remotesensing based models for the estimation of latent heat flux over India, Hydrological SciencesJournal, 62:16, 2705-2719, DOI: 10.1080/02626667.2017.1404067 Goldreich Y. (1992). Urban climate studies in Johannesburg, a subtropical city located on a ridge – a review. Atmospheric Environment 26B: 407–420. Grimmond, C.S.B., (1992). The suburban energy balance: methodological considerations and results for a mid-latitude west coastcity under winter and spring conditions. Int. J. Climatol. 12, 481–497. 40 Grimmond, C.S.B., Oke, T.R. (1999). Aerodynamic properties of urban areas derived from analysis of surface form. J. Appl. Meteorol., 38, 1262-1292. Grimmond, C.S.B.; Salmond, J.A.; Oke, T.R.; Offerle, B.; Lemonsu, A. (2004). Flux and turbulence measurements at a densely build-up site in Marseille: Heat, mass (water and carbon dioxide), and momentum. J. Geophys. Res., 109, doi: 10.1029/2004JD004936. Grimmond, S., (2007). Urbanization and global environmental change: Local effects of urban warming, Geogr. J., 173(1), 83–88, doi:10.1111/j.1475-4959.2007.232_3.x. Grimmond, C.S.B., Ward, H.C. and Kotthaus, S., (2016) How is urbanization altering local and regional climate? In: Seto, K.C., Solecki, W. D. and Griffith, C. A. (eds.). The Routledge Handbook of Urbanization and Global Environmental Change. Routledge. ISBN 9780415732260 Grimmond, C.S.B., Ward, H.C. and Kotthaus, S., (2016) How is urbanization altering local and regional climate? In: Seto, K.C., Solecki, W. D. and Griffith, C. A. (eds.). The Routledge Handbook of Urbanization and Global Environmental Change. Routledge. ISBN 9780415732260 Hanna, S., Marciotto, E., Britter, R., (2011) Urban Energy Fluxes in Build-Up Downtown Areas and Variations across the Urban Area, for Use in Dispersion Models.J. Appl. Meteor. Climatol., 50 (6), 1341–1353. References DOI:10.1175/2011JAMC2555.1 Hanna, S., Marciotto, E., Britter, R., (2011) Urban Energy Fluxes in Build-Up Downtown Areas and Variations across the Urban Area, for Use in Dispersion Models.J. Appl. Meteor. Climatol., 50 (6), 1341–1353. DOI:10.1175/2011JAMC2555.1 Hrisko, J., Ramamurthy, P., Gonzalez J. E., (2021) Estimating heat storage in urban areas using multispectral satellite data and machine learning. Remote Sensing of Environment, 252, 112125. DOI: 10.1016/j.rse.2020.112125 Kato, S.; Yamaguchi, Y., (2005) Analysis of urban heat-island effect using ASTER and ETM+ data: Separation of anthropogenic heat discharge and natural heat radiation from sensible heat flux. Remote Sens. Environ.,99, 44-54. Kato, S.; Yamaguchi, Y., (2007) Estimation of storage heat flux in an urban area using ASTER data. Remote Sens. Environ.,110, 1-17. 41 41 Kato, S., Yamaguchi, Y., Liu, C. C. & Sun, C. Y., (2008).Surface Heat Balance Analysis of Tainan City on March 6, 2001 Using ASTER and Formosat-2 Data. Sensors 8, 6026–6044. Kotthaus, S., Grimmond, C.S.B., (2014). Energy exchange in a dense urban environment – Part I: Temporal variability of long-term observations in central London, Urban Climate, 10, 261–280. Kuang, W., Y. Dou, C. Zhang, W. Chi,A. Liu, Y. Liu, R. Zhang, and J. Liu (2015). Quantifying the heat flux regulation ofmetropolitan land use/land covercomponents by coupling remotesensing modeling with in situmeasurement, J. Geophys. Res. Atmos.,120, 113–130, doi:10.1002/2014JD022249. Kato, S., Yamaguchi, Y., Liu, C. C. & Sun, C. Y., (2008).Surface Heat Balance Analysis of Tainan City on March 6, 2001 Using ASTER and Formosat-2 Data. Sensors 8, 6026–6044. Kotthaus, S., Grimmond, C.S.B., (2014). Energy exchange in a dense urban environment – Part I: Temporal variability of long-term observations in central London, Urban Climate, 10, 261–280. Kuang, W., Y. Dou, C. Zhang, W. Chi,A. Liu, Y. Liu, R. Zhang, and J. Liu (2015). Quantifying the heat flux regulation ofmetropolitan land use/land covercomponents by coupling remotesensing modeling with in situmeasurement, J. Geophys. Res. Atmos.,120, 113–130, doi:10.1002/2014JD022249. Kuang, W., Y. Dou, C. Zhang, W. Chi,A. Liu, Y. Liu, R. Zhang, and J. Liu (2015). Quantifying the heat flux regulation ofmetropolitan land use/land covercomponents by coupling remotesensing modeling with in situmeasurement, J. Geophys. Res. Atmos.,120, 113–130, doi:10.1002/2014JD022249. Landsat 7 (L7) Data Users Handbook, version 2.0, November 2019. Document owner: Ihlen, V., LSRD Project Manager, U.S. Geological Survey. Approved by: Zanter, K., LSDS CCB Chair, U.S. Geological Survey. https://www.usgs.gov/media/files/landsat-7-data-users- handbook Landsat 8 (L8) Data Users Handbook, version 5.0, November 2019, Document owner: Ihlen, V., LSRD Project Manager, U.S. Geological Survey. References Approved by: Zanter, K., LSDS CCB Chair, U.S. Geological Survey. https://www.usgs.gov/land-resources/nli/landsat/landsat-8- data-users-handbook Liang, S, Wang D, He T, Yu Y, (2019) Remote sensingof earth’s energy budget: synthesis and review, Int. J. of Digital Earth, 12:7, 737-780,DOI: 10.1080/17538947.2019.1597189 Macdonald, R.W., Griffiths, R.F., Hall, D.J. (1998) An improved method for the estimation of surface roughness of obstacle arrays. Atmos.Environ., 32, 1857-1864 42 Masson, V., C.S.B. Grimmond and T.R. Oke, (2002) Evaluation of the Town Energy Balance (TEB) scheme with direct measurements from dry districts in two cities. J. Appl. Meteorol., 41, 1011-1026. Mathew, A., Khandelwal, S., Kaul N., (2016) Spatial and temporal variations of urban heat island effect and theeffect of percentage impervious surface area and elevation on landsurface temperature: Study of Chandigarh city, India. Sustainable Cities and Society, 26, 264–277. http://dx.doi.org/10.1016/j.scs.2016.06.018 Mathew, A., Khandelwal, S., Kaul N., (2017) Investigating spatial and seasonal variations of urban heat island effect overJaipur city and its relationship with vegetation, urbanization and elevation parameters. Sustainable Cities and Society, 35, 157–177. http://dx.doi.org/10.1016/j.scs.2017.07.013. Mauder, M., Foken, T., Cuxart, J., (2020) Surface-Energy-Balance Closure over Land: A Review. Boundary-Layer Meteorology. https://doi.org/10.1007/s10546-020-00529-6 Moriwaki, R., and Kanda, M., (2004) Seasonal and Diurnal Fluxes of Radiation, Heat, Water Vapor, and Carbon Dioxide over a Suburban Area. Journal of Applied Meteorology, 43, 1700 - 1710. Munn, R.E., (1966) Descriptive micrometeorology. New York: Academic Press. Myrup, L.O. (1969) A numerical model of the urban heat island. Journal of Applied Meteorology 8, 896-907. Naserikia, M., Shamsabadi, E. A., Rafieian, M., Filho,W. L., (2019) The urban heat island in an urban context: a case study of Mashhad, Iran. Int. J. Environ. Res.Public Health, 16, 313.doi:10.3390/ijerph16030313. 43 Nishida, K., Nemani, R. R., Running, S. W. & Glassy, J. M. (2003) An operational remote sensing algorithm of land surface evaporation.J. Geophys. Res. 108, 4720. Offerle B, Jonsson P, Eliasson I, Grimmond C. S. B., (2005) Urban modification of the surface energy balance in the West African Sahel: Ouagadougou, Burkina Faso. Journal of Climate 18: 3983–3995. Oke, T.R., Yap, D. and Fuggle, R.F. (1972) Determination of urban sensible heat fluxes. In Adams, W.P. and Helleiner, F.M., editors, International geography, University of Toronto: Toronto Press, 176-78. Oke, T. R. (1982). The Energetic Basis of the Urban Heat Island.Quarterly Journal of the Royal Meteorological Society 108: 1–24. Oke, T.R., (1988). The urban energy balance.Prog. Phys. Geogr. 12: 471–508. Oke T R, Zeuner G, Jauregui E. (1992). References The surface energy balance in Mexico City.Atmospheric Environment 26B: 433–444. Oke T.R.; Spronken-Smith, R.A.; Jáuregui, E.; Grimmond, C.S.B., (1999).The energy balance of central Mexico City during the dry season.Atmos.Environ.,33, 3919-3930. central Mexico City during the dry season.Atmos.Environ.,33, 3919-3930. Paulson C. A., (1970). “The mathematical representation of wind speed and temperature profiles in the unstable atmospheric surface layer.” J. Appl. Meteorol., 9, 857–861. Pearlmutter D, Bitan A, Berliner P. (1999). Microclimatic analysis of “compact” urban canyons in an arid zone.Atmospheric Environment33: 4143–4150. 44 Pearlmutter D, Berliner P, Shaviv E. (2005). Evaluation of Urban surface energy fluxes using an open-air scale model. Journal of Applied Meteorology, 44, 532–545. Pearlmutter D, Berliner P, Shaviv E. (2006). Physical modeling of the pedestrian energy exchange within the urban canopy. Building an dEnvironment, 41, 783–795. , , ( ) y g p gy exchange within the urban canopy. Building an dEnvironment, 41, 783–795. Probald, F., (1971): The energy balance as the basis of the urban climate of Budapest. Annales U i i i S i i B d i i S i G hi 7 51 68 Universitatis Scientiarum Budapestinensis, Sectio Geographica 7, 51-68. Prueger, John H. and Kustas, William P. (2005). Aerodynamic Methods for Estimating Turbulent Fluxes.Publications from USDAARS / UNL Faculty. 1394. Rahman, M., M., and Zhang, W., (2019).Review on estimation methods of the Earth’s surface energy balance components from ground and satellite measurements. J. Earth Syst. Sci. 128:84.https://doi.org/10.1007/s12040-019-1098-5. Roth M., (2007).Review of urban climate research in (sub)tropical regions. Int. J. Climatol.27: 1859–1873. DOI: 10.1002/joc.1591 Roth, M., (2013). Handbook of Environmental Fluid Dynamics, Volume Two, edited by Harindra Joseph Shermal Fernando. ISBN: 978-1-4665-5601-0, pp. 143–159. Roupioz L., Jia L., Nerry F. and Menenti M., (2016). Estimation of Daily Solar Radiation Budget at Kilometer Resolution over the Tibetan Plateau by Integrating MODIS Data Products and a DEM, Remote Sens., 8, 504; doi:10.3390/rs8060504 Roupioz L., Jia L., Nerry F. and Menenti M., (2016). Estimation of Daily Solar Radiation Budget at Kilometer Resolution over the Tibetan Plateau by Integrating MODIS Data Products and Salmond J. (1999). Bibliography of urban climate 1996–1999, Available at: http://www.urban- climate.org (last accessed: 20 April 2020). Salmond J. (2005). Bibliography of urban climate 2000–2004, Available at: http://www.urban- climate.org (last accessed: 20 April 2020). 45 Seto, K. C.,Güneralp, B., and Hutyra, L. R., (2012).Global forecasts of urban expansion to 2030 and direct impacts on biodiversity and carbon pools, Proc. Natl. Acad. Sci. References U.S.A., 109(40), 16,083–16,088, doi:10.1073/pnas.1211658109. Silva, B.B.d., Braga, A.C., Braga, C.C., Oliveira, L. M. M. d., Montenegro, S.M.G.L., Barbosa Junior, B. (2016). Procedures for calculation of the albedo with OLI-Landsat 8 images: Application to the Brazilian semi-arid. Revista Brasileira de EngenhariaAgrícola e Ambiental, 20, 3. Silva, B.B.d., Braga, A.C., Braga, C.C., Oliveira, L. M. M. d., Montenegro, S.M.G.L., Barbosa Junior, B. (2016). Procedures for calculation of the albedo with OLI-Landsat 8 images: Application to the Brazilian semi-arid. Revista Brasileira de EngenhariaAgrícola e Ambiental, 20, 3. Sobrino, J.A., Oltra-Carrió, R., Sòria, G., Bianchi, R., Paganini, M., (2012). Impact of spatial resolution and satellite overpass time on evaluation of the surface urban heat island effects. Remote Sens. Environ. 117, 50–56. Spronken-Smith, R.A., (2002).Comparison of summer- and winter-time suburban energy fluxes in Christchurch, New Zealand. Int. J. Climatol., 22, 979-992. Sultana, S., and A.N.V. Satyanarayana. (2018). Urban heat island intensity during winter over metropolitan cities of India using remote-sensing techniques: impact of urbanization, Int. J. Rem. Sens., 39, 6692–6730. DOI:10.1080/01431161.2018.1466072 Sultana, S., and A.N.V. Satyanarayana. (2020). Assessment of urbanisation and urban heat island intensities using Landsat imageries during 2000–2018 over a sub-tropical Indian City. Sustainable Cities and Society, 52, 101846. https://doi.org/10.1016/j.scs.2019.101846 Sundborg, A., (1951). Climatological studies in Uppsala with special regard to the temperature conditions in the urban area. Geographica 22. Geographical Institute, Uppsala University. 46 Tag, P.M. (1968). Surface temperatures in an urban environment. Pennsylvania State University, unpublished thesis. Tag, P.M. (1968). Surface temperatures in an urban environment. Pennsylvania State University, unpublished thesis. Templeton, N.P., Vivoni, E.R., Wang, Z.H., Schreiner-McGraw, A.P., (2018). Quantifying water and energy fluxes over different urban land covers in Phoenix, Arizona. Journal of Geophysical Research: Atmospheres, 123, 2111 – 2128. DOI: 10.1002/2017JD027845. Voogt, J.A., and Oke, T.R., (2003). Thermal remote sensing of urban climates.Remote Sensing of Environment. 86, 370–384. Templeton, N.P., Vivoni, E.R., Wang, Z.H., Schreiner-McGraw, A.P., (2018). Quantifying water and energy fluxes over different urban land covers in Phoenix, Arizona. Journal of Geophysical Research: Atmospheres, 123, 2111 – 2128. DOI: 10.1002/2017JD027845. Voogt, J.A., and Oke, T.R., (2003). Thermal remote sensing of urban climates.Remote Sensing of Environment. 86, 370–384. Webb, E. K. (1970).“Profile relationships: the log-linear range, and extensionto strong stability.”Q. J. R. Meteorol. Soc., 96, 67–90. Weng, Q., Hu, X., Quattrochi, D. & Liu, H., (2014). Assessing intra-urban surface energy fluxes using remotely sensed aster imagery and routine meteorological data: A case study in Indianapolis, USA. IEEE J. Yang J., Menenti M., Krayenhoff E., S., Wu Z., Shi Q., Ouyang X., (2019). Parameterization of Urban Sensible Heat Flux from Remotely Sensed Surface Temperature: Effects of Surface Structure, Remote Sens., 11, 1347; doi:10.3390/rs11111347 Yang J., Menenti M., Krayenhoff E., S., Wu Z., Shi Q., Ouyang X., (2019). Parameterization of Urban Sensible Heat Flux from Remotely Sensed Surface Temperature: Effects of Surface Structure, Remote Sens., 11, 1347; doi:10.3390/rs11111347 Yang J., Menenti M., Krayenhoff E., S., Wu Z., Shi Q., Ouyang X., (2019). Parameterization of Urban Sensible Heat Flux from Remotely Sensed Surface Temperature: Effects of Surface Structure, Remote Sens., 11, 1347; doi:10.3390/rs11111347 References Sel. Top.Appl. Earth Obs. Remote Sens.7, 4046–4057. Weng, Q., Hu, X., Quattrochi, D. & Liu, H., (2014). Assessing intra-urban surface energy fluxes using remotely sensed aster imagery and routine meteorological data: A case study in Indianapolis, USA. IEEE J. Sel. Top.Appl. Earth Obs. Remote Sens.7, 4046–4057. Wetherley, E. B., Roberts, D. A., Tague, C. L., Jones, C., Quattrochi, D. A., McFadden, J. P., (2021). Remote sensing and energy balance modeling of urban climate variability across a semi-arid megacity, Urban Clim., 35, 100757. https://doi.org/10.1016/j.uclim.2020.100757 Wetherley, E. B., Roberts, D. A., Tague, C. L., Jones, C., Quattrochi, D. A., McFadden, J. P., (2021). Remote sensing and energy balance modeling of urban climate variability across a semi-arid megacity, Urban Clim., 35, 100757. https://doi.org/10.1016/j.uclim.2020.100757 World Bank, 2017. The World Bank Annual Report (2017). Washington, DC: World Bank. https://openknowledge.worldbank.org/handle/10986/27986 License: CC BY-NC-ND 3.0 IGO. http://hdl.handle.net/10986/27986. World Bank, 2017. The World Bank Annual Report (2017). Washington, DC: World Bank. https://openknowledge.worldbank.org/handle/10986/27986 License: CC BY-NC-ND 3.0 IGO. http://hdl.handle.net/10986/27986. World Bank, 2017. The World Bank Annual Report (2017). Washington, DC: World Bank. https://openknowledge.worldbank.org/handle/10986/27986 License: CC BY-NC-ND 3.0 IGO. http://hdl.handle.net/10986/27986. Yang J., Wong M. S., Menenti M., (2016). Effects of Urban Geometry on Turbulent Fluxes: A Remote Sensing Perspective, IEEE Geoscience and Remote Sensing Letters, 13 (12), 1767–1771, Dec., doi: 10.1109/LGRS.2016.2607759. Yang J., Wong M. S., Menenti M., (2016). Effects of Urban Geometry on Turbulent Fluxes: A Remote Sensing Perspective, IEEE Geoscience and Remote Sensing Letters, 13 (12), 1767–1771, Dec., doi: 10.1109/LGRS.2016.2607759. 47 Figure Captions Figure Captions Fig. 1 Map of India and location of study regions, the Indian metropolitan cities Fig. 2 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Kolkata for year 2002, 2007, 2012 and 2017 Fig. 1 Map of India and location of study regions, the Indian metropolitan cities Fig. 2 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Kolkata for year 2002, 2007, 2012 and 2017 48 Fig. 3 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Visakhapatnam for year 2001, 2008, 2012 and 2017 Fig. 3 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Visakhapatnam for year 2001, 2008, 2012 and 2017 Fig. 4 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Mumbai for year 2002, 2008, 2012 and 2018 Fig. 5 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Kochi for year 2002, 2007, 2013 and 2018 Fig. 6 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Delhi for year 2002, 2007, 2013 and 2017 Fig. 7 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Chandigarh for year 2000, 2005, 2010, 2013 and 2018 Fig. 8 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Jaipur for year 2002, 2007, 2012 and 2017 Fig. 9 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Lucknow for year 2002, 2007, 2012 and 2017 Fig. 10 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Nagpur for year 2002, 2007, 2013 and 2018 Fig. 11 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Hyderabad for year 2002, 2007, 2012 and 2017 Fig. 12 (a) Net radiation flux, (b) sensible heat flux, (c) latent heat flux, and (d) residual heat flux over Bengaluru for year 2002, 2008, 2013 and 2018 49 Fig. Figure Captions 13 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Kolkata, (b) Visakhapatnam, (c) Mumbai and (d) Kochi Fig. 14 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Delhi, (b) Chandigarh, (c) Jaipur and (d) Lucknow Fig. 14 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Delhi, (b) Chandigarh, (c) Jaipur and (d) Lucknow Fig. 15 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Nagpur, (b) Hyderabad, and (c) Bengaluru Fig. 15 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Nagpur, (b) Hyderabad, and (c) Bengaluru Table Captions 51 51 Figures Figures Table Captions Table Captions Table 1 Details of Landsat images used in the study. Table 2 Typical Parameters for different surface cover types. 50 Table 3 Changes in the urban energy balance (UEB) components during the study period over the buildup area, dry lands and vegetated areas of the study regions. Table 3 Changes in the urban energy balance (UEB) components during the study period over the buildup area, dry lands and vegetated areas of the study regions. Table 4 Comparison of the land surface temperature (LST) ranges with the ranges of the sensible heat flux (SHF) and the latent heat flux (LHF) for Kolkata and Visakhapatnam. Table 5 Comparison of the land surface temperature (LST) ranges with the ranges of the sensible heat flux (SHF) and the latent heat flux (LHF) for Mumbai and Kochi. Table 6 Comparison of the land surface temperature (LST) ranges with the ranges of the sensible heat flux (SHF) and the latent heat flux (LHF) for Delhi and Jaipur. Table 7 Comparison of the land surface temperature (LST) ranges with the ranges of the sensible heat flux (SHF) and the latent heat flux (LHF) for Chandigarh. Table 7 Comparison of the land surface temperature (LST) ranges with the ranges of the sensible heat flux (SHF) and the latent heat flux (LHF) for Chandigarh. Table 8 Comparison of the land surface temperature (LST) ranges with the ranges of the sensible heat flux (SHF) and the latent heat flux (LHF) for Lucknow and Nagpur. Table 9 Comparison of the land surface temperature (LST) ranges with the ranges of the sensible heat flux (SHF) and the latent heat flux (LHF) for Hyderabad and Bengaluru. Table 9 Comparison of the land surface temperature (LST) ranges with the ranges of the sensible heat flux (SHF) and the latent heat flux (LHF) for Hyderabad and Bengaluru. Table 10 Ratios of sensible heat flux (QH), latent heat flux (QE) and residual heat flux (QR) to the net radiation flux (Q) over the built up area of the study regions. Table 10 Ratios of sensible heat flux (QH), latent heat flux (QE) and residual heat flux (QR) to the net radiation flux (Q) over the built up area of the study regions. Table 11 Ratios of heat flux to the net radiation flux as estimated in the present study over urban areas for winter seasons and comparison with the corresponding earlier studies. Figure 1 Map of India and location of study regions, the Indian metropolitan cities Map of India and location of study regions, the Indian metropolitan cities Figure 2 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residu year 2002, 2007, 2012 and 2017 Figure 2 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Kolkata for year 2002, 2007, 2012 and 2017 Figure 2 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Kolkata for year 2002, 2007, 2012 and 2017 Figure 3 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Visakhapatnam for year 2001, 2008, 2012 and 2017 Figure 3 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Visakhapatnam for year 2001, 2008, 2012 and 2017 Figure 4 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) r Figure 4 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Mumbai for year 2002, 2008, 2012 and 2018 Figure 4 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Mumbai for year 2002, 2008, 2012 and 2018 Figure 5 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over K year 2002, 2007, 2013 and 2018 Figure 5 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Kochi for year 2002, 2007, 2013 and 2018 Figure 6 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Delhi for year 2002 2007 2013 and 2017 Figure 6 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Delhi for year 2002, 2007, 2013 and 2017 Figure 7 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Chandiga for year 2000, 2005, 2010, 2013 and 2018 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Chandigarh for year 2000, 2005, 2010, 2013 and 2018 Figure 8 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Jaipur for year 2002, 2007, 2012 and 2017 Figure 8 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Jaipur for year 2002, 2007, 2012 and 2017 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Jaipur for year 2002, 2007, 2012 and 2017 Figure 9 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Lucknow for year 2002, 2007, 2012 and 2017 Figure 9 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Lucknow for year 2002, 2007, 2012 and 2017 Figure 10 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Nagpur fo year 2002, 2007, 2013 and 2018 Figure 10 Figure 10 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Nagpur for year 2002, 2007, 2013 and 2018 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Nagpur for year 2002, 2007, 2013 and 2018 Figure 11 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Hyderaba for year 2002, 2007, 2012 and 2017 Figure 11 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Hyderabad for year 2002, 2007, 2012 and 2017 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Hyderabad for year 2002, 2007, 2012 and 2017 Figure 12 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Bengaluru for year 2002, 2008, 2013 and 2018 Figure 12 (a) Net radiation §ux, (b) sensible heat §ux, (c) latent heat §ux, and (d) residual heat §ux over Bengaluru for year 2002, 2008, 2013 and 2018 Figure 13 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Kolkata, (b) Visakhapatnam, (c) Mumbai and (d) Kochi Figure 13 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Kolkata, (b) Visakhapatnam, (c) Mumbai and (d) Kochi Figure 14 The variation of surface energy components with respect to build up area, dry lands and vegetation ove (a) Delhi, (b) Chandigarh, (c) Jaipur and (d) Lucknow Figure 14 The variation of surface energy components with respect to build up area, dry lands and vegetation over Figure 14 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Delhi, (b) Chandigarh, (c) Jaipur and (d) Lucknow Figure 15 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Nagpur, (b) Hyderabad, and (c) Bengaluru Figure 15 The variation of surface energy components with respect to build up area, dry lands and vegetation over (a) Nagpur, (b) Hyderabad, and (c) Bengaluru
https://openalex.org/W2069249538	https://bmcnephrol.biomedcentral.com/counter/pdf/10.1186/1471-2369-11-31	English	null	Bone Morphogenetic Protein (BMP)-7 expression is decreased in human hypertensive nephrosclerosis	BMC nephrology	2,010	cc-by	9,226	© 2010 Bramlage et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH ARTICLE Open Access Abstract Background: Bone Morphogenetic Protein (BMP)-7 is protective in different animal models of acute and chronic kidney disease. Its role in human kidneys, and in particular hypertensive nephrosclerosis, has thus far not been described. Methods: BMP-7 mRNA was quantified using real-time PCR and localised by immunostaining in tissue samples from normal and nephrosclerotic human kidneys. The impact of angiotensin (AT)-II and the AT-II receptor antagonist telmisartan on BMP-7 mRNA levels and phosphorylated Smad 1/5/8 (pSmad 1/5/8) expression was quantified in proximal tubular cells (HK-2). Functional characteristics of BMP-7 were evaluated by testing its influence on TGF-b induced epithelial-to-mesenchymal transition (EMT), expression of TGF-b receptor type I (TGF-bRI) and phosphorylated Smad 2 (pSmad 2) as well as on TNF-a induced apoptosis of proximal tubular cells. Results: BMP-7 was predominantly found in the epithelia of the distal tubule and the collecting duct and was less abundant in proximal tubular cells. In sclerotic kidneys, BMP-7 was significantly decreased as demonstrated by real-time PCR and immunostaining. AT-II stimulation in HK-2 cells led to a significant decrease of BMP-7 and pSmad 1/5/8, which was partially ameliorated upon co-incubation with telmisartan. Only high concentrations of BMP-7 (100 ng/ml) were able to reverse TNF-a-induced apoptosis and TGF-b-induced EMT in human proximal tubule cells possibly due to a decreased expression of TGF-bRI. In addition, BMP-7 was able to reverse TGF-b-induced phosphorylation of Smad 2. Conclusions: The findings suggest a protective role for BMP-7 by counteracting the TGF-b and TNF-a-induced negative effects. The reduced expression of BMP-7 in patients with hypertensive nephrosclerosis may imply loss of protection and regenerative potential necessary to counter the disease. 3) anti-inflammatory effects by decreasing the accumula- tion of inflammatory cells [3,6] and an amelioration of TNF-a-induced expression of pro-inflammatory cytokines in proximal tubular cells [7]. Bone Morphogenetic Protein (BMP)-7 expression is decreased in human hypertensive nephrosclerosis Carsten P Bramlage1, Björn Tampe1, Michael Koziolek1, Imad Maatouk1, Jelena Bevanda1, Peter Bramlage2, Katharina Ahrens2, Katharina Lange3, Holger Schmid4, Clemens D Cohen5, Matthias Kretzler6, Gerhard A Müller1 Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Correspondence: c.bramlage@med.uni-goettingen.de 1Department of Medicine, Nephrology and Rheumatology, Georg-August- University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany Full list of author information is available at the end of the article Cell culture experiments The human proximal tubular epithelial cell line HK-2 was cultured in serum free complete Quantum 286 medium (PAA, Pasching, Germany) [15]. Cells were made quiescent 24 hours prior to stimulation by incuba- tion with DMEM medium without additives (Invitrogen, Carlsbad, USA). Background Bone Morphogenetic Protein (BMP)-7 has been found to be renoprotective and to promote kidney regeneration in several animal models [1]. This finding was also observed in acute renal injury of the adult kidney as well as in chronic kidney disease [2-4]. The following mechanisms have been found to play a role in the effect of BMP-7: 1) regeneration of tubular epithelial cells by reversal of the epithelial-to-mesenchymal transition (EMT) [4], 2) decrease of apoptosis in tubular epithelial cells [5] and However, these results have usually been obtained in animal models, and data from human kidneys are scarce. The available experiments in human tissue have shown a tubular expression pattern of BMP-7 in normal kidneys [8] and a reversibility of TGF-b-induced EMT by BMP-7 in proximal tubular cells [9], which was similar to the results obtained in rodents. On the other hand, in contrast with some prior animal data, some research has shown an increased expression of BMP-7 in proximal tubular cells in patients with proteinuria [10] and a failure to attenuate Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 2 of 14 a TGF-b-induced EMT in primary or immortalised human proximal tubule epithelial cells in vitro [11]. identify the proximal and distal tubule, and counter- staining with Aquaporin-2 (AQ-2, diluted 1:50, rabbit polyclonal IgG, Calbiochem, San Diego, USA) was used to identify the collecting duct [16]. Double staining was performed using a combination of rhodamine red for BMP-7, and FITC (green) for AQ-1 and -2. Double- labelled cells resulted in orange staining. Negative controls were included in all experiments by exclusive incubation with the second antibody. The aim of the present study was to comprehensively investigate BMP-7 expression as well as its regulation and function in normal and hypertensive sclerotic human kidneys [12,13]. Immunofluorescence Double immunofluorescence (DIF) with anti BMP-7 (goat polyclonal IgG, Santa Cruz, USA) was performed to identify the expression localisation of BMP-7 inside the normal and nephrosclerotic kidney. Counterstaining with Aquaporin-1 (AQ-1, diluted 1:50; rabbit polyclonal IgG, Alpha Diagnostic, San Antonio, USA) was used to Patients and Tissues To analyse EMT, HK-2 cells (10,000/ml) were stained for the epithelial marker zona occludens-1 (ZO-1, 1:25, rabbit polyclonal IgG, Santa Cruz, Santa Cruz, USA) and the mesenchymal marker a-smooth muscle actin (a-sm actin, 1:250, rabbit monoclonal IgG, Novus Biolo- gicals, Littleton, USA) after stimulation for 48 hours with TGF-b (10 ng/ml) in the presence or absence of BMP-7 (10, 100 ng/ml). Bioptic kidney samples for immunostaining were obtained from 12 patients with clinically confirmed nephrosclerosis. Control tissue was obtained from patients undergoing surgical nephrectomy for neoplastic kidney disease (n = 10). Kidney tissue for real-time PCR was obtained from the European Renal cDNA Bank [14]. We included 32 sam- ples from patients with nephrosclerosis and 10 samples from pretransplant biopsies of living and deceased donors. None of the donors had relevant proteinuria, decreased renal function or arterial hypertension [14]. The characteristics of patients with nephrosclerosis are displayed in Table 1. To analyse the influence of BMP-7 on cell death, HK-2 cells (10,000/ml) were stained for annexin-V (Annexin-V-FLUOS Staining Kit, Roche, Mannheim, Germany) after stimulation with TNF-a (20 ng/ml) for 24 hours in the presence or absence of BMP-7 (1, 10, 100 ng/ml, R&D, Minneapolis, USA). Staining for TGF-b receptor type I (TGF-bRI, 1:50, rabbit polyclonal IgG, Santa Cruz, Santa Cruz, USA) was performed after incubation with BMP-7 (100 ng/ml) for 48 hours. The use of human samples was approved by the ethi- cal committee of the Georg August University Göttin- gen (Ref-No #11/10/04). In both kidney slices and HK-2 cells, cell nuclei (blue) were identified by counterstaining with 4,6-diamino- 2-phenylindolyl-dihydrochloride (DAPI; Vector Labs, Burlingame, USA). Staining was visualised by epifluores- cence microscopy (Zeiss Axiovert S100TV, Oberkochen, Germany). Quantification of cell staining intensity was given in grey values by the software AnalySIS (Olympus Soft Imaging Solutions GmbH, Münster, Germany). For comparison between the single stimulation experiments, the same number of cells was evaluated. Immunohistochemistry Immunohistochemistry To identify differences in the BMP-7 expression pattern of sclerotic and normal kidneys, immunohistochemistry (IMH) was performed. The IMH of BMP-7 was performed Table 1 Characteristics of patients with nephrosclerosis Table 1 Characteristics of patients with nephrosclerosis Real time PCR (European Renal cDNA Bank) Immunohisto-chemistry Number of patients 32 12 Age in years (median, IQR) 55 (48-64) 64.5 (58.5-67.5) Male/female (n) 25/7 9/3 Disease duration (years) n.a. 14 (7-25.8) Creatinine Clearance (ml/min) (median, IQR) 51.8 (32.8-61.8) 39.0 (15.5-56.4) Proteinuria (g/day) (median, IQR) 2.3 (0.4-3.1) 0.5 (0.2-1.4) Degree of interstitial fibrosis (%) (median, IQR) n.a. 43.6 (39.8.-48.5) Legend: IQR, interquartile range; n.a., not available Reference values: Creatinine Clearance: 80-140 ml/min., Proteinuria: <150 mg/day Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 3 of 14 as described [16]. An antibody against human BMP-7 (see DIF) was used as the primary antibody, and rabbit anti- goat biotinylated Ig antibody (DAKO, Glostrup, Denmark) was used as the secondary antibody. Negative controls were included in all experiments, and for these, the first antibody was omitted. Nuclear counterstaining was per- formed using hematoxylin. Intensity of BMP-7 expression in tubules were evaluated by an established semiquantita- tive score as follows: 0 = no staining; 1 = weak staining; 2 = moderate staining and 3 = strong staining [17]. For evaluation, five visual field areas per kidney slice were analysed, and the results are presented as the mean ± standard error. microdissection [14]. Thereby, tubulointerstitial and glomerular samples were from the same individuals. Quantification of BMP-7 transcription in HK-2 cells by real-time PCR was done after stimulation with angioten- sin-II (AT-II, 10-7, 10-5 and 10-3 M, Sigma Aldrich, St. Louis, USA), with the angiotensin-II receptor antagonist telmisartan (10, 20 and 30 μM, Boehringer Ingelheim, Germany) or both in combination (10-3 M AT-II + 10, 20 and 30 μM telmisartan) for 12 hours [14,16]. To evaluate EMT in human HK-2 cells, quantification of the epithelial marker ZO-1, and E-cadherin as well as the mesenchymal marker a-sm actin and S100A4 was done after stimulation with TGF-b (10 ng/ml), BMP-7 (100 ng/ml) or both in combination (10 ng/ml TGF-b + 100 ng/ml BMP-7). Moreover, TGF-bRI was determined after stimulation with 1, 10 and 100 ng/ml BMP-7 in HK-2 cells. Total RNA from both tissue and cells was extracted using the Qiagen RNeasy Mini Kit, including a treatment with RNase-Free DNase (both Qiagen, Hilden, Germany) and reverse transcribed using random priming. Oligonu- cleotides were obtained from Primerdesign (Southampton, UK) and are listed in Table 2. Table 1 Characteristics of patients with nephrosclerosis The housekeeping gene Peptidyl-Prolyl-Isomerase-A (PPIA) was chosen because its expression level was comparable to BMP-7 and vali- dated in kidney tissue with a constant expression [18]. Real-time PCR Real-time RT-PCR was performed on a TaqMan ABI 7700 Sequence Detection System (PE Biosystems, Wei- terstadt, Germany) using heat-activated TaqDNA poly- merase (Amplitaq Gold; PE Biosystems). After an initial hold of two minutes at 50°C and ten minutes at 95°C, the samples were cycled 40 times at 95°C for 15 seconds and 60°C for 60 seconds. Quantification was performed using the standard curve approach applying serial dilu- tions of standard cDNA for each target and reference gene. All samples, controls and patients, were analysed in the same run in technical replicates with high repro- ducibility (threshold cycle (Ct) difference <0.4; mean Ct of each sample <32) [14,16]. To exclude the possibility of genomic contamination, we conducted a “no-RT con- trol” in every single PCR using non-reverse transcribed mRNA instead of cDNA. Only real-time PCRs with a negative “no-RT control” were evaluated. FACS analysis of apoptosis FACS analysis was performed to determine the rate of apoptosis in HK-2 cells [19]. Cells were stimulated with either TNF-a (20 ng/ml, R&D, Minneapolis, USA), BMP-7 (1, 10, 100 ng/ml), or both for 24 hours. Apop- totic cells were classified as annexin-V positive and pro- pidium iodide negative using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Levels of glomerular BMP-7 mRNA did not signifi- cantly differ among patients with nephrosclerosis (0.26 (0.14 - 0.54)) compared to those without kidney diseases (0.19 (0.13 - 0.30), p = 0.561) (Figure 1). Statistical analysis Statistical analysis was performed using SigmaStat (Systat Software Inc., San Jose, USA). Real-time PCR data of BMP-7 expression in the whole kidney were evaluated using the Kruskal-Wallis test followed by the Mann-Whitney-U test for comparison. These data and the clinical data were presented in median with quar- tiles (25th and 75th percentile). The results of the cell culture experiments were presented as the means with standard errors. Significant changes were evaluated using Student’s t-test due to the sample sizes (mini- mum: n = 3) and can only be regarded as descriptive. For both statistical methods, p-levels of < 0.05 were regarded to be significant. Figure 1 Real-time PCR analysis: the BMP-7 mRNA level is decreased in the tubulointerstitium but not in the glomeruli of patients with sclerotic compared to normal kidney tissue. Real time PCR with BMP-7 in normal kidneys (n = 10) and those from patients with nephrosclerosis (n = 32), housekeeping gene PPIA. Relative BMP-7 mRNA transcription in the tubulointerstitium and glomeruli of the kidneys are shown. BMP-7 mRNA was significantly decreased in the tubulointerstitium of patients with nephrosclerosis compared to normal kidneys (p < 0.01; Kruskal-Wallis test followed by Mann-Whitney-U test). The BMP-7 mRNA level in the glomeruli did not differ significantly. The results are displayed as medians with quartiles. Figure 1 Real-time PCR analysis: the BMP-7 mRNA level is decreased in the tubulointerstitium but not in the glomeruli of patients with sclerotic compared to normal kidney tissue. Real time PCR with BMP-7 in normal kidneys (n = 10) and those from patients with nephrosclerosis (n = 32), housekeeping gene PPIA. Relative BMP-7 mRNA transcription in the tubulointerstitium and glomeruli of the kidneys are shown. BMP-7 mRNA was significantly decreased in the tubulointerstitium of patients with nephrosclerosis compared to normal kidneys (p < 0.01; Kruskal-Wallis test followed by Mann-Whitney-U test). The BMP-7 mRNA level in the glomeruli did not differ significantly. The results are displayed as medians with quartiles. Figure 1 Real-time PCR analysis: the BMP-7 mRNA level is decreased in the tubulointerstitium but not in the glomeruli of patients with sclerotic compared to normal kidney tissue. Real time PCR with BMP-7 in normal kidneys (n = 10) and those from patients with nephrosclerosis (n = 32), housekeeping gene PPIA. Relative BMP-7 mRNA transcription in the tubulointerstitium and glomeruli of the kidneys are shown. BMP-7 is mainly expressed in the distal tubule and collecting duct In normal kidneys, BMP-7 protein was found to be loca- lised in the epithelium of the distal tubule and the col- lecting duct but was less abundant in the proximal tubular cells or glomeruli. Expression in the distal tubules was suggested by parallel binding of BMP-7 and AQ-1 in tubules with smaller cuboidal cells and larger lumen. Proximal tubules with prominent cuboidal epithelial cell lining and the smaller lumen showed higher AQ-1 than BMP-7 expression (Figure 2). Double immunofluorescence (DIF) with BMP-7 and AQ-2 showed parallel expression, revealing expression of BMP-7 in the collecting duct (Figure 3). As illustrated by double immunofluorescence (Figure 2, 3, 4) and immunohistochemistry (Figure 5), localisation of the BMP-7 expression was unchanged in patients with BMP-7 mRNA level is decreased in nephrosclerotic compared to normal kidneys BMP-7 mRNA level is decreased in nephrosclerotic compared to normal kidneys BMP-7 mRNA was significantly reduced in the tubuloin- terstitium of patients with nephrosclerosis (relative med- ian quantity with quartiles: 0.25 (0.14 - 0.42)) compared to normal kidneys (0.69 (0.28 - 0.83), p < 0.01) (Figure 1). Levels of glomerular BMP-7 mRNA did not signifi- cantly differ among patients with nephrosclerosis (0.26 (0.14 - 0.54)) compared to those without kidney diseases (0.19 (0.13 - 0.30), p = 0.561) (Figure 1). BMP-7 mRNA was significantly reduced in the tubuloin- terstitium of patients with nephrosclerosis (relative med- ian quantity with quartiles: 0.25 (0.14 - 0.42)) compared to normal kidneys (0.69 (0.28 - 0.83), p < 0.01) (Figure 1). Protein extraction and immunoblotting Western blot analysis of phosphorylated Smad 1/5/8 (pSmad 1/5/8, diluted 1:1000; rabbit polyclonal IgG, Cell Signalling, Beverly, USA) and phosphorylated Smad 2 (pSmad 2, diluted 1:1000; rabbit polyclonal IgG, Cell Signaling, Beverly, USA) was performed as previously described [17]. Polyclonal goat anti-rabbit IgG antibody (1:2000, DAKO, Glostrup, Denmark) was used as secondary antibody. Phosphorylated Smad 1/5/8 was Quantification of BMP-7 mRNA transcription in kid- ney tissue samples was determined separately for glo- meruli and for the tubulointerstitium after manual Table 2 Oligonucleotides mRNA Gene Bank accession number Oligonucleotide (5’ - 3’) (up/down) bp PPIA NM 021130 TGG GCA ACA TAG TGA GAC G 139 TGT ACA GTG GCA TGA TAA TAG C BMP-7 NM 001719 CCT CCA TTG CTC GCC TTG 114 TAT GCT GCT CAT GTT TCC TAA TAC E-cadherin NM 00436 CAT GAG TGT CCC CCG GTA TC 89 CAG TAT CAG CCG CTT TCA GA ZO-1 NM 003257 AAA CAA GCC AGC AGA GAC C 95 CGC AGA CGA TGT TCA TAG TTT C a-sm-actin NM 001613 AAG CAC AGA GCA AAA GAG GAA T 76 ATG TCG TCC CAG TTG GTG AT S100A4 NM 002961 TCT TTC TTG GTT TGA TCC TGA CT 130 AGT TCT GAC TTG TTG AGC TTG A TGF-bRI NM 004612 TGA CTG AAG GCT GCT CTG G 125 CAT CTG CTC AAT CTC CAA ACT TG Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 4 of 14 determined in HK-2 cells (1,000,000/ml, 25 μg/blot) after stimulation with AT-II (10-3 M) for 36 hours. Phosphory- lated Smad 2 was determined in HK-2 cells (1,000,000/ ml, 25 μg/blot) after stimulation with 10 ng/ml TGF-b alone and in combination with BMP-7 (100 ng/ml). Statistical analysis BMP-7 mRNA was significantly decreased in the tubulointerstitium of patients with nephrosclerosis compared to normal kidneys (p < 0.01; Kruskal-Wallis test followed by Mann-Whitney-U test). The BMP-7 mRNA level in the glomeruli did not differ significantly. The results are displayed as medians with quartiles. Page 5 of 14 Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 BMP-7 & Aquaporin-1 (x200) Aquaporin-1 (x200) BMP-7 (x200) Negative Control A B C D Figure 2 Immunofluorescence of BMP-7 and Aquaporin 1 in normal kidneys revealing expression in distal tubules and proximal tubular cells. Double immunofluorescence of BMP-7 with aquaporin-1 (AQ-1): A) AQ-1 is green (FITC), B) BMP-7 staining is red (rhodamine red), C) double labelling of BMP-7 and AQ-1 is orange and nuclei is blue (DAPI). Parallel binding of BMP-7 and AQ-1 is seen mainly in tubules with smaller cuboidal cells and larger lumen indicating expression in distal tubules (small arrow) and less in proximal tubule cells (bulky arrow). Staining was low in the glomeruli. Original magnification: × 200. D) Negative control × 100. Aquaporin-1 (x200) A BMP-7 (x200) B Aquaporin-1 (x200) BMP-7 (x200) BMP-7 (x200) B A BMP-7 & Aquaporin-1 (x200) Negative Control BMP-7 & Aquaporin-1 (x200) C Negative Control D D C Figure 2 Immunofluorescence of BMP-7 and Aquaporin 1 in normal kidneys revealing expression in distal tubules and proximal tubular cells. Double immunofluorescence of BMP-7 with aquaporin-1 (AQ-1): A) AQ-1 is green (FITC), B) BMP-7 staining is red (rhodamine red), C) double labelling of BMP-7 and AQ-1 is orange and nuclei is blue (DAPI). Parallel binding of BMP-7 and AQ-1 is seen mainly in tubules with smaller cuboidal cells and larger lumen indicating expression in distal tubules (small arrow) and less in proximal tubule cells (bulky arrow). Staining was low in the glomeruli. Original magnification: × 200. D) Negative control × 100. stimulation with 10-3 M AT-II compared to unstimulated cells (95% confidence interval (CI) 66.1 - 103.3, p < 0.001) and increased after stimulation with 30 μM telmisartan (95% CI 29.6 - 146.2, p < 0.01). The stimulation with a combination of AT-II and telmisartan showed higher BMP-7 mRNA levels compared to AT-II alone; the differ- ence was, however, not significant. nephrosclerosis, but the mean intensity was significantly lower (0.86 ± 0.17) compared to controls (2.5 ± 0.07) (95% confidence interval (CI) 0.23 - 3.10, p < 0.05). Regulation of BMP-7 expression by the renin-angiotensin system To examine the interaction of BMP-7 with the renin- angiotensin system, BMP-7 mRNA level in HK-2 cells was quantified by real-time PCR (18 μg/ml cDNA; 25 - 30 cycles) after stimulation with AT-II, telmisartan or both in combination for 12 hours. As shown in Figure 6, the tran- scription of BMP-7 was significantly decreased after BMP-7 is able to reverse EMT in human proximal tubule cells (HK-2) To analyse the influence of BMP-7 on EMT in human proximal tubule cells, the expression of epithelial and Page 6 of 14 Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Figure 3 Immunofluorescence of BMP-7 and Aquaporin 2 in normal kidneys showing expression in the collecting duct. Double immunofluorescence of BMP-7 with aquaporin-2 (AQ-2): A) AQ-2 is green (FITC), B) BMP-7 staining is red (rhodamine red), C, D) double-labelling of BMP-7 and AQ-2 is stained orange (C: × 200, D: × 400) and nuclei are blue (DAPI). Parallel binding of BMP-7 and AQ-2 revealing expression of BMP-7 in the collecting duct. Negative control: see Figure 2D. Original magnification: × 200 and × 400. Figure 3 Immunofluorescence of BMP-7 and Aquaporin 2 in normal kidneys showing expression in the collecting duct. Double immunofluorescence of BMP-7 with aquaporin-2 (AQ-2): A) AQ-2 is green (FITC), B) BMP-7 staining is red (rhodamine red), C, D) double-labelling of BMP-7 and AQ-2 is stained orange (C: × 200, D: × 400) and nuclei are blue (DAPI). Parallel binding of BMP-7 and AQ-2 revealing expression of BMP-7 in the collecting duct. Negative control: see Figure 2D. Original magnification: × 200 and × 400. mRNA levels of E-cadherin (95% CI: 32.2 - 17.6, p < 0.001), by a tendency for increased mRNA levels of ZO-1 (95% CI: 0.5 - 56.5, p = 0.05) and by significantly decreased levels of a-sm-actin (95% CI: 28.1 - 93.8, p < 0.001) and S100A4 (95% CI: 9.6 - 34.7, p < 0.05). In analogy, similar changes could be detected for the protein level of ZO-1 (95% CI: 5.7 - 69.1, p < 0.01) and a-sm-actin (95% CI: 284.7 - 859.6, p < 0.05) (Figure 8). Both the expression of ZO-1 and a-sm-actin showed dose-dependent effects. Sig- nificant changes in both protein and mRNA level were observed only after stimulation with a concentration of 100 ng/ml BMP-7. BMP-7 alone did not substantially influence the mRNA level of ZO-1, E-cadherin, a-sm- actin and S100A4 (Figure 8), nor did it effect the protein mesenchymal marker were determined after stimulation with BMP-7 alone or in combination with TGF-b. Stimulation of human proximal tubule cells (HK-2) with TGF-b (10 ng/ml) alone induced EMT as demonstrated by morphological transformations of epithelial into fibroblastoid cells (Figure 7). BMP-7 is able to reverse EMT in human proximal tubule cells (HK-2) This morphological obser- vation is underscored by the lower mRNA level of the epithelial markers ZO-1 and E-cadherin and higher transcription of the mesenchymal markers a-sm-actin and S100A4 (Figure 8). Co-stimulation of TGF-b and BMP-7 led to a reversal of the prior morphological transformation with a predomi- nant epithelial phenotype (Figure 7). The reversal of EMT was further demonstrated by significantly increased Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 7 of 14 Figure 4 Immunofluorescence of BMP-7 and Aquaporin 1 and Aquaporin 2 in nephrosclerotic kidneys showing decreased expression of BMP-7. Double immunofluorescence of BMP-7 with aquaporin-1 (AQ-1) and aquaporin-2 (AQ-2): BMP-7 staining is red (rhodamine red, A, B), double-labeling of BMP-7 and AQ-1 (C) or AQ-2 (D) is stained organge. Expression of BMP-7 was lower than in the control kidneys demonstrated by the lower staining intensity of BMP-7 despite a longer exposure time and therewith a stronger intensity of AQ-1 and AQ-2. Original magnification: × 200. Figure 4 Immunofluorescence of BMP-7 and Aquaporin 1 and Aquaporin 2 in nephrosclerotic kidneys showing decreased expression of BMP-7. Double immunofluorescence of BMP-7 with aquaporin-1 (AQ-1) and aquaporin-2 (AQ-2): BMP-7 staining is red (rhodamine red, A, B), double-labeling of BMP-7 and AQ-1 (C) or AQ-2 (D) is stained organge. Expression of BMP-7 was lower than in the control kidneys demonstrated by the lower staining intensity of BMP-7 despite a longer exposure time and therewith a stronger intensity of AQ-1 and AQ-2. Original magnification: × 200 Figure 5 Immunohistochemistry of BMP-7 with unchanged localisation, but lower expression levels in patient with nephrosclerosis compared to normal kidneys. Immunostaining for BMP-7 was done in 10 normal kidneys (A - C) and in 12 kidneys from patients with nephrosclerosis (D - F). Expression levels of BMP-7 were low in all examined kidney slices. BMP-7 is located mainly endoluminally in the normal kidney (A: × 400; B: × 1000). In kidneys of patients with nephrosclerosis, localisation of BMP-7 expression was unchanged, but lower than in the control kidneys (D: × 400; E: × 1000). Negative controls did not show any staining (C, F). Figure 5 Immunohistochemistry of BMP-7 with unchanged localisation, but lower expression levels in patient with nephrosclerosis compared to normal kidneys. Immunostaining for BMP-7 was done in 10 normal kidneys (A - C) and in 12 kidneys from patients with nephrosclerosis (D - F). BMP-7 is able to reverse EMT in human proximal tubule cells (HK-2) Expression levels of BMP-7 were low in all examined kidney slices. BMP-7 is located mainly endoluminally in the normal kidney (A: × 400; B: × 1000). In kidneys of patients with nephrosclerosis, localisation of BMP-7 expression was unchanged, but lower than in the control kidneys (D: × 400; E: × 1000). Negative controls did not show any staining (C, F). Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 8 of 14 Figure 6 BMP-7 mRNA level in HK-cells is decreased after stimulation with angiotensin-II and increased after stimulation with telmisartan. BMP-7 mRNA level in the proximal tubule cell line HK-2 was determined by real-time PCR (housekeeping gene PPIA) after stimulation with angiotensin-II (10-7, 10-5, 10-3 M) and telmisartan (10, 20, 30 μM) or with both combined for 12 hours (n = 12). BMP-7 mRNA level is significantly decreased after stimulation with 10-3 M AT-II (p < 0.001) and increased after stimulation with 30 μM telmisartan (p < 0.01). Co-stimulation of AT-II and telmisartan showed non-significantly increased BMP-7 mRNA levels compared to stimulation with AT-II alone. The results are presented as the mean % of unstimulated cells ± standard error. Figure 6 BMP-7 mRNA level in HK-cells is decreased after stimulation with angiotensin-II and increased after stimulation with telmisartan. BMP-7 mRNA level in the proximal tubule cell line HK-2 was determined by real-time PCR (housekeeping gene PPIA) after stimulation with angiotensin-II (10-7, 10-5, 10-3 M) and telmisartan (10, 20, 30 μM) or with both combined for 12 hours (n = 12). BMP-7 mRNA level is significantly decreased after stimulation with 10-3 M AT-II (p < 0.001) and increased after stimulation with 30 μM telmisartan (p < 0.01). Co-stimulation of AT-II and telmisartan showed non-significantly increased BMP-7 mRNA levels compared to stimulation with AT-II alone. The results are presented as the mean % of unstimulated cells ± standard error. level of ZO-1 and of a-sm-actin compared to unstimu- lated cells (= 100%) (Figure 7). 21.60%, 10 ng/ml: 39.29 ± 2.60%, 100 ng/ml: 25.71 ± 8.37%, data not shown). Thereby, stimulation with 100 ng/ml leads to a significant decrease of TGF-bRI expres- sion at both the protein (95% CI: 27.3 - 75.6, p < 0.01) and the mRNA level (95% CI: 53.5 - 95.1, p < 0.01) com- pared to unstimulated HK-2 cells (Figure 10). Decreased expression of pSmad 1/5/8 after stimulation with BMP-7 To further verify the decrease of BMP-7 by angiotensin- II, we examined the expression of phosphorylated Smad 1/5/8 as intracellular pathway mediators of BMP-7 by western blotting (n = 3). In concordance with the decreased BMP-7 expression by AT-II, phosphorylated Smad 1/5/8 was significantly decreased in HK-2 cells after stimulation with 10-3 mM AT-II to 54.8 ± 9.8% of the unstimulated controls (= 100%, 95% CI: 21.0 - 70.2, p < 0.05) (Figure 9A). BMP-7 is able to reverse EMT in human proximal tubule cells (HK-2) Moreover, the expression pattern of TGF-bRI changed after stimula- tion with BMP-7 to a localisation that was more intracel- lular than on the cell surface. Decreased pSmad-2 expression in HK-2 cells after stimulation with BMP-7 To further test the hypothesis that the decreased expression of the TGF-b receptor type I by BMP-7 may have func- tional consequences, phosphorylated Smad 2 was deter- mined by western blotting after stimulation with TGF-b in presence or absence of BMP-7. Compared to unstimulated HK-2 cells, phosphorylated Smad 2 was increased after sti- mulation with TGF-b alone (323.9 ± 96.4%) and signifi- cantly decreased after co-stimulation with TGF-b and BMP-7 (95% CI: 34.8 - 52.8, p < 0.05; Figure 9B). BMP-7 decreases TGF-b Receptor type I in HK-2 cells To test the hypothesis that reversal of EMT by BMP-7 may be mediated by the down-regulation of TGF-b Receptor type I (TGF-bRI) expression, this expression was analysed after stimulation of HK-2 cells with BMP-7. Expression of TGF-bRI was decreased after stimulation with BMP-7 in a dose-dependent manner compared to unstimulated cells (= 100%). This was demonstrated at the protein level by immunofluorescence (Figure 10) and at the mRNA level by real-time PCR (1 ng/ml: 92.98 ± BMP-7 influence on TNF-a induced apoptosis 7 on cell death and apoptosis in proximal tubule cells (HK-2) after stimulation with TNF-a alone and in com- bination with BMP-7. Apoptotic cells were detected by FACScan (FACS) counting annexin-V positive and pro- pidium iodide negative cells (Figure 11A): TNF-a (20 ng/ml): 386.6 ± 41.3%; TNF-a (20 ng/ml) + 1 ng/ml BMP-7: 356.1 ± 53.3%; + 10 ng/ml: 272.0 ± 28.9%; + 100 ng/ml: 173.2 ± 48.4%, 95% CI: 57.0 - 369.8%, p < 0.05), each compared to unstimulated cells. 7 on cell death and apoptosis in proximal tubule cells (HK-2) after stimulation with TNF-a alone and in com- bination with BMP-7. Apoptotic cells were detected by FACScan (FACS) counting annexin-V positive and pro- pidium iodide negative cells (Figure 11A): TNF-a (20 ng/ml): 386.6 ± 41.3%; TNF-a (20 ng/ml) + 1 ng/ml BMP-7: 356.1 ± 53.3%; + 10 ng/ml: 272.0 ± 28.9%; + 100 ng/ml: 173.2 ± 48.4%, 95% CI: 57.0 - 369.8%, p < 0.05), each compared to unstimulated cells. BMP-7 alone did not substantially induce cell death (DIF, 100 ng/ml: 97.5 ± 3.9%) or apoptosis (FACS, 100 ng/ml: 152.34 ± 5.9%). BMP-7 influence on TNF-a induced apoptosis BMP-7 influence on TNF-a induced apoptosis Since BMP-7 is known to have protective effects on tub- ular epithelial cells, we examined the influence of BMP- Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 9 of 14 Figure 7 BMP-7 reverses TGF-b-induced EMT detected by immunofluorescence. BMP-7 reverses TGF-b-induced EMT as demonstrated by immunofluorescence of ZO-1 (A) and a-sm actin (B) in human proximal tubular cells (HK-2, n = 3). Stimulation with TGF-b alone (10 ng/ml) led to a distinctive morphological transformation into fibroblastoid cells in combination with a lowered expression of ZO-1 (A) and a higher expression of a-sm actin (B). Reversal of EMT was demonstrated by co-stimulation of HK-2 cells with TGF-b (10 ng/ml) and BMP-7 (10, 100 ng/ ml) showing epithelial morphology. Co-Stimulation with 10 ng/ml TGF-b and 100 ng/ml BMP-7 were leading to a significantly higher expression of ZO-1 (p < 0.01; A) and lower expression of a-sm-actin (p < 0.001; B) compared to TGF-b stimulation alone. BMP-7 alone did not substantially influence the expression of ZO-1 and a-sm-actin. Values were evaluated by determination of the staining intensity per cell using Student’s t-test and are presented as % of unstimulated cells. Figure 7 BMP-7 reverses TGF-b-induced EMT detected by immunofluorescence. BMP-7 reverses TGF-b-induced EMT as demonstrated by immunofluorescence of ZO-1 (A) and a-sm actin (B) in human proximal tubular cells (HK-2, n = 3). Stimulation with TGF-b alone (10 ng/ml) led to a distinctive morphological transformation into fibroblastoid cells in combination with a lowered expression of ZO-1 (A) and a higher expression of a-sm actin (B). Reversal of EMT was demonstrated by co-stimulation of HK-2 cells with TGF-b (10 ng/ml) and BMP-7 (10, 100 ng/ ml) showing epithelial morphology. Co-Stimulation with 10 ng/ml TGF-b and 100 ng/ml BMP-7 were leading to a significantly higher expression of ZO-1 (p < 0.01; A) and lower expression of a-sm-actin (p < 0.001; B) compared to TGF-b stimulation alone. BMP-7 alone did not substantially influence the expression of ZO-1 and a-sm-actin. Values were evaluated by determination of the staining intensity per cell using Student’s t-test and are presented as % of unstimulated cells. TNF-a stimulation alone (95% CI: 52.7 - 189.3%, p < 0.05). Thus, the effect was dose-dependent: TNF-a + 1 ng/ml BMP-7: 318.0 ± 2.1%; + 10 ng/ml BMP-7: 283.4 ± 6.4%; +100 ng/ml BMP-7: 181.2 ± 0.7%). Discussion The influence of BMP-7 on TNF-a induced cell death of HK-2 cells was determined by immunofluorescence after staining for annexin-V (Figure 11B). Stimulation of the proximal tubule cells with 20 ng/ml TNF-a led to cell death rates of 302.2 ± 1.5% compared to unstimulated controls. Co-stimulation with a combination of TNF-a (20 ng/ml) and BMP-7 (100 ng/ml) resulted in signifi- cantly lower cell death rates than those observed with In this study, expression, regulation and function of BMP-7 was compared in human hypertensive nephro- sclerotic versus normal kidneys. Our results demon- strated the following: 1) renal BMP-7 is decreased in human hypertensive nephrosclerosis; 2) BMP-7 tran- scription is regulated by the renin-angiotensin system (AT-II vs. telmisartan) in vitro; 3) BMP-7 is able to reverse TNF-a- and TGF-b-induced effects in human Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 10 of 14 Figure 8 Real time PCR indicating reversal of TGF-b induced EMT by BMP-7. The mRNA level of the epithelial marker ZO-1 and E-cadherin and the mesenchymal marker a-sm-actin and S100A4 were analysed after stimulation of HK-2 cells with TGF-b alone, BMP-7 alone or both in combination (n = 3). There was a decrease of ZO-1 and E-cadherin and an increase of a-sm-actin and S100A4 mRNA after stimulation with TGF- b alone, while no significant effect was seen after stimulation with BMP-7 alone. Co-stimulation of TGF-b and BMP-7 led to a significant increase of ZO-1 (p < 0.001) and E-cadherin (p = 0.05) as well as a decrease of a-sm-actin (p < 0.001) and S100A4 mRNA (p < 0.05) compared to TGF-b alone. The results were analysed by Student’s t-tests and are displayed as % of unstimulated cells. Figure 8 Real time PCR indicating reversal of TGF-b induced EMT by BMP-7. The mRNA level of the epithelial marker ZO-1 and E-cadherin Figure 8 Real time PCR indicating reversal of TGF-b induced EMT by BMP-7. The mRNA level of the epithelial marker ZO-1 and E-cadherin and the mesenchymal marker a-sm-actin and S100A4 were analysed after stimulation of HK-2 cells with TGF-b alone, BMP-7 alone or both in combination (n = 3). There was a decrease of ZO-1 and E-cadherin and an increase of a-sm-actin and S100A4 mRNA after stimulation with TGF- b alone, while no significant effect was seen after stimulation with BMP-7 alone. Discussion Co-stimulation of TGF-b and BMP-7 led to a significant increase of ZO-1 (p < 0.001) and E-cadherin (p = 0.05) as well as a decrease of a-sm-actin (p < 0.001) and S100A4 mRNA (p < 0.05) compared to TGF-b alone. The results were analysed by Student’s t-tests and are displayed as % of unstimulated cells. proximal tubule cells, albeit only high concentrations; and 4) BMP-7 decreased TGF-bRI expression. acute ischemic renal injury, tubulointerstitial fibrosis and diabetic nephropathy [2,20,21]. In contrast, pre- vious investigations by Rudnicki et al. in proteinuric patients showed an increase of BMP-7 expression in proximal tubule cells [10]. Due to the heterogeneity of the possible underlying kidney disease [10] in the dif- ferent studies, it is difficult to assess and compare these findings. Moreover, a reason for the discrepancy of the results could be disparities in the stage of exam- ined kidney disease. Thus, progression of kidney fibro- sis is associated with a concomitant loss of BMP-7 expression in later stages of kidney disease [22]. Experiments with streptozotocin-induced diabetes in rodents demonstrated decreased renal expression of BMP-7 to 50% of its original level at week 15, and up to 10% of the control animals by week 30 [22]. This, in BMP-7 localisation in normal kidneys and nephrosclerosis BMP-7 was found to be expressed predominantly in the epithelium of the distal tubule, the collecting duct and less in the proximal tubular cells in normal kidneys. This expression pattern is concordant with recently published data, demonstrating the same expression pattern in normal human kidneys [8]. We found that localisation of BMP-7 expression was unchanged in human hypertensive nephrosclerosis, but expression was reduced, as demonstrated by immunostaining and real- time PCR. Our results are in accordance with findings in animals, where the renal BMP-7 expression was reduced in several kidney disease models, including Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 11 of 14 Figure 9 Figure 9A - Smad 1/5/8 is decreased by angiotensin-II. Western Blotting was performed in HK-2 cells for phosphorylated Smad 1/5/8 after stimulation with AT-II (10-3 mM, n = 3). Phosphorylated Smad 1/5/8 was significantly decreased in HK-2 cells after stimulation with AT-II (p < 0.05). Figure 9B - BMP-7 attenuates TGF-b induced pSmad 2. Western blotting was performed in HK-2 cells for phosphorylated Smad 2 after stimulation with TGF-b alone (10 ng/ml) and TGF-b in combination with BMP-7 (100 ng/ml, n = 3). Discussion Phosphorylated Smad 2 was increased after stimulation with TGF-b alone and significantly decreased after stimulation with TGF-b in combination with BMP-7 (p < 0.05). Figure 9 Figure 9A - Smad 1/5/8 is decreased by angiotensin-II. Western Blotting was performed in HK-2 cells for phosphorylated Smad 1/5/8 after stimulation with AT-II (10-3 mM, n = 3). Phosphorylated Smad 1/5/8 was significantly decreased in HK-2 cells after stimulation with AT-II (p < 0.05). Figure 9B - BMP-7 attenuates TGF-b induced pSmad 2. Western blotting was performed in HK-2 cells for phosphorylated Smad 2 after stimulation with TGF-b alone (10 ng/ml) and TGF-b in combination with BMP-7 (100 ng/ml, n = 3). Phosphorylated Smad 2 was increased after stimulation with TGF-b alone and significantly decreased after stimulation with TGF-b in combination with BMP-7 (p < 0.05). turn, could be due to the TGF-b induced down-regula- tion of BMP-7 as demonstrated in proximal tubule cells [22]. BMP-7 mRNA in primary and immortalised (HK-2) proximal tubule cells [8], BMP-7 mRNA was detected by Rudnicki et al. [10]. In our experiments, BMP-7 tran- scription in HK-2 cells was present, but copy number was found to be low. Despite the low mRNA level, BMP-7 was significantly decreased after stimulation with AT-II, which could explain the lower BMP-7 expression in the kidneys of patients with hypertensive nephro- sclerosis. The effect may be evident from the decreased expression of the pSmads 1/5/8 after stimulation with angiotensin-II. However, the Smad complex 1/5/8 is also activated by pro-fibrotic BMPs (e.g., BMP-2, -4) and the exact regulation is due to several confounding factor [24,25]. BMP-7 and the role of the renin-angiotensin system To further test our hypothesis of BMP-7 involvement in the development of human hypertensive nephrosclerosis, we performed tissue culture experiments. In preliminary experiments, we were able to detect BMP-7 mRNA in primary isolated human distal tubule cells [23], but the number of cells were limited; moreover, we were not able to cultivate these cells long enough to perform stimulation experiments. Due to the unavailability of human distal tubule cell lines, we used the human prox- imal cell line HK-2, although results on BMP-7 mRNA transcription in proximal tubular epithelial cells are con- troversial. Whereas Wetzel et al. failed to show any Increased BMP-7 transcription after co-stimulation with AT-II and the angiotensin-II receptor antagonist telmisartan may, in part, explain the protective effects Page 12 of 14 Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Figure 10 BMP-7 decreases expression of TGF-b receptor type I. Expression of TGF-b receptor type I after stimulation with BMP-7 (1, 10, 100 ng/ml) detected by double immunofluorescence: quantification of cell staining intensity was given in grey values by the software AnalySIS. Expression rate is changed compared to unstimulated cells, concomitant with reduced staining intensity (100 ng/ml: p < 0.01). Original magnification: × 1000. Results were analysed by Student’s t-test and are displayed as the % of unstimulated cells in the mean ± standard error. Figure 10 BMP-7 decreases expression of TGF-b receptor type I. Expression of TGF-b receptor type I after stimulation with BMP-7 (1, 10, 100 ng/ml) detected by double immunofluorescence: quantification of cell staining intensity was given in grey values by the software AnalySIS. Expression rate is changed compared to unstimulated cells, concomitant with reduced staining intensity (100 ng/ml: p < 0.01). Original magnification: × 1000. Results were analysed by Student’s t-test and are displayed as the % of unstimulated cells in the mean ± standard error. of these drugs in kidneys. However, significantly increased BMP-7 mRNA level was seen after stimula- tion with telmisartan alone, revealing an AT-II inde- pendent effect of telmisartan on BMP-7 expression. These findings are in line with former studies, although most authors have focused primarily on the telmisartan-induced decrease of inflammatory cyto- kines and less on protective growth factors. BMP-7 and the role of the renin-angiotensin system Hence, AT-II independent effects of telmisartan were described to decrease TNF-a induced expression of interleukin-6 in vascular smooth muscle cells [26], as well as the expression of monocyte chemoattractant protein 1 (MCP-1, CCL-2) and its receptor CCR2 in peripheral blood monocytes [27]. primary and immortalised human proximal tubule cells [11]. Our functional data showed that BMP-7 was able to reverse the TGF-b-induced EMT in human proximal tubule cells by using high concentrations of recombinant BMP-7 (100 ng/ml). One possible mode of action can be the decreased expression of the TGF-b receptor type I, which may also contribute to the reversal of EMT by BMP-7. Thereby, the attenuated TGF-b signalling may be caused by the decreased expression level as well as by the decreased receptor expression on the cell surface. This hypothesis may be in line with previous findings suggest- ing that TGF-b R1 is internalised in clathrin-coated vesi- cles and that the process of being expressed on the cell surface plays an important regulatory role in TGF-b sig- nalling [29]. The functional aspect may be demonstrated by the decreased expression of phosphorylated Smad 2 after stimulation with TGF-b and BMP-7 compared to TGF-b alone. BMP-7 effect on EMT Thus far, the data on the reversibility of EMT by BMP-7 in human kidneys are controversial. Although research by Xu [9] and Veerasamy [28] showed that BMP-7 attenuated TGF-b-induced EMT in human proximal tubule cells (HK-2), Dudas was not able to confirm these results in In contrast to Dudas [11], Xu et al. and our working group used concentrations as high as 100 ng/ml BMP- 7 to attenuate TGF-b-induced EMT in HK-2 cells [9]. Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Page 13 of 14 Figure 11 BMP-7 deceases TNF-a induced apoptosis. Induction of apoptosis (A) and cell death (B) in proximal tubular cells (HK-2) after stimulation with 20 ng/ml TNF-a alone and/or in combination with BMP-7 (1, 10, 100 ng/ml). Apoptotic cells were detected by FACS analysis counting annexin-V positive and propidium iodide negative cells (n = 3). Cell death was detected after staining with annexin-V (n = 3). TNF-a alone induced high rates of apoptosis and cell death. Co-stimulation with TNF-a and BMP-7 resulted in significant dose-dependent lower rates of apoptosis and cell death compared to TNF-a alone (DIF: p < 0.05, FACS: p < 0.05). The results were analysed by Student’s t-test and are displayed as the % of unstimulated cells. Figu stimu coun alone of ap displ Figure 11 BMP-7 deceases TNF-a induced apoptosis. Induction of apoptosis (A) and cell death (B) in proximal tubular cells (HK-2) after stimulation with 20 ng/ml TNF-a alone and/or in combination with BMP-7 (1, 10, 100 ng/ml). Apoptotic cells were detected by FACS analysis counting annexin-V positive and propidium iodide negative cells (n = 3). Cell death was detected after staining with annexin-V (n = 3). TNF-a alone induced high rates of apoptosis and cell death. Co-stimulation with TNF-a and BMP-7 resulted in significant dose-dependent lower rates of apoptosis and cell death compared to TNF-a alone (DIF: p < 0.05, FACS: p < 0.05). The results were analysed by Student’s t-test and are displayed as the % of unstimulated cells. This high dose of BMP-7 may have inhibited the sys- temic administration as a therapeutic option for chronic kidney disease. However, of course, in vitro studies in cell lines are not necessarily transferable to in vivo studies in organisms. the reduced expression of BMP-7 in patients with hypertensive nephrosclerosis may imply a loss of protec- tion and regenerative potential necessary to counter the disease. BMP-7 effect on EMT The decrease may be in part induced by angio- tensin-II and attenuated by telmisartan. Acknowledgements Th h h k Ch In addition, data from our study revealed that BMP-7 counteracts TNF-a-induced apoptosis in human proximal tubule cells. These results in human cells are in accor- dance with those reported by Vukicevic and co-workers, who demonstrated decreases in apoptotic cells in BMP-7 treated subjects in rodents with acute renal failure [3]. However, again, high concentrations of BMP-7 were necessary to achieve significant changes. The authors thank Christina Lauterberg, Swantje Wehn, Gabi Wolf and Sonja Wook for excellent technical assistance. This work was supported by a grant from Boehringer Ingelheim Pharma GmbH & Co. KG to CB, by P30 DK081943-01 (Applied System Biology Core) to MK and Else Kröner-Fresenius Foundation to CDC. We thank all participating centers of the European Renal cDNA Bank-Kroener-Fresenius biopsy bank (ERCB-KFB) and their patients for their cooperation. Active members at the time of the study: C. D. Cohen, H. Schmid, M. Fischereder, L. Weber, M. Kretzler, D. Schlöndorff, Munich/Zurich/AnnArbor/New York; J. D. Sraer, P. Ronco, Paris; M. P. Rastaldi, G. D’Amico, Milano; P. Doran, H. Brady, Dublin; D. Mönks, C. Wanner, Würzburg; A. J. Rees, Aberdeen; F. Strutz, G. A. Müller, Göttingen; P. Mertens, J. Floege, Aachen; N. Braun, T. Risler, Tübingen; L. Gesualdo, F. P. Schena, Bari; J. Gerth, G. Wolf, Jena; R. Oberbauer, D. Kerjaschki, Vienna; B. Banas, B. K. Krämer, Regensburg; M. Saleem, Bristol; R. P. Wüthrich, Zurich; W. Samtleben, Munich; H. Peters, H. H. Neumayer, Berlin; M. Daha, Leiden; K Ivens, B. Grabensee, Düsseldorf; F. Mampaso(†), Madrid; J. Oh, F. Schaefer, M. Zeier, H.- J. Gröne, Heidelberg; P. Gross, Dresden; G. Tonolo; Sassari; V. Tesar, Prague; H. Rupprecht, Bayreuth; H.-P. Marti, Bern. The authors thank Christina Lauterberg, Swantje Wehn, Gabi Wolf and Sonja Wook for excellent technical assistance. This work was supported by a grant from Boehringer Ingelheim Pharma GmbH & Co. KG to CB, by P30 DK081943-01 (Applied System Biology Core) to MK and Else Kröner-Fresenius Foundation to CDC. We thank all participating centers of the European Renal cDNA Bank-Kroener-Fresenius biopsy bank (ERCB-KFB) and their patients for their cooperation. Active members at the time of the study: C. D. Cohen, H. Schmid, M. Fischereder, L. Weber, M. Kretzler, D. Schlöndorff, Munich/Zurich/AnnArbor/New York; J. D. Sraer, P. Ronco, Paris; M. P. Rastaldi, G. D’Amico, Milano; P. Doran, H. Brady, Dublin; D. Mönks, C. Wanner, Würzburg; A. J. Rees, Aberdeen; F. Strutz, G. A. Müller, Göttingen; P. Mertens, J. Floege, Aachen; N. Braun, T. Risler, Tübingen; L. Gesualdo, F. P. Schena, Bari; J. Gerth, G. Wolf, Jena; R. Oberbauer, D. Kerjaschki, Vienna; B. Banas, B. K. Krämer, Regensburg; M. Saleem, Bristol; R. P. Wüthrich, Zurich; W. Samtleben, Munich; H. Peters, H. H. Neumayer, Berlin; M. Daha, Leiden; K Ivens, B. Grabensee, Düsseldorf; F. Mampaso(†), Madrid; J. Oh, F. Schaefer, M. Zeier, H.- J. Gröne, Heidelberg; P. Gross, Dresden; G. Tonolo; Sassari; V. Tesar, Prague; H. Rupprecht, Bayreuth; H.-P. Marti, Bern. References Dai Q, Xu M, Yao M, Sun B: Angiotensin AT1 receptor antagonists exert anti-inflammatory effects in spontaneously hypertensive rats. Br J Pharmacol 2007, 152(7):1042-1048. 6. Kaps C, Bramlage C, Smolian H, Haisch A, Ungethum U, Burmester GR, Sittinger M, Gross G, Haupl T: Bone morphogenetic proteins promote cartilage differentiation and protect engineered artificial cartilage from fibroblast invasion and destruction. Arthritis Rheum 2002, 46(1):149-162. 6. Kaps C, Bramlage C, Smolian H, Haisch A, Ungethum U, Burmester GR, Sittinger M, Gross G, Haupl T: Bone morphogenetic proteins promote cartilage differentiation and protect engineered artificial cartilage from fibroblast invasion and destruction. Arthritis Rheum 2002, 46(1):149-162. 28. Veerasamy M, Nguyen TQ, Motazed R, Pearson AL, Goldschmeding R, Dockrell ME: Differential regulation of E-cadherin and alpha-smooth muscle actin by BMP 7 in human renal proximal tubule epithelial cells and its implication in renal fibrosis. Am J Physiol Renal Physiol 2009, 297(5):F1238-1248. 7. Gould SE, Day M, Jones SS, Dorai H: BMP-7 regulates chemokine, cytokine, and hemodynamic gene expression in proximal tubule cells. Kidney Int 2002, 61(1):51-60. 7. Gould SE, Day M, Jones SS, Dorai H: BMP-7 regulates chemokine, cytokine, and hemodynamic gene expression in proximal tubule cells. Kidney Int 2002, 61(1):51-60. 29. Chen Y: Endocytic regulation of TGF-beta signaling. Cell Research 2009, 19(1):58-70. 29. Chen Y: Endocytic regulation of TGF-beta signaling. Cell Research 2009, 19(1):58-70. 8. Wetzel P, Haag J, Campean V, Goldschmeding R, Atalla A, Amann K, Aigner T: Bone morphogenetic protein-7 expression and activity in the human adult normal kidney is predominantly localized to the distal nephron. Kidney international 2006, 70(4):717-723. Author details 1 14. Cohen CD, Frach K, Schlondorff D, Kretzler M: Quantitative gene expression analysis in renal biopsies: a novel protocol for a high- throughput multicenter application. Kidney Int 2002, 61(1):133-140. 1Department of Medicine, Nephrology and Rheumatology, Georg-August- University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany. 2Institute for Cardiovascular Pharmacology and Epidemiology, Menzelstrasse 21, 15831 Mahlow, Germany. 3Department of Medical Statistics, Georg- August-University Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany. 4Department of Nephrology, Medical Policlinic, Ludwig Maximilians University Munich, Pettenkoferstr. 8a, D - 80336 Munich, Germany. 5Division of Nephrology and Institute of Physiology, University Zürich, Rämistr. 100, 8091 Zürich, Switzerland. 6Internal Medicine - Nephrology University of Michigan 1150 W Medical Centre Ann Arbor 15. Ryan MJ, Johnson G, Kirk J, Fuerstenberg SM, Zager RA, Torok-Storb B: HK- 2: an immortalized proximal tubule epithelial cell line from normal adult human kidney. Kidney Int 1994, 45(1):48-57. 16. Koziolek MJ, Schmid H, Cohen CD, Blaschke S, Hemmerlein B, Zapf A, Muller GA, Strutz F: Potential role of fractalkine receptor expression in human renal fibrogenesis. Kidney Int 2007, 72(5):599-607. 17. Koziolek MJ, Riess R, Geiger H, Thevenod F, Hauser IA: Expression of multidrug resistance P-glycoprotein in kidney allografts from cyclosporine A-treated patients. Kidney Int 2001, 60(1):156-166. Nephrology, University of Michigan, 1150 W. Medical Centre, Ann Arbor, USA. 18. Cui X, Zhou J, Qiu J, Johnson MR, Mrug M: Validation of endogenous internal real-time PCR controls in renal tissues. Am J Nephrol 2009, 30(5):413-417. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. 21. Dube PH, Almanzar MM, Frazier KS, Jones WK, Charette MF, Paredes A: Osteogenic Protein-1: gene expression and treatment in rat remnant kidney model. Toxicol Pathol 2004, 32(4):384-392. Received: 1 August 2010 Accepted: 16 November 2010 Published: 16 November 2010 Received: 1 August 2010 Accepted: 16 November 2010 Published: 16 November 2010 22. Wang SN, Lapage J, Hirschberg R: Loss of tubular bone morphogenetic protein-7 in diabetic nephropathy. J Am Soc Nephrol 2001, 12(11):2392-2399. Conclusions Our findings suggest a protective role of BMP-7 by counteracting TGF-b and TNF-a negative effects in vitro, although high concentrations of BMP-7 are neces- sary. In agreement with the findings in animal studies, Page 14 of 14 Bramlage et al. BMC Nephrology 2010, 11:31 http://www.biomedcentral.com/1471-2369/11/31 Authors’ contributions CPB and GAM outlined the study design; MK, MK, KL, HS and CDC gave important input into the study design; CPB, BT, JB and IM were responsible for conducting the experiments; CPB, PB, and KA did the statistical evaluations and drafted the manuscript. All authors revised the manuscript for important intellectual content and approved the final manuscript. 19. Heeg MH, Koziolek MJ, Vasko R, Schaefer L, Sharma K, Muller GA, Strutz F: The antifibrotic effects of relaxin in human renal fibroblasts are mediated in part by inhibition of the Smad2 pathway. Kidney Int 2005, 68(1):96-109. for conducting the experiments; CPB, PB, and KA did the statistical evaluations and drafted the manuscript. All authors revised the manuscript for important intellectual content and approved the final manuscript. 20. Biyikli NK, Tugtepe H, Cakalagaoglu F, Ilki A, Alpay H: Downregulation of the expression of bone morphogenetic protein 7 in experimental pyelonephritis. Pediatr Nephrol 2005, 20(9):1230-1236. References 1. Bramlage CP, Haupl T, Kaps C, Bramlage P, Muller GA, Strutz F: [Bone morphogenetic proteins in the skeletal system]. Z Rheumatol 2005, 64(6):416-422. 23. Baer PC, Nockher WA, Haase W, Scherberich JE: Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note. Kidney Int 1997, 52(5):1321-1331. 2. Mitu G, Hirschberg R: Bone morphogenetic protein-7 (BMP7) in chronic kidney disease. Front Biosci 2008, 13:4726-4739. 2. Mitu G, Hirschberg R: Bone morphogenetic protein-7 (BMP7) in chronic kidney disease. Front Biosci 2008, 13:4726-4739. 24. Roxburgh SA, Kattla JJ, Curran SP, O’Meara YM, Pollock CA, Goldschmeding R, Godson C, Martin F, Brazil DP: Allelic depletion of grem1 attenuates diabetic kidney disease. Diabetes 2009, 58(7):1641-1650. 3. Vukicevic S, Basic V, Rogic D, Basic N, Shih MS, Shepard A, Jin D, Dattatreyamurty B, Jones W, Dorai H, et al: Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat. J Clin Invest 1998, 102(1):202-214. 3. Vukicevic S, Basic V, Rogic D, Basic N, Shih MS, Shepard A, Jin D, Dattatreyamurty B, Jones W, Dorai H, et al: Osteogenic protein-1 (bone morphogenetic protein-7) reduces severity of injury after ischemic acute renal failure in rat. J Clin Invest 1998, 102(1):202-214. 25. Oxburgh L: Control of the bone morphogenetic protein 7 gene in developmental and adult life. Curr Genomics 2009, 10(4):223-230. 26. Tian Q, Miyazaki R, Ichiki T, Imayama I, Inanaga K, Ohtsubo H, Yano K, Takeda K, Sunagawa K: Inhibition of tumor necrosis factor-alpha-induced interleukin-6 expression by telmisartan through cross-talk of peroxisome proliferator-activated receptor-gamma with nuclear factor kappaB and CCAAT/enhancer-binding protein-beta. Hypertension 2009, 53(5):798-804. 4. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, Kalluri R: BMP-7 counteracts TGF-beta1-induced epithelial-to- mesenchymal transition and reverses chronic renal injury. Nat Med 2003, 9(7):964-968. 4. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F, Kalluri R: BMP-7 counteracts TGF-beta1-induced epithelial-to- mesenchymal transition and reverses chronic renal injury. Nat Med 2003, 9(7):964-968. 5. Yanagita M: Modulator of bone morphogenetic protein activity in the progression of kidney diseases. Kidney Int 2006, 70(6):989-993. 5. Yanagita M: Modulator of bone morphogenetic protein activity in the progression of kidney diseases. Kidney Int 2006, 70(6):989-993. 27. Dai Q, Xu M, Yao M, Sun B: Angiotensin AT1 receptor antagonists exert anti-inflammatory effects in spontaneously hypertensive rats. Br J Pharmacol 2007, 152(7):1042-1048. 27. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2369/11/31/prepub The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2369/11/31/prepub 9. Xu Y, Wan J, Jiang D, Wu X: BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition in human renal proximal tubular epithelial cells. J Nephrol 2009, 22(3):403-410. doi:10.1186/1471-2369-11-31 Cite this article as: Bramlage et al.: Bone Morphogenetic Protein (BMP)-7 expression is decreased in human hypertensive nephrosclerosis. BMC Nephrology 2010 11:31. doi:10.1186/1471-2369-11-31 Cite this article as: Bramlage et al.: Bone Morphogenetic Protein (BMP)-7 expression is decreased in human hypertensive nephrosclerosis. BMC Nephrology 2010 11:31. 10. Rudnicki M, Eder S, Perco P, Enrich J, Scheiber K, Koppelstatter C, Schratzberger G, Mayer B, Oberbauer R, Meyer TW, et al: Gene expression profiles of human proximal tubular epithelial cells in proteinuric nephropathies. Kidney Int 2007, 71(4):325-335. 11. Dudas PL, Argentieri RL, Farrell FX: BMP-7 fails to attenuate TGF-{beta}1- induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells. Nephrol Dial Transplant 2008. 11. Dudas PL, Argentieri RL, Farrell FX: BMP-7 fails to attenuate TGF-{beta}1- induced epithelial-to-mesenchymal transition in human proximal tubule epithelial cells. Nephrol Dial Transplant 2008. 12. Islam TM FC, Mann D, Munter P: Age-related associations of hypertension and diabetes mellitus with chronic kidney disease. BMC Nephrology 2009, 10:17. 12. Islam TM FC, Mann D, Munter P: Age-related associations of hypertension and diabetes mellitus with chronic kidney disease. BMC Nephrology 2009, 10:17. 13. Davies MR, Lund RJ, Hruska KA: BMP-7 is an efficacious treatment of vascular calcification in a murine model of atherosclerosis and chronic renal failure. J Am Soc Nephrol 2003, 14(6):1559-1567.
https://openalex.org/W1823312867	https://journals.library.ualberta.ca/csp/index.php/csp/article/download/18984/14707	Latin	null	Reviewers for Volume 39	Canadian studies in population	2,013	cc-by	373	“Historical Studies on Mortality,” special issue, Canadian Studies in Population 39, no. 3–4 (Fall/Winter 2012) “Historical Studies on Mortality,” special issue, Canadian Studies in Population 39, no. 3–4 (Fall/Winter 2012) Reviewers for Volume 39 146 Nurul Alam, International Centre for Diarrhoeal Disease Research, Dhaka (Bangladesh) Kirill F. Andreev, United Nations Department of Economic and Social Affairs, New York (US Asaduzzaman, University of Dhaka (Bangladesh) Roderic Beaujot, University of Western Ontario, London (Canada) Alain Bélanger, INRS - Urbanisation, culture et société, Montreal (Canada) Kwame Boadu, Government of Alberta, Edmonton (Canada) K. Steve Brown, University of Waterloo (Canada) Thomas K. Burch, University of Victoria (Canada) Marie-Soleil Cloutier, INRS - Urbanisation, culture et société, Montreal (Canada) Anton de Craen, Leiden University Medical Center (Netherlands) Frank Denton, McMaster University, Hamilton (Canada) Bertrand Desjardins, Université de Montréal (Canada) Sven Drefahl, Stockholm University (Sweden) Rajulton Fernando, University of Western Ontario, London (Canada) Maxime Fougère, Human Resources and Skills Development Canada, Ottawa (Canada) Danielle Gauvreau, Concordia University, Montreal (Canada) Hani Guend, INRS - Urbanisation, culture et société, Montreal (Canada) Susanne Huber, University of Veterinary Medicine, Vienna (Austria) M. Mazharul Islam, Sultan Qaboos University, Muscat (Oman) Charles Jones, University of Toronto (Canada) Don Kerr, University of Western Ontario, London (Canada) Anastasia Kostaki, Athens University of Economics and Business (Greece) Parameswara Krishnan, University of Alberta, Edmonton (Canada) Tracey LaPierre, University of Kansas, Lawrence (USA) Évelyne Lapierre-Adamcyk, Université de Montréal (Canada) Katherine Marshall, Statistics Canada, Ottawa (Canada) Ryan Mazan, University of Western Ontario, London (Canada) Michael E. Mercier, McMaster University, Hamilton (Canada) Andrew Noymer, University of California, Irvine (USA) Margaret Penning, University of Victoria (Canada) Victor Piché, Université de Montréal (Canada) Amélie Quesnel-Vallée, McGill University, Montreal (Canada) K.V. Rao, India Network Foundation (USA) James C. Raymondo, Tennessee Technological University, Cookeville (USA) Arzu Sardarli, First Nations University of Canada, Regina (Canada) Reto Schumacher, University of Geneva (Switzerland) Steven J. Stack, Wayne State University, Detroit (USA) Zongli Tang, Auburn University at Montgomery (USA) Marc Tremblay, Université du Québec à Chicoutimi (Canada) Bart Van de Putte, University of Ghent (Belgium) Andrew Wister, Simon Fraser University, Vancouver (Canada) Xuelin Zhang, Statistics Canada, Ottawa (Canada) K.V. Rao, India Network Foundation (USA) 146 “Historical Studies on Mortality,” special issue, Canadian Studies in Population 39, no. 3–4 (Fall/Winter 2012) 147
https://openalex.org/W3033813856	https://equityhealthj.biomedcentral.com/track/pdf/10.1186/s12939-020-01212-5	English	null	Extent of and trends in inequalities in child stunting in Sierra-Leone from 2005 to 2013: evidence from demographic and health surveys and multiple indicator cluster surveys	International journal for equity in health	2,020	cc-by	9,304	(2020) 19:88 (2020) 19:88 Shibre et al. International Journal for Equity in Health (2020) 19:88 https://doi.org/10.1186/s12939-020-01212-5 Shibre et al. International Journal for Equity in Health https://doi.org/10.1186/s12939-020-01212-5 RESEARCH Open Access Extent of and trends in inequalities in child stunting in Sierra-Leone from 2005 to 2013: evidence from demographic and health surveys and multiple indicator cluster surveys Gebretsadik Shibre1* , Betregiorigis Zegeye2 and Jemal Haidar3 Gebretsadik Shibre1* , Betregiorigis Zegeye2 and Jemal Haidar3 Correspondence: gebretshh@gmail.com 1Department of Reproductive, Family and Population Health, School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia Full list of author information is available at the end of the article Abstract Background: Comprehensive assessment of stunting disparity in Sierra-Leone has not been done so far. We aimed to document extent and over time dynamics of inequality in stunting in Sierra-Leone using approaches that facilitate implementation of interventions aim to eliminate non-justified stunting disparity in the country. Methods: The data for the study were derived from two rounds of the Sierra Leone Demographic and Health Survey conducted in 2008 and 2013, and two rounds of the Sierra Leone Multiple Indicator Cluster Survey done in 2005 and 2010. We used the 2019 update WHO Health Equity Assessment Toolkit (HEAT) to do the analysis. The toolkit makes use of data stored in the WHO Health Equity Monitor database. We analyzed stunting inequality using summary measures: Population Attributable Risk, Population Attributable Fraction, Difference and Ratio. The summary measures were computed for five equity stratifers: wealth, education, child’s sex, place of residence and subnational region. We computed 95% Confidence Interval (CI) for each point estimate to show whether or not observed stunting inequalities are statistically significant, and whether or not the disparity changed over time statistically significantly. Results: The findings demonstrated stark inequalities in stunting in all the studied dimensions of inequality. While residence and subnational regional related inequalities remain unchanged over time, wealth and educational inequality had seen slight improvement during the same time period. Large sex related stunting inequality remained during the first three surveys time points, but it disappeared in 2013. Conclusions: Huge stunting disparities occurred in Sierra Leone, and the disparity disproportionately affects disadvantaged subpopulations and male children. Nutrition interventions that specifically target the subgroups which suffer more from the burden of stunting are required. Keywords: Stunting, Inequality, Sierra Leone, DHS, MICS Background risk of stunting [12–16]. Moreover, it has been shown that male children are at a higher risk of stunting than their female counterparts [17, 18]. Stunting is a form of childhood undernutrition used to refer to population of children who are too short for their age and is an unambiguous sign of not developing well physically and mentally inside the first 1000 days [1]. Not only is stunting labeled as the “best overall indi- cator” of children’s well-being, but it is recognized as an “accurate reflection” of disparities among populations [2]. Stunting is also known to be “both a symptom of past deprivation and a predictor of future poverty” [3]. p Measuring extent of disparities around a health care indicator is a decisive first step to locate where interven- tion is required, and design how health-related policies and strategies are implemented properly to help narrow the gap by addressing people who are left behind. Such endeavors could be achieved through analysis of data disaggregated by population subcategories into how nu- merous aspects of health are experienced in the entire population. The call for “data disaggregation” under the Sustainable Development Goal [19] has led to the prolif- eration of inequality studies on health care service globally. However, there is no in-depth evaluation of stunting inequality in Sierra Leone so far. We located few studies done on stunting in the country [20–25]. However, our study substantially updates the existing knowledge in many aspects. First, we looked at the over time dynamics of stunting disparity between 2005 and 2013 with respect to five different equity stratifiers. This would help policy makers and planners to learn the government’s past endeavors on stunting and to reframe future stunting interventions to end the disparity by focusing on those subpopulations suffering higher burden of stunting. Second, the analysis was performed using the high-quality WHO Health Equity Monitor (HEM) data. Finally, following the WHO recommenda- tion for equity studies, we carried out the inequality analysis using simple and complex, as well as absolute and relative measures of inequality. This approach is important not to miss out disparities when they actually exist and also to capture disparities from different perspectives [26]. The study is specifically designed to document whether or not stunting inequality exist in Sierra Leone with respect to different dimensions of in- equality with different inequality measures, and whether or not the disparity changed over time. Background Child stunting is associated with numerous attributes such as poor socioeconomic status, insufficient nutri- tional intake, infectious diseases, and other environment factors [4, 5]. The lasting damage of stunting is not just confined to those who are stunted, but it also has serious consequences on the socioeconomic development of a nation [1]. Finally, stunting is associated with poor school performance and subsequently, leads to income inequality, high fertility, poor caring for children and to perpetuation of poverty across generations [6, 7]. World- wide, 21.3% of under 5 children suffered from stunting in 2019 [8]. South Asia and Sub-Saharan Africa are the hardest hit places in the world, where one in three under five children is stunted [1]. In Africa, two in every five under five children are stunted, with large variations across the five sub-regions; it varied between 2% in Southern Africa to 23.1% in Eastern Africa [8]. Further- more, not only is Africa home to a significant proportion of stunted children, but it is the only region that saw increased number of stunted children between 2000 and 2018 [1]. Like the situation in SSA, Sierra Leone experi- ences nutritional problems, and many people suffer from food insufficiency. Evidence shows that more than 3 million people in Sierra Leone are estimated to be deprived of access to food and among them, 170, 000 are estimated to be severely food insecure. Chronic malnutrition is a widespread problem in the country, with nearly one in three children suffers from stunting, and Sierra Leone is under the WHO “high” threshold category for stunting burden [9, 10]. © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Shibre et al. International Journal for Equity in Health (2020) 19:88 Page 2 of 10 Page 2 of 10 Shibre et al. International Journal for Equity in Health (2020) 19:88 Methods Study area It is worth noting at this junction that decline in na- tional stunting prevalence is not necessarily associated with absence of within country stunting inequality across different subpopulation. Past studies observed the possi- bility for stunting disparity to still prevail in that country even when its overall prevalence is low [11]. This evi- dence points to the practical contributions of stunting inequality studies to the combat against its burden both nationally and worldwide. To this end, many studies have been conducted to determine whether within coun- try variations exist in the prevalence of stunting between different subgroups. Children born to mothers who are economically worse-off, uneducated, residing in rural areas and in certain geographic locations are at elevated y Sierra Leone has a total population of 7,092,113 with slightly more females than male population (3,601,135 females and 3,490,178 males, and nearly 6 in 10 people live in rural area [27]. The average annual population growth rate was 3.2% between 2004 and 2015 with a total fertility rate (TFR) of 5.2 children per woman. Sierra Leone has a young population with nearly 41% of the population being 15 years old or under [27]. Economy grew at 3.5% in 2018, and poverty is a widespread problem in the country, with more than half of the population living in the poverty zone. Until Ebola outbreak occurred in 2014, Sierra Leone was working to move into middle-income country by 2035, but problems of high youth unemployment, corruption and Page 3 of 10 Shibre et al. International Journal for Equity in Health (2020) 19:88 Shibre et al. International Journal for Equity in Health area. Populations living in collective housing units such as hotels, hospitals, work camps, prisons, or boarding schools were excluded. The MICS follows a sampling strategy similar to that of DHS [24, 25]. weak governance continue to pose greatest impediments to the country’s development [28]. The 2019 Global Hunger Index (GHI) report showed that Sierra Leone ranked 103rd with a score of 30.4, suggesting a serious levels of hunger in the country [29]. Data In addition, valid height and weight data were taken from 2770 and 5090 children under five in 2008 and 2013 SDHS respectively. The SLDHS sample is designed to produce reliable estimates for important indicators for the entire country, as well as for urban and rural areas. Sample allocation and participant selection in the SLDHSs have been described in detail in the ‘appendix’ section of the SDHSs pdf final reports [22, 23]. Briefly, the samples were selected through stratified cluster sampling tech- nique that happened in two stages. Each district in the country was first stratified in to urban and rural areas. Then samples were drawn independently from each stratum in two stages. In the first stage, enumeration areas (also called clusters or primary sampling units) were selected through probability proportional to size technique. Information about the enumeration areas was taken from the 2004 Sierra Leone Population and Housing Census (PHC) and the 2004 PHC provided sampling frame for the first stage. In the second stage, a fixed num- ber of households were drawn from each enumeration In 2005 and 2010 Sierra Leone MICS, 7654 and 13,416 women age 15 to 49 were interviewed respectively, and information was gathered from mothers or care givers of 5246 and 8619 under five children respectively. Similarly, in 2008 and 2013 SDHS, 7422 and 16,658 females age 15 to 49 respectively were interviewed. In addition, valid height and weight data were taken from 2770 and 5090 children under five in 2008 and 2013 SDHS respectively. Data We measured inequality for child stunting among children who were born 5 years prior to the respective household surveys. Stunting is defined in standard deviation units (z-scores) from the median of the refer- ence population. Children whose height-for-age-z score fall below minus 2 SD from the median of the WHO reference population are either moderately or severely stunted, and this definition is used in this study. We disaggregated stunting prevalence using five dimensions of inequality represented in the WHO Health Equity Monitor [32] which included wealth, education, place of residence, subnational region and child’s sex. Education is measured as three subgroups (no education, primary school and secondary school and higher). Wealth is classified into five categories: poorest, poor, middle, rich and richest. Wealth index has been used as a proxy for economic status in house- hold surveys and is computed based on household assets and possessions using a statistical technique called Principal Component Analysis [33]. Residence is a binary variable and is measured as urban or rural and sex as male vs. female. Subnational region is mea- sured as North, South, East and West. We used two rounds of the Sierra-Leone Demographic and Health Survey (SLDHS) data conducted in 2008, and 2013, and two rounds of the Sierra-Leone Multiple Indicator Cluster Survey (MICS) carried out in 2005 and 2010. The DHS and MICS are the major sources of in- formation for monitoring key demographic and public health indicators and are highly comparable household surveys that permit direct comparison between them [30, 31]. They provide data on different background characteristics of respondents of a survey and health indicators including indicators of maternal, newborn, and child health in low-and-middle-income countries. The surveys cover different topics which include inter alia child mortality; maternal mortality; maternal health care services; child immunization coverage; childhood fever, acute respiratory infections, and diarrhea; HIV (knowledge of transmission and prevention, prior test- ing, stigma, and discrimination); and biomarkers (height and weight). Women age 15 to 49 and men age 15 to 59 were eligible for the surveys, and information was also gathered for under five children. g In 2005 and 2010 Sierra Leone MICS, 7654 and 13,416 women age 15 to 49 were interviewed respectively, and information was gathered from mothers or care givers of 5246 and 8619 under five children respectively. Similarly, in 2008 and 2013 SDHS, 7422 and 16,658 females age 15 to 49 respectively were interviewed. Statistical analysis d The larger the absolute value of PAF and PAR, the larger the degree of inequality. They become zero if no further improvement can be achieved. On the other hand, D and R are simple measures of inequality that show gap between just two subgroups of an equity strati- fier. If there is no inequality, D takes the value of zero and R becomes one. The further the value of D and R from zero and 1, respectively, the higher the level of inequality becomes. μ D is calculated as the difference between two subgroups: D = yhigh - ylow. For binary dimensions (residence and sex for our study), yhigh refers to the subgroup with the highest estimate (rural and male) and ylow to the subgroup with the lowest estimate (urban and female). For ordered dimen- sions, yhigh refers to the most-disadvantaged subgroup (poorest and illiterate subgroups) and ylow to the most- advantaged subgroup (richest and secondary education subgroups). For subnational region, yhigh refers to the sub- group with the highest estimate and ylow to the subgroup with the lowest estimate. The calculation of R parallels that of D except that we divide estimate in one category by an- other category. See Table 1 in the result section to identify subgroups with highest and lowest estimates of stunting. While D and PAR are absolute measures, PAF and R are relative measures of inequality. Absolute measures show the absolute difference or gap in health or health care indicators between subgroups considered and retain the original units of the health indicators studied. Rela- tive measures, on the other hand, measures disparities in relative terms. Complex measures are able to account for sizes of all subgroups of an equity stratifer, not just two groups. This feature of complex measures of inequality makes them robust choice especially in the event of population shift. However, simple measures do not possess this property. While complex measures have the drawbacks of somehow challenging interpretation and calculation, The PAR and PAF are complex measures of inequality that show potential for improvement in the national level of a health indicator if all sub-groups are perform- ing equally with the best performing sub-group. Statistical analysis d We assessed stunting inequalities following recommen- dation on health equity study of the WHO. Prevalence of stunting was first disaggregated by the five equity stratifiers discussed above. Then, the inequality was fur- ther assessed using four inequality measures: Difference (D), Population Attributable risk (PAR), Ratio (R) and Population Attributable Fraction (PAF). p y The SLDHS sample is designed to produce reliable estimates for important indicators for the entire country, as well as for urban and rural areas. Sample allocation and participant selection in the SLDHSs have been described in detail in the ‘appendix’ section of the SDHSs pdf final reports [22, 23]. Briefly, the samples were selected through stratified cluster sampling tech- nique that happened in two stages. Each district in the country was first stratified in to urban and rural areas. Then samples were drawn independently from each stratum in two stages. In the first stage, enumeration areas (also called clusters or primary sampling units) were selected through probability proportional to size technique. Information about the enumeration areas was taken from the 2004 Sierra Leone Population and Housing Census (PHC) and the 2004 PHC provided sampling frame for the first stage. In the second stage, a fixed num- ber of households were drawn from each enumeration p Calculations for each summary measure have been de- tailed elsewhere [26, 34]. Briefly, PAR is calculated as the difference between the estimate for the reference subgroup yref and the national average μ: PAR = yref - μ. The selection of the reference subgroup yref depends on the type of health indicator and on the characteristics of the dimension of inequality, for which PAR is calculated. The health care indicator in this study is stunting and is unfavorable. Thus, for residence and sex dimensions, yref refers to the subgroups with the lowest estimate, which are respectively urban and female subcategories. For ordered dimensions (wealth and education in this study), yref refers to the most-advantaged subgroup (wealthiest and secondary education groups). For subnational region, yref refers to the subgroups with the lowest estimate which are different in the four rounds of the household surveys. Shibre et al. International Journal for Equity in Health (2020) 19:88 Page 4 of 10 Page 4 of 10 PAF is calculated by dividing the population attributable risk (PAR) by the national average μ and multiplying the fraction by 100: PAF = [PAR / μ] * 100. stunting. Notes: popn Population share of each subgroup, CI Confidence Interval Statistical analysis d They take positive values for favorable health intervention indicators and negative for adverse health indicators like Table 1 Prevalence of childhood stunting across different dimensions of inequality in Sierra-Leone from 2005 to 2013 Dimensions of inequality Year 2005 2008 2010 2013 %(95% CI) Popn %(95% CI) Popn %(95% CI) Popn %(95% CI) Popn Wealth Poorest 49.95 (46.40, 53.50) 1034 36.45 (31.88, 41.27) 604 46.84 (43.65, 50.06) 1725 42.63 (39.08, 46.26) 1182 Poor 52.53 (49.20, 55.84) 1150 43.59 (38.57, 48.76) 580 48.91 (45.73, 52.10) 1743 40.41 (36.52, 44.43) 1196 Middle 48.57 (45.15, 51.99) 1079 37.71 (32.54, 43.17) 615 47.84 (44.90, 50.78) 1611 38.14 (34.30, 42.14) 1121 Rich 45.53 (42.37, 48.72) 952 36.52 (30.91, 42.52) 589 41.46 (38.28, 44.72) 1519 35.02 (31.43, 38.78) 945 Richest 32.76 (29.03, 36.71) 697 22.65 (18.06, 28.01) 373 32.78 (29.13, 36.66) 1129 28.11 (23.49, 33.24) 647 Education No education 49.06 (47.19, 50.93) 3957 37.91 (34.96, 40.96) 1701 46.49 (44.55, 48.43) 5644 38.96 (36.60, 41.37) 2873 Primary school 40.08 (35.88, 44.43) 506 30.09 (23.74, 37.31) 234 40.33 (36.77, 43.99) 1024 38.39 (33.34, 43.70) 577 Secondary school + 35.80 (31.70, 40.10) 445 22.88 (17.53, 29.28) 212 37.27 (33.91, 40.75) 1061 32.54 (27.74, 37.73) 658 Residence Rural 49.21 (47.29, 51.14) 3874 38.87 (36.05, 41.76) 2013 45.74 (43.84, 47.64) 5620 40.31 (37.99, 42.66) 3923 Urban 38.54 (35.14, 42.05) 1040 29.70 (24.63, 35.32) 750 40.86 (37.05, 44.78) 2110 29.66 (26.47, 33.06) 1169 Sex Female 44.97 (42.77, 47.19) 2485 34.27 (31.44, 37.22) 1422 41.66 (39.56, 43.79) 3876 36.90 (34.42, 39.46) 2633 Male 48.99 (46.79, 51.19) 2429 38.62 (35.10, 42.26) 1341 47.17 (45.05, 49.29) 3854 38.89 (36.58, 41.24) 2460 Regions 01 east 44.11 (40.38, 47.92) 1199 33.56 (29.09, 38.35) 507 41.50 (38.35, 44.73) 2067 42.17 (37.81, 46.66) 1182 02 north 52.39 (49.68, 55.09) 1887 39.51 (35.62, 43.54) 1327 48.64 (45.76, 51.53) 2930 35.40 (32.61, 38.30) 2226 03 south 45.79 (42.96, 48.64) 1390 38.05 (32.81, 43.57) 546 42.71 (39.99, 45.48) 1944 42.19 (38.26, 46.21) 1164 04 west 34.96 (30.65, 39.52) 436 26.89 (22.09, 32.29) 382 40.44 (34.24, 46.97) 787 28.91 (23.68, 34.76) 520 Notes: popn Population share of each subgroup CI Confidence Interval f childhood stunting across different dimensions of inequality in Sierra-Leone from 2005 to 2013 Shibre et al. International Journal for Equity in Health (2020) 19:88 Shibre et al. International Journal for Equity in Health Page 5 of 10 Page 5 of 10 simple measures have the strength of straightforward interpretation and calculation. Statistical analysis d WHO recommends adoption of simple and complex, as well as absolute and relative measures in an inequality study to allow for examination of an inequality from different per- spectives [26]. overlap, making it difficult to comment on the rate of re- duction of stunting. Similarly, while stunting burden in the no-education group had decreased by 10 percentage points over the study time periods, the other two cat- egories of education had seen no statistically significant change over time. We found that prevalence of stunting was consistently higher among male children and rural residents throughout the studied years. See Table 1 for detail. We measured the over time dynamics of stunting dis- parity using the 95% Confidence Intervals (CI) approach. We declared that a statistically significant difference was observed when the different CIs do not overlap. On the other hand, when the intervals do overlap, we declared that there was no a statistically significant difference in inequality level between any two survey years. For all our analyses, we used the offline version of the WHO’s HEAT software application updated in 2019 [34]. The WHO HEM database stores re-analyzed data derived from DHS and MICS that have been conducted in many low-and-middle-income countries. The HEAT then uses the re-analyzed data in a way that facilitate interpret- ation of findings and is updated regularly to incorporate new surveys. Given that experts proficient in the area analyzed the data, papers written based on this source is deemed reliable and comparable both over time and be- tween countries. Inequalities in stunting as measured by different inequality measures As shown in Table 2, there were both absolute and relative wealth related inequalities in stunting in Sierra-Leone in all the four rounds of the surveys and with all the measures of disparity, with the inequality occurred consistently favour- ing economically better-off groups. For example, in 2005, based on just a point estimate, the prevalence of stunting among children born to poorest women was nearly 17 points higher than the prevalence in children born to women who fell in the richest wealth quintile The R showed that children from poorest households had stunting that is between 1.3 to 1.7 times higher than that of children from richest households in 2005. The pro-rich condition of stunting has also been sup- ported by the complex measures. For example, the PAF finding showed that stunting was more concentrated among the poor in all the four survey periods, and that prevalence of stunting in the country in 2008 would have been fallen between roughly 27 to 49% if the poor- est, poor, middle and rich subgroups of wealth were on par with the richest subgroup with respect to stunting burden. Although the disparity do not changed notice- ably by the simple measures, the complex measures indi- cated that the wealth related disparity in stunting improved slightly over time though overlap of the CIs complicate interpretation of the change. Ethical consideration Since the analyses were made using the publicly available data stored in the WHO HEM, we did not require eth- ical permissions. Ethical procedures were the responsi- bility of the institutions that commissioned, funded, or managed the surveys. Prevalence of childhood stunting disaggregated by different equity stratifiers Table 1 presents the prevalence of stunting disaggre- gated by subcategories of the five dimensions of inequal- ity for the four waves of the household surveys (two MICS-2005 and 2010, and two DHS-2008 and 2013). The stunting disaggregation is presented along with the population share of each subgroup in absolute numbers. The total children population participated in all the four rounds were 20, 500. Of these, 50.8, 75.2 and 69.1% were females, rural residents and born to illiterate women, respectively. We presented stunting point estimate in each subgroup along with the corresponding 95% CIs to allow comparison between subgroups and over time. For example, the prevalence of stunting varied significantly between wealth categories (the 95% CIs of the five wealth categories did not overlap), with the richest cat- egory contributed the smallest portion of stunting bur- den. While stunting among the poorest subgroup had fallen by about 7 percentage points between 2005 and 2013, the CIs for the richest subgroup had substantial Likewise, educational status inequality in childhood stunting was observed across all the four rounds. We found education related disparity in 2005, 2008 and 2010 schools by all the summary measures and in 2013 by PAR, PAF and D, favouring children born to mothers with secondary or higher schools. Based on the point es- timates, stunting among children born to non-educated women was roughly 13 points higher than stunting in children born to women who completed secondary education or higher in 2005. This difference was halved in 2013. Over the surveyed years, there had been small decline in the educational disparity of stunting. For example, over the period of 8 years, stunting decreased by nearly 10 points according to the point estimate of the PAF measure. Our findings also demonstrated that, except in 2010 by the R measure, where there was no disparity, there Shibre et al. Prevalence of childhood stunting disaggregated by different equity stratifiers International Journal for Equity in Health (2020) 19:88 Page 6 of 10 Table 2 Extent and trends of inequalities in childhood stunting across different measures of inequalities from 2005 to 2013 Dimensions of inequality Year 2005 2008 2010 2013 Measure %(95% CI) %(95% CI) %(95% CI) %(95% CI) Wealth D 17.19 (11.97, 22.41) 13.79 (6.96, 20.61) 14.05 (9.11, 18.99) 14.52 (8.47, 20.56) PAF −30.23 (−37.20, −23.26) −37.72 (−48.86, −26.59) −26.16 (−31.93, −20.39) −25.76(−34.41, −17.10) PAR −14.19 (−17.47, −10.92) −13.72 (−17.77, −9.67) −11.61 (−14.18, −9.05) −9.75 (−13.03, −6.47) R 1.52 (1.31, 1.73) 1.60 (1.20, 2.01 1.42 (1.23, 1.61) 1.51 (1.22, 1.80) Education D 13.26 (8.67, 17.85) 15.03 (8.45, 21.60) 9.21 (5.28, 13.14) 6.42 (0.89, 11.95) PAF −23.72 (−32.83, −14.61) −35.67 (−51.07, −20.26) −16.07 (−22.19, −9.94) −14.03 (−22.77, −5.30) PAR −11.13 (−15.40, −6.85) −12.68 (−18.16, −7.20) −7.13 (−9.85, −4.41) −5.31 (−8.62, −2.00) R 1.37 (1.20, 1.53) 1.65 (1.21, 2.10) 1.24 (1.12, 1.37) 1.19 (0.99, 1.39) Residence D 10.67 (6.72, 14.62) 9.16 (3.11, 15.22) 4.87 (0.57, 9.17) 10.64 (6.61, 14.67) PAF −17.92 (−23.56, −12.27) −18.35 (−26.18, −10.52) −7.98 (−12.03, −3.93) −21.66(−27.84, −15.47) PAR −8.41 (−11.06, −5.76) −6.67 (−9.52, −3.82) −3.54 (−5.34, −1.74) −8.20 (−10.54, −5.85) R 1.27 (1.15, 1.40) 1.30 (1.05, 1.56) 1.11 (1.00, 1.23) 1.35 (1.18, 1.52) Sex D 4.01 (0.90, 7.12) 4.34 (−0.23, 8.93) 5.50 (2.51, 8.49) 1.98 (−1.44, 5.41) PAF −4.22 (−7.17, −1.28) −5.80 (−10.59, −1.00) −6.18 (−8.67, −3.69) −2.52 (−5.93, 0.87) PAR −1.98 (−3.36, −0.60) −2.11 (−3.85, −0.36) −2.74 (−3.85, −1.63) −0.95 (−2.24, 0.33) R 1.08 (1.01, 1.16) 1.12 (0.98, 1.26) 1.13 (1.05, 1.20) 1.05 (0.95, 1.14) Regions D 17.43 (12.25, 22.61) 12.62 (6.17, 19.06) 8.19 (1.19, 15.20) 13.27 (6.46, 20.09) PAF −25.55 (−34.71, −16.38) −26.08 (−37.61, −14.56) −8.92 (−16.25, −1.58) −23.64 (−33.49, −13.79) PAR −11.99 (−16.30, −7.69) −9.49 (−13.68, −5.29) −3.96 (−7.21, −0.70) −8.95 (−12.68, −5.22) R 1.49 (1.29, 1.70) 1.46 (1.15, 1.78) 1.20 (0.99, 1.40) 1.45 (1.14, 1.77) Notes: D Difference, PAF Population Atrributable Fraction, PAR Population Attributable Risk, R Ratio, CI Confidence Interval Table 2 Extent and trends of inequalities in childhood stunting across different measures of inequalities from 2005 to 2013 both absolute and relative subnational regional inequal- ities in childhood stunting across all the four rounds. The regional variations of stunting had decreased between 2005 and 2010 by D, PAF and PAR measures, and increased between 2010 and 2013 by the complex measures. Prevalence of childhood stunting disaggregated by different equity stratifiers was place of residence inequality in stunting in all the rounds and the disparity disproportionately affected the rural children. The point estimates from D showed that children in rural areas had nearly 11, 9, 5 and 11 per- centage points higher stunting burden than that of their counterparts in urban areas in 2005, 2008, 2010 and 2013 respectively, yielding more like a ‘U’ shape pattern. The urban-rural stunting disparity had seen fluctuations; it decreased between 2005 and 2010 by 10 points, and increased afterwards by 14 points as evidenced by the PAF. Discussion The study attempted to shed light on the over time dynamics of inequalities in the prevalence of stunting in Sierra-Leone among children aged below 5 years using the WHO HEM database. While we did not record any male-female differential in the occurrence of stunting in 2013, we highlighted large sex related inequality in stunting in the first three household surveys, where we observed the disparity in 2005 and 2010 by all the measures and in 2008 by the complex measures only. If there was no male-female gap in stunting (or equivalently, if stunting level in male chil- dren was reduced to a level in females), then stunting in the country would have been decreased by between nearly 1 to 7 points in 2005, nearly 1 to 11 points in 2008, and nearly 4 to 9 points in 2010. Our findings demonstrated that both absolute and relative economic inequalities in childhood stunting were prevalent in all the four household surveys. The stunting burden was more pronounced among the most disadvantaged sub-groups who fell under the poorest wealth categories. The poor-rich disparity in stunting could be due to dissimilarity in the distribution of income, education, and ethnicity that are directly or in- directly related with food security [24]. The fact that the pro-rich situation of stunting inequality did not improve significantly over time raises a concern on whether the country could achieve the stunting targets set for 2025 In terms of subnational region, except in 2010 by sim- ple measure (R), where there was disparity, there were Page 7 of 10 Shibre et al. International Journal for Equity in Health (2020) 19:88 Page 7 of 10 Shibre et al. International Journal for Equity in Health and 2030 SDG [35, 36]. The present findings are comparable with that of previous studies done in other African countries which reported that childhood stunt- ing was greater among families who had low family in- come [37–39]. children born to women who fell in the richest wealth category had, on average, a 47% lower odd of experien- cing stunting compared with children born to women in the poorest wealth quintile [39]. Discussion Although exploring drivers of the change in stunting inequality over time is beyond the remit of the study, our finding argues that the differential pace of reduction of stunting over time between the poorest and richest may explain the fact that the poor-rich disparity narrowed in 2013. While the poorest subgroup recorded a 7 percentage point decline in the prevalence of stunting, the richest subgroup did not see any change in the prevalence of stunting during the same time (Table 1). With regard to time trend of wealth driven stunting disparity, we confirmed that the pro-rich inequality of stunting had seen mixed patterns based on the type of summary measures calculated; while simple measures showed no sign of improvement, complex measures demonstrated that the wealth related inequality of stunt- ing had slightly narrowed with time. As is clearly de- tailed in the method section of this paper, simple measures of inequality account for just two subgroups of a dimension of inequality, and ignore the remaining sub- populations in the middle. On the other hand, complex measures take into account the entire subgroups of an equity stratifer and this statistical property allows them to overcome the inherent blemish of simple measures when one wants to observe distribution of a health indi- cator along the entire rungs of a dimension of inequality like wealth and education. This difference between them could explain for the emergence of differing conclusion on the over time change of stunting disparity. In our study, the PAR showed that the wealth related stunting inequality decreased, based on the point estimate, by 4.4 percentage points between the first and the last surveys, without a statistically significant difference between the first three rounds. The PAR finding further showed that Sierra Leone would have reduced the national stunting prevalence by between, based on the point estimates, nearly 10 to 14 points had the levels of stunting in the other wealth quintiles been reduced to the level in the wealthiest quintile. Subsequently, the significant drop in the national prevalence of stunting could have been accompanied by large number of children escaped from the suffering of stunting. This in turn would have big effect on attainment of the national and global goals of stunting set for 2025 and 2030. Discussion Currently, one in 3 chil- dren in the nation lives with stunting [9], making Sierra Leone one of the countries with the hardest hit of stunt- ing and it is unlikely for the country to hit the global targets for stunting unless pro-poor interventions are designed and promoted. This is because reducing income inequality and increasing health care spending have been shown to reduce stunting level [40]. Similarly, we documented that the relative disparity (according to the PAF finding) narrowed between the first and the last surveys studied at least based on the point estimates. Similar wealth related disparity in stunting was docu- mented in literature [41]. A multi-country analysis based on DHSs conducted in 35 SSA countries showed that stunting was more concentrated among the poorer households [42]. Akombi BJ et al. (2019) indicated that As all the summary measures calculated indicated, the burden of stunting in our study was highly concentrated among children born to women with no formal educa- tion than children born to women who had completed primary and secondary educations. Unfortunately, the education driven disparity persisted though evidence of improvement was observed. The simple measures indi- cated a statistically significant reduction of the education driven disparity over time including total disappearance in 2013 (by the measure of R). The complex measures also supported the finding that stunting disparity be- tween educated and non-educated groups narrowed over time. We observed that over the period of the study, on average, the gap narrowed by 6 points and this is slightly higher than recorded in the wealth-based inequality. The absolute inequality based on the PAR finding showed that, it would have been possible to drop the country’s 2013 stunting prevalence by approximately 2 to 9 points if the prevalence of stunting among the non-educated subgroup had been reduced to the level in the secondary or above category. In terms of relative inequality finding derived from PAF, Sierra Leone was to reduce the na- tional stunting level in 2013 by between closely 5 to 23% if there were no education related disparity in stunting. The finding points to the demonstrated practical impli- cation of education on ensuring health care equity by breaking the vicious cycle of poverty [43]. In the effort to curb the threat of stunting by removing within country disparities, closing gap in secondary or more education is important. Discussion In this regard, Sierra Leone would be benefited from accelerated education program as the net enrollment rate is only 38.33% for secondary education [44]. Our finding that stunting is more common among children of women with low educational attainment is aligned with similar prior findings [16, 39, 42]. The possible explanation for this is that a higher maternal education could lead to improved health care use which, in turn, affects health-related decisions that improve child nutritional outcomes [45]. Like wealth related in- equality, educational disparity in stunting had shown slight improvement over the course of the studied years. Page 8 of 10 Shibre et al. International Journal for Equity in Health (2020) 19:88 Shibre et al. International Journal for Equity in Health (2020) 19:88 Shibre et al. International Journal for Equity in Health (2020) 19:88 Shibre et al. International Journal for Equity in Health than any other parts of the country; identifying the drivers of this could pave the way to implementing context specific interventions that would help eliminate the subnational region related stunting disparity. Also, experiences that worked in the West of the country could be applied to the other settings in order to nar- row the gap between regions and subsequently decrease the national stunting prevalence. The absolute measure indicated that Sierra Leone would have declined the 2005 stunting level in country between nearly 8 to 16 points if the prevalence of stunting in the North had reduced to the level in the west. Further, the nation was to reduce the 2013 stunting level between nearly 5 to 13 points if stunting prevalence in the South fell to the level in the West. The finding points to the need to launch specific policy relevant equity interventions that work best in the context of each region in the country if the aim is to accelerate attainment of global goals for stunting set for 2025 and 2030 [35, 36]. The present finding is in concordance with that of prior study which reported within country variations of stunting with re- spect to subnational regions [39]. Discussion The same reason that we used to justify the improvement of wealth based disparity can be used to justify the im- provement in educational inequality; variation in the rate of reduction of stunting between these subpopulations causes for the disparity to shrink; stunting decreased significantly over time in the non-educated category while it remained relatively unchanged among higher or more educated group (Table 1). We showed that more stunted children were from rural than urban settings in all the four rounds and this evi- dence is supported by all measures. Our finding is similar with past evidence [39]. Such variations are likely to be due to the differences in the distribution of socioeconomic conditions [46]. The study highlighted not only was stunt- ing highly widespread in rural areas, but the country had not seen any sign of narrowing in the urban-rural disparity over the studied years. We observed a roughly U shape pattern in the residence related inequality of stunting; by all but one measures, the disparity was lowest in 2010 with the first and the last survey years experiencing the highest disparity. More interestingly, the disparity disappeared in 2010 altogether by the R measure. It is not coincidence that all the measures identified 2010 as most equitable year than others, and further studies may be required to explore the reason. If rural areas were on par with urban settings, then there were substantial improvements in bur- den of stunting in the country. In 2013, for instance, Sierra Leone would have decreased the national stunting level by about 6 to 11 percentage points. In terms of the relative gains, the 2013 national stunting prevalence would have been reduced by nearly 15 to 28%. This finding empha- sizes the need to draw increased attention towards rural areas if the aim is to close the urban-rural gap in terms of stunting burden. Policy makers and programmers should prioritize pro-rural and pro-poor nutritional interventions. Another important finding that emerges from our study is that variations in the level of stunting were observed among sub-national regions by all measures of inequality across all studied years, except that the disparity vanished in 2010 by the R measure. Generally, the regional inequal- ity had shown fluctuation over time by the different measures of inequality; it fluctuated with complex measures; it decreased until 2010 and increased again afterwards, resulting in a U shape nature of the disparity. Conclusions 1Department of Reproductive, Family and Population Health, School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia. 2Birehan for mothers and children morbidity and mortality surveillance project, Debre Brehan, Ethiopia. 3Department of Nutrition and Dietetics, School of Public Health, Addis Ababa University, Addis Ababa, Ethiopia. Stark inequalities in stunting in Sierra-Leone remained to the disadvantage of children of poor and uneducated mothers, those living rural residents and certain regions. We also wed that stunting disproportionately impacted male children. Not only did we show the occurrence of substantial inequality to the disadvantage of certain sub- populations, we highlighted that the inequality persisted throughout the study periods though some signs of im- provements were observed for some dimensions and the male-female stunting gap disappeared in 2013. Received: 21 February 2020 Accepted: 2 June 2020 Received: 21 February 2020 Accepted: 2 June 2020 Abbreviations CI: Confidence Interval; D: Difference; DHS: Demographic and Health Survey; EA: Enumeration Area; GHI: Global Hunger Index; HEAT: Health Equity Assessment Toolkit; ICF: Inner City Fund; MICS: Multiple Indicator Cluster Survey; PAR: Population Attributable Fraction; PAR: Population Attributable Risk; PCA: Principal Component Analysis; PSUs: Primary Sampling Units; PPS: Probability Proportional to Size; R: Ratio; SDG: Sustainable Development Goal; SDHS: Sierra Leone Demographic and Health Survey; WHO: World Health Organization Acknowledgments The study has a few strengths. We investigated the stunting disparity using the WHO HEM database through the HEAT software which allows us to do the inequality analysis with high standard of quality. The reason being, the re-a-analysis of the DHS and MICS data in the HEM was carried out by experts in the sector of the health equity and with huge care. Findings analyzed based on the HEM database are easily comparable across countries and over time. Finally the use of different summary measures in a single study has the potential to capture inequality from different ethical positions and perspectives, and this in turn affects the type of policy decisions and programmes to be made. The study also suffers a limitation. We did not explore the reason why stunt- ing disparity remained in the country. Once it is known that stunting disparity exists in the country across different population groups, further researches are important to help explore factors that led to the inequality. We acknowledge the WHO for making the software available to the public domain for free. Authors’ contributions GS did the literature review, carried out the analysis and interpreted the findings. BZ contributed to the literature review and carried out the analysis. JH contributed to the interpretation of the findings and reviewed the manuscript for its content. All authors approved the final version of the manuscript. Discussion International Journal for Equity in Health (2020) 19:88 Page 9 of 10 Page 9 of 10 needs to increase the observed stunting average annual rate of reduction by nearly 8 fold if the aim is to achieve the 14.7% stunting level by 2025 [36]. We argue that removing the large within country disparities around stunting between different subpopulations could signifi- cantly reduce the overall stunting prevalence, which in turn would translate to achievement of the global goals of stunting. Funding g No funding was received for this work. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Discussion This is similar with pattern of wealth based disparity eluci- dated above. The fluctuation with the simple measure (D) was that it first decreased between 2005 and 2010, and remained unchanged afterwards. g Congruent with prior works [39, 47], our finding demon- strated glaring sex related stunting inequality favouring female children. A study revealed that female children had 16 to 25% lower odd of experiencing stunting than their male counterparts [39], supporting the pro-female nature of stunting observed in this study. While a decomposition analysis would be warranted to learn factors that contrib- uted to the observed male-female gap with respect to stunt- ing distribution, available evidence showed that mothers may be motivated to start Complementary Feeding (CF) early for boys when they are born small or “weak” due to pre-existing nutritional status, and weaker babies are likely to be breast fed for longer period without having CF when it should be initiated at a recommended age [37, 48]. This, in turn, results in weaker babies experiencing higher odd of stunting. However, the sex differential of stunting ended in 2013, where stunting equally affected both sexes. Even if the disappearance of pro-female nature of stunting in the recent survey might reflect the country’s commitment to end male-female disparity of nutritional problems, this issue warrants further investigation and the strategies that work for sex driven disparity can be applied to the socio- economic and residence related disparities of stunting to avoid within country variations in stunting burden. The WHO nutrition targets call for 40 and 50% reduc- tion of stunting in 2025 and 2030 respectively from the 2012 stunting level. On average, stunting has to be reduced by 4% annually to attain the stated targets [35, 36]. However, Sierra Leone is far removed from the stated average annual rate of reduction, with stunting decreased by a meager of only 0.25% annually between 2005 and 2013 [23, 24]. This means that the country What is particularly impressing is that the Northern and West regions host respectively the highest and low- est prevalence of stunting in the nation, causing stark regional variations in stunting to exist. It is important to explore why the Northern Sierra Leone contributed to the unacceptably high clustering of stunted children Shibre et al. International Journal for Equity in Health (2020) 19:88 Shibre et al. International Journal for Equity in Health (2020) 19:88 Shibre et al. Availability of data and materials The datasets generated and/or analyzed during the current study are available in the WHO’s HEAT version 3.1 [https://www.who.int/gho/health_ equity/assessment_toolkit/en/]. Ethics approval and consent to participate Ethics approval was not required since the data is available to the public domain. Ethics approval and consent to participate Ethics approval was not required since the data is available to the public domain. References Onis MD, Borghi E, Arimond M, Webb P, Croft T, Saha K, et al. Prevalence thresholds for wasting, overweight and stunting in children under 5 years. Public Health Nutr. 2019;22(1):175–9. 32. Hosseinpoor AR, Bergen N, Schlotheuber A, Victora C, Boerma T, Barros AJD. Data resource profile: WHO Health Equity Monitor (HEM). Int J Epidemiol. 2016;45(5):1404–1405e. 11. WHO. State of Inequality: Reproductive, maternal, newborn and child health. Geneva: WHO; 2015. 33. Rutstein SO And Johnson K. The DHS wealth index. http://www. measuredhs.com/pubs/pdf/CR6/CR6.pdf. Accessed 3 June 2020. 33. Rutstein SO And Johnson K. The DHS wealth index. http://www. measuredhs.com/pubs/pdf/CR6/CR6.pdf. Accessed 3 June 2020. 11. WHO. State of Inequality: Reproductive, maternal, newborn and child health. Geneva: WHO; 2015. 34. Health Equity Assessment Toolkit (HEAT). Software for exploring and comparing health inequalities in countries. Built-in database edition. Version 3.1. Geneva: World Health Organization; 2019. 12. Domínguez GG, Victora C, Barro AJD. Ethnic inequalities and trends in stunting prevalence among Guatemalan children: an analysis using national health surveys 1995–2014. Int J Equity Health. 2019;18(1):110. 12. Domínguez GG, Victora C, Barro AJD. Ethnic inequalities and trends in stunting prevalence among Guatemalan children: an analysis using national health surveys 1995–2014. Int J Equity Health. 2019;18(1):110. 35. World Health Organization. Nutrition. Global Targets 2025. To improve maternal, infant and young child nutrition. Available at: http://www.who.int/ nutrition/global-target-2025/en/. Accessed Feb 15 2020. 13. Nepali S, Simkhada P, Davie I. Trends and inequalities in stunting in Nepal: a secondary data analysis of four Nepal demographic health surveys from 2001 to 2016. BMC Nutrition. 2019;5:19. 36. WHO/UNICEF Discussion paper. The extension of the 2025 Maternal, Infant and Young Child nutrition targets to 2030. Accessed from: https://www. who.int/nutrition/global-target-2025/discussion-paper-extension-targets-203 0.pdf?ua=1. Accessed on April 28. 2020. 14. Menon P, Headey D, Avula R, Nguyen PH. Understanding the geographical burden of stunting in India: a regression-decomposition analysis of district- level data from 2015-16. Matern Child Nutr. 2018;14(4):e12620. 0.pdf?ua=1. Accessed on April 28. 2020. 15. Flores-Quispe MDP, Restrepo-Mendez MC, MF SM, Ferreira LZ, Wehrmeister FC. Trends in socioeconomic inequalities in stunting prevalence in Latin America and the Caribbean countries: differences between quintiles and deciles. Int J Equity Health. 2019;18(1):156. 37. Tiwari R, Ausman LM, Agho KE. Determinants of stunting and severe stunting among under-fives: evidence from the 2011 Nepal demographic and health survey. BMC Pediatr. 2014, 14:239. 38. Jonah CMP, Sambu WC, May JD. References Accessed on April 29, 2020. 45. Abuya BA, Ciera J, Kimani-Murage E. Effect of mother's education on child's nutritional status in the slums of Nairobi. BMC Pediatr. 2012;12:80. 22. Statistics Sierra Leone (SSL) and ICF Macro. Sierra Leone demographic and health survey 2008. Calverton: Statistics Sierra Leone (SSL) and ICF Macro; 2009. 46. Fotso JC. Urban–rural differentials in child malnutrition: trends and socioeconomic correlates in sub-Saharan Africa. Health Place. 2007;13(1):205–23. socioeconomic correlates in sub-Saharan Africa. Health Place. 2007;13(1):205–23. 47. Bork KA, Diallo A. Boys are more stunted than girls from early infancy to 3 years of age in rural Senegal. J Nutr. 2017;147(5):940–7. 23. Statistics Sierra Leone (SSL) [Sierra Leone] and ICF International.2014. Sierra Leone Demographic and Health survey. Freetown, Sierra Leone, and Rockville, Maryland, USA: SSL and ICF International; 2013. 23. Statistics Sierra Leone (SSL) [Sierra Leone] and ICF International.2014. Sierra Leone Demographic and Health survey. Freetown, Sierra Leone, and Rockville, Maryland, USA: SSL and ICF International; 2013. 47. Bork KA, Diallo A. Boys are more stunted than girls from early infancy to 3 years of age in rural Senegal. J Nutr. 2017;147(5):940–7. 48. Inge van der Knaap ,. Jeroen Smits . The determinants of sex differences in child stunting in Sub Saharan Africa: a multilevel logistic regression analysis. Thesis 2018.Accessed on Feb 13, 2020. 24. Statistics Sierra Leone and UNICEF-Sierra Leone. Sierra Leone multiple Indicator cluster survey 2005, final report. Freetown, Sierra Leone: Statistics Sierra Leone and UNICEF-Sierra Leone; 2007. 24. Statistics Sierra Leone and UNICEF-Sierra Leone. Sierra Leone multiple Indicator cluster survey 2005, final report. Freetown, Sierra Leone: Statistics Sierra Leone and UNICEF-Sierra Leone; 2007. 25. tatistics Sierra Leone and UNICEF-Sierra Leone. Sierra Leone multiple Indicator cluster survey 2010, final report. Freetown, Sierra Leone: Statistics Sierra Leone and UNICEF-Sierra Leone; 2011. 25. tatistics Sierra Leone and UNICEF-Sierra Leone. Sierra Leone multiple Indicator cluster survey 2010, final report. Freetown, Sierra Leone: Statistics Sierra Leone and UNICEF-Sierra Leone; 2011. References A comparative analysis of socioeconomic inequities in stunting: a case of three middle-income African countries. Arch Public Health. 2018;76:77. 16. Krishna A, Mejia-Guevara I, McGovern M, Aguayo VM, Subramanian SV. Trends in inequalities in child stunting in South Asia. Matern Child Nutr. 2018;14(Suppl 4):e12517. 39. Akombi BJ, Agho KE, Renzaho AM, Hall JJ, Merom DR. Trends in socioeconomic inequalities in child undernutrition: Evidence from Nigeria Demographic and Health Survey (2003–2013). PLoS One. 2019. 17. Mzumara B, Bwembya P, Halwiindi H, Mugode R, Banda J. Factors associated with stunting among children below five years of age in Zambia: evidence from the 2014 Zambia demographic and health survey. BMC Nutr. 2018;4:51. 40. Bowser DM, Wu Z, Akobirshoev I, Morill TC, Nandakumar AK. The impact of health care spending and income inequality on stunting prevalence. Int J Healthc. 18. Yaya S, Oladimeji O, Odusina EK, Bishwajit G. Household structure, maternal characteristics and children’s stunting in sub-Saharan Africa: evidence from 35 countries[published online ahead of print, 2020 Jan 13]. Int Health. 2020; ihz105. https://doi.org/10.1093/inthealth/ihz105. 41. Restrepo-Méndez MC, Barros AJD, Black RE, Victora CG. Time trends in socio- economic inequalities in stunting prevalence: analyses of repeated national survey. Public Health Nutr. 2014;18(12):2097–104. 19. UN General Assembly, Transforming our world: the 2030 Agenda for Sustainable Development, 2015, A/RES/70/1, available at: https://www. refworld.org/docid/57b6e3e44.html [accessed 25 November 2019]. 42. Ekholuenetale M, Tudeme G, Onikan A, Ekholuenetale CE. Socioeconomic inequalities in hidden hunger, undernutrition, and overweight among under-five children in 35 sub-Saharan African countries. J Egypt Public Health Assoc. 2020;95. 42. Ekholuenetale M, Tudeme G, Onikan A, Ekholuenetale CE. Socioeconomic inequalities in hidden hunger, undernutrition, and overweight among under-five children in 35 sub-Saharan African countries. J Egypt Public Health Assoc. 2020;95. 20. Government of Sierra Leone. Action against Hunger IA. Sierra Leone National Nutrition Survey 2017. Available from: https://reliefweb.int/report/ sierra-leone/sierra-leone-national-nutrition-survey-2017-august-28-october-1 0-2017. Accessed on Feb 17, 2020. 43. Hahn RA, Truman BI. Education improves public health and promotes health equity. Int J Health Serv. 2015;45(4):657–78. 43. Hahn RA, Truman BI. Education improves public health and promotes health equity. Int J Health Serv. 2015;45(4):657–78. 21. Buisman LR, Van de Poel E, O'Donnell O, van Doorslaer EKA. What explains the fall in child stunting in sub-Saharan Africa? SSM Popul Health. 2019;8:100384. 44. UNESCO Institute for Statistics. Sierra Leone Education and Literacy Available from: http://uis.unesco.org/en/country/sl. Accessed on April 29, 2020. 44. UNESCO Institute for Statistics. Sierra Leone Education and Literacy Available from: http://uis.unesco.org/en/country/sl. References 1. UNICEF. The State of the World’s Children 2019. Children, food and nutrition: growing well in a changing world. New York: UNICEF; 2019. p. 258. 2. Deonis M, Branca F. Childhood stunting: A global perspective. Matern Child Nutr. 2016;12(Supplement 2):12–26. We recommend implementation of equitable nutri- tion interventions focusing on the sub-populations suffering higher burden of stunting. The country needs to increase the distribution and coverage of secondary or higher educations to close gaps in stunt- ing and other SDG indicators between educated and non-educated individuals. Also, the glaring wealth dis- parity in the country should be the government’s top priority area to make economic schemes pro-poor. Finally, we recommend that the nation should work aggressively to ensure that people living in the pov- erty zone are benefiting from the country’s economic growth to ensure that they are not left behind. 3. Development Initiatives. The P20 initiative: data to leave no one behind, baseline report march 2017. Washington DC: Development Initiatives; 2017. 4. Robert E, Black LHA, Bhutta ZA, Caulfield LE, De Onis M, MajidEzzati CM, et al. Maternal and child under nutrition: global and regional exposures and health consequences. Elsevier. 2008;371(9608):260. 5. Ikeda NIY, Shibuya K. Determinants of reduced child stunting in Cambodia: analysis of pooled data from three demographic and health surveys. Bull World Health Organ. 2013;91(5):349. 6. Grantham McGregor SCY, Cueto S, Glewwe P, Richter L, Strupp B. International Child Development Steering Group. Developmental potential in the first 5 years for children in developing countries. Lancet. 2007; 369(9555):70. 7. WY CL, Coyte PC. Income-related children’s health inequality and health achievement in China. Int J Equity Health. 2014;13(1):102. 8. UNICEF, WHO and World Bank. Levels and trends in child malnutrition: key findings of the 2020 Edition of the joint child malnutrition estimates. Geneva: WHO; 2020. Page 10 of 10 Shibre et al. International Journal for Equity in Health (2020) 19:88 (2020) 19:88 9. World food Programme. Sierra Leone. Available at: https://www.org/ countries/sierra-, leaone. Accessed 3 June 2020. 9. World food Programme. Sierra Leone. Available at: https://www.org/ countries/sierra-, leaone. Accessed 3 June 2020. health-equity-rmnch-potential-for-improvement.pdf?sfvrsn=1fba8883_2. Accessed on April 25, 2020. health-equity-rmnch-potential-for-improvement.pdf?sfvrsn=1fba8883_2. Accessed on April 25, 2020. 10. Onis MD, Borghi E, Arimond M, Webb P, Croft T, Saha K, et al. Prevalence thresholds for wasting, overweight and stunting in children under 5 years. Public Health Nutr. 2019;22(1):175–9. 10. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 26. World Health Organization. Handbook on health inequality monitoring with a special focus on low and middle income countries. Geneva: World Health Organization; 2013. 26. World Health Organization. Handbook on health inequality monitoring with a special focus on low and middle income countries. Geneva: World Health Organization; 2013. 27. Samuel Beresford Weekes and Silleh Bah. Statistics Sierra Leone. Sierra Leone 2015 Population and Housing Census Thematic Report on Population Structure and Population Distribution. 2017. Available from: https://sierraleone.unfpa.org/sites/default/files/pub-pdf/Population%2 0structure%20Report_1.pdf. Accessed at Feb 17, 2020. 28. World Bank. Sierra Leone Overview. Available from: https://www.worldbank. org/en/country/sierraleone/overview. Accessed 3 June 2020. 29. GLOBAL Hunger index. Sierra Leone Available from: https://www. globalhungerindex.org/sierra-leone.html. Accessed 3 June 2020. 30. Hancioglu A, Arnold F. Measuring Coverage in MNCH: Tracking Progress in Health for Women and Children Using DHS and MICS Household Survey. PLoS Med. 2013;10(5):e1001391. 31. World Health Organization. Technical Notes. Potential for improvement in RMNCH interventions. Retrieved from: https://www.who.int/docs/default- source/gho-documents/health-equity/state-of-inequality/technical-notes/
https://openalex.org/W4380562383	https://zenodo.org/records/8036033/files/17..pdf	English	null	Impact of Digitalisation on the Indian Stock Market	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	4,646	Abstract: According to research conducted by 5paisa.com in July 2020, 2/3rd of the cash and application of software programmes and hardware tools to make financial services and processes more efficient in terms of time and cost and to enable finance managers to make better decisions.Technological advancements like Machine Learning, Deep Learning,Blockchain Technologies, Distributed Ledger Systems, Smart Contracts, Robo advisors, and Algo Trading arerevolutionisinghow stock trading used to happen in the last few years. The depository participants are launching their own online trading platforms to serve their clients better and increase client engagement. This is one of the main reasons for an increase in retail investor participation in the Indian stock market. According to research conducted by 5paisa.com in July 2020, 2/3rd of the cash and application of software programmes and hardware tools to make financial services and processes more efficient in terms of time and cost and to enable finance managers to make better decisions.Technological advancements like Machine Learning, Deep Learning,Blockchain Technologies, Impact of Digitalisation on the Indian Stock Market Ms. Anusha S Sangondimath1, Prof. S. B Kamashetty2 1Research Scholar, Dept of PG Studies and Research in Management, Karnataka State Akka Mahadevi Women’s University, Vijayapura, Karnataka 2Professor, Department of Management, Karnataka State Akka Mahadevi Women’s University, Vijayapura, Karnataka Corresponding Author- Ms. Anusha S Sangondimath Email: anushasangondimath3@gmail.com Ms. Anusha S Sangondimath1, Prof. S. B Kamashetty2 1Research Scholar, Dept of PG Studies and Research in Management, Karnataka State Akka Mahadevi Women’s University, Vijayapura, Karnataka 2Professor, Department of Management, Karnataka State Akka Mahadevi Women’s University, Vijayapura, Karnataka Corresponding Author- Ms. Anusha S Sangondimath Email: anushasangondimath3@gmail.com Ms. Anusha S Sangondimath1, Prof. S. B Kamashetty2 1Research Scholar, Dept of PG Studies and Research in Management, Karnataka State Akka Mahadevi Women’s University, Vijayapura, Karnataka 2Professor, Department of Management, Karnataka State Akka Mahadevi Women’s University, Vijayapura, Karnataka Corresponding Author- Ms. Anusha S Sangondimath Email: anushasangondimath3@gmail.com Abstract: Digitalisation is bringinga significant change in all domains. The onset of the Digitalization of the Indian Stock Market can be identified through NSE’s NEAT (National Exchange for Automated Trading) system. In the year 1995, BSE moved from an open outcry method of trading to aScreen-based system. This made the transactions faster and reduced the transaction cost along withenhancing the transparency of the system.The increase in Internet and Mobile usage and Accessibility have accelerated retail participation in the stock market.Today we have AI which is almost nullifying human intervention in placing orders and post-trade settlements through its Machine Learning and ANN (Artificial Neural Networks) concepts.In this context, this study is undertaken to trace the path of digitalization of the Stock Market in India and analyse the applications of AI in stock market trading. The study finds that though the extended use of technology and applications makes trading convenient and faster it still has some flaws like a lack of robust technical infrastructure, data protection etc., which needs to be addressed by an efficient regulatory framework. We all are well aware of the NSE Co-location Scam which made the regulatory bodies rethink on thesufficiency of existing regulations for trading and information sharing and protecting investor interests.Hence, the adoption of advanced technology like blockchain, distributed ledger technologies etc., is rising the need for modification of the role of regulatory bodies.Also, the study finds that the number of dormant Demat accounts is more in Indian Stock Market and the stockbrokers have to take measures to convert these into active accounts so that they can meaningfully contribute to the development of the economy. Keywords: AI,Behavioural Finance, Blockchain Technology, Digitalization,Smart Contracts, Stock Markets. and application of software programmes and hardware tools to make financial services and processes more efficient in terms of time and cost and to enable finance managers to make better decisions.Technological advancements like Machine Learning, Deep Learning,Blockchain Technologies, Distributed Ledger Systems, Smart Contracts, Robo advisors, and Algo Trading arerevolutionisinghow stock trading used to happen in the last few years. The depository participants are launching their own online trading platforms to serve their clients better and increase client engagement. This is one of the main reasons for an increase in retail investor participation in the Indian stock market. International Journal of Advance and Applied Research www.ijaar.co.in ISSN – 2347-7075 Impact Factor – 7.328 Peer Reviewed Bi-Monthly Vol.4 No.20 May – June 2023 International Journal of Advance and Applied Research www.ijaar.co.in A. Discussion: Prateek Rani and Adithya Srinivasan (2015), conducted a study to analyse the impact and future of digitalization of financial markets. They studied different electronic trading platforms andthe digitalization of the US Market. They found that US Markets are highly digitalised and algo-trading will be the future of electronic trading and the success of algo trading depends on the transparency of the system.Jiya Goel et al., (2020) conducted a study on concepts applications and limitations of AI in Stock Markets with the aim of understanding and evaluating the prospects of integrating AI in Stock Trading. They explored various ways in which stock trading can be done using AI. However, they recognised that TMA and SMA should be used as a part of Machine Learning AI models rather than MACD and RSI. They also found that lack of sufficient high-quality data to feed and train into the system would cause a problem for these models. Siddharth Nair and Dr. Garima Malik (2020), undertook a study on the application of AIto stock market prediction with the intention to understand the various software available forstock prediction while analysing its benefits to investors. They reached the conclusion that AI can be used for predicting the stock markets and sooner it will be the new way of trading in the stock market. Nevertheless, they stressed on the need to protect the investors’ money and data privacy. Gone are the days when trading used to happen by agents meeting face-to-face and using hand signals. In earlier days the transactions needed to be placed and get settled manually.There was no system where the investors could get real-time information about share prices. One should always be dependent on the broker for information as well as the execution of trades. Though the share price information can be seen in the newspaper that would be the previous day’s price, the investors need real-time prices to make investment decisions. The physical transmission and execution had increased the share transaction cost, which would affect the final profits. Perhaps, this is the reason, common men did not prefer investments in the stock market. The beginning of digitalization in Indian Financial Markets can be traced back to 1991-92 with National Stock Exchange. NSE is the first stock exchange to introduce an Electronic Trading System through NEAT (National Exchange for Automated Trading). Introduction: Technology has always been a change agent and it has led to the progressive transformation of the business and society. With the influx of the internet, the world is heading towards a digital transformation. As a result, we are in a digitalised era, and witnessing technological disruption. According to Gartner, “Digitalisation is the use of digital technologies to change a business model and provide new revenue and value-producing opportunities. It is the process of moving to a digital business.” All the business areas are experiencing the effect of digitalisation and so is thefinancial markets and services industry in the form of Financial Technologies which is shortly known as FinTech. FinTech refers to the use Distributed Ledger Systems, Smart Contracts, Robo advisors, and Algo Trading arerevolutionisinghow stock trading used to happen in the last few years. The depository participants are launching their own online trading platforms to serve their clients better and increase client engagement. This is one of the main reasons for an increase in retail investor participation in the Indian stock market. According to research conducted by 5paisa.com in July 2020, 2/3rd of the cash 81 A. Discussion: NEAT is a fully automated electronic and screen-based trading system where members can enter into the computers the quantity and the price they want to trade and the transaction is automatically executed as soon as it finds a matching sale or buy order from the counter-party. This provided depth and liquidity to the market along with offering the convenience of trading for the investors.Under this system, the orders are matched based on time priority.Unmatched ordersremain in the system until they are modified or a fresh order is placed for the same share.NEAT generates and maintains the audit trails of the orders that are executed by providing the unique order number to every order that has been placed on the NEAT system. This makes the tracking of the order and settlement more efficient. Order processing has picked up speed andbecome moretransparent after the introduction ofthe Screen-Based Trading System.Then also the trades had to be IJAAR 1. To understand the purview digitalizationconcerningFinancial Markets. 1. To understand the purview o digitalizationconcerningFinancial Markets. market volumeswasaccounted for retail investor trades in the respective month.Retail shareholding in Indian markets has been raised to anearly 15-year high in June 2022(Ravi Kumar, 2023).In this light, this study is conducted to understand the impact of digitalization on the Indian financial markets. 2. To know the applications of AI in Indian Financial Markets. 2. To know the applications of AI in Indian Financial Markets. 3. To analyze the impact of digitalization on retail investor participation in Indian Stock Markets. IJAAR channelised through the stock brokers.As technology progressed the brokers started seeking digital methods to reach out to their clients. These digital methods involve company sites, web portals, social media, smartphone applications, SMS, e-mail, After the adoption of NEAT screen-based trading, another major milestone in the Digitalization of the Indian Stock Market was the dematerialization of securities.SEBI introduced the dematerialization of securities in the year 1996, under The Depositories Act, 1996.Dematerialization of securities is the process by which the physical share certificates are converted into digital form. The digitalized shares are held in the Demat account of the client. Just like we have bank accounts for holding cash our digital shares are held in the Demat account.Thus, having a Demat account is a must for every trader.These accounts are maintained by the depositories approved by the SEBI viz., NSDL and CDSL. It has reduced the transaction cost, fastened the settlement and made the transactions more reliable,contrary to physical share trading. etc.,Brokerage companies started using their websites to provide price information and market movements.Fig(1) indicates that Internet-based trading is increasing year on year. etc.,Brokerage companies started using their websites to provide price information and market movements.Fig(1) indicates that Internet-based trading is increasing year on year. etc.,Brokerage companies started using their websites to provide price information and market movements.Fig(1) indicates that Internet-based trading is increasing year on year. strategy can be used by the investors to limit the potential losses that can be occurred on a share. The applications now have an extended feature called GTT (Good Till Triggered) which means the investors can placeorders at a limit price and the order will be active even if the price does not hit by the end of the day unlike, the earlier system which used to get cancelledif the price does not reach the set limit price. Below given fig(1) indicates that mobile and internet- based trading is gradually increasing with the passing years. The stock market is known for its vulnerability and unpredictability. This can be attributed to the involvement of the human emotional quotient. As a major field of behavioural finance, it is highly dependent on the greed and fear of investors and these are the driving forces behind the price changes. This opened a wide scope for studies on market prediction. Researchers and investment practitioners have also found many technical tools which can work better for predicting the market. b. Research Gap: p The literature review shows that while there are many studies which are concerned towards the use of AI to predict share prices and technology, this study aims at making the common man aware of the digitalization of the stock market and the AI-based applications which can be used by the investor while trading to make informed trading decisions. c. Objectives of the Study: c. Objectives of the Study: This study is undertaken with the following objectives: Ms. Anusha S Sangondimath, Prof. S. B Kamashetty Ms. Anusha S Sangondimath, Prof. S. B Kamashetty 82 Vol.4 No.20 ISSN – 2347-7075 IJAAR pace because of two reasons. One is, because the computer executes the trades automatically when the predefined criteria are met, there will be no room for human greed for “some more profits” or fear of “What if the price goes still down?”. The algorithms will stick to the trading rules set by the investors. Another important reason is, the processing speed of computers is more than human beings. So, they can execute the trades at afaster speed and can crack the deals at better prices than manual trades. intervention as soon as the instructions are made good. For example, if an investor sets an instruction to buy 100 shares of ABC company if the Daily and Weekly RSI crosses above 70, the computer will automatically buy 100 shares of ABC company as soon as the daily and weekly RSI crosses 70. Algo trading can be understood just as an extension of limit orders. But the difference between these two is limit order uses only quantity and price parameters whereas the algo trading algorithms use combinations of technical tools along with the price and quantity.This is being adopted at a faster intervention as soon as the instructions are made good. For example, if an investor sets an instruction to buy 100 shares of ABC company if the Daily and Weekly RSI crosses above 70, the computer will automatically buy 100 shares of ABC company as soon as the daily and weekly RSI crosses 70. Algo trading can be understood just as an extension of limit orders. But the difference between these two is limit order uses only quantity and price parameters whereas the algo trading algorithms use combinations of technical tools along with the price and quantity.This is being adopted at a faster quantity.This is being adopted at a faster The invention ofDLT (Distributed Ledger Technology), Smart contracts andBlockchain technology ischanging the way of trade execution out and out. Smart Contracts are self-executing programmes which will enforce the contracts on fulfilment of the terms and conditions mentioned thereof. They are used in the clearing and settlement of transactions in the stock market. Also, with the use of DLT transactions can be made more secure and cost-effective. DLT is a digital system which records the transactions along with their details at multiple places at the same time. This validates, securesand updates the transactions on a real-time basis. IJAAR This is the reason the SEBI could adopt a T+1-day settlement cycle and now, it is planning to use blockchain for mitigate the need for a third-party regulator to an extent. The rules and regulations would be built within the smart contracts and can be automatically enforced and the post-trade transactions and transfer of legal ownership can be performed through the use of DLT. The Robo Advisors are being developed for customising investment plans. “It is a digital platform that provides automated, algorithm-driven financial planning and investment services with little or no human intervention.” (Jake Frankenfield, 2023). These advisors work based on the information collected by the registered investor regarding the investment profile and risk tolerance ability of the investor and investment goals. The necessary information is collected through the questionnaires and then the robo advisors will prepare a customised investment schedule accordingly. These are programmed to work on a Modern Portfolio Theory where the portfolios are rebalancedin line with the set benchmark index. According to an article by Ashish Rukhaiyar in Business Today, there were nearly 80 Robo advisory Firms in Indiain 2021. In India, this industry is still in the gestation stage. automatingpre-trading and post-trading activities in Indian Stock Market. In fact, SEBI has formed an advisory Committee on Financial and Regulatory Technologies (CFRT) toresearch the blockchain platform and other technologies that are used in fundraising, asset management and post- trade settlement.Blockchain technology can be used to make stock exchanges efficient through automation and decentralisation and automatingpre-trading and post-trading activities in Indian Stock Market. In fact, SEBI has formed an advisory Committee on Financial and Regulatory Technologies (CFRT) toresearch the blockchain platform and other technologies that are used in fundraising, asset management and post- trade settlement.Blockchain technology can be used to make stock exchanges efficient through automation and decentralisation and will prepare a customised investment schedule accordingly. These are programmed to work on a Modern Portfolio Theory where the portfolios are rebalancedin line with the set benchmark index. According to an article by Ashish Rukhaiyar in Business Today, there were nearly 80 Robo advisory Firms in Indiain 2021. In India, this industry is still in the gestation stage. Fi 1 G h h i th t f t d th h diff t d f t di i Ms. Anusha S Sangondimath, Prof. S. IJAAR However, technological advancement has led to theemployment of AIfor more accurate stock predictions and faster trade executions. Concepts of Machine learning and Artificial Neural Networks are being used to simulate the human brain to understand and mark human behaviour in a particular situation and thus try to make the markets more predictable.These systems are capable of handling huge volumes of data and use historical pricedata and event information to make predictions.Investors can prepare their own models or a committee of models (combinations of different models)by adjusting specific parameters to seek trend- relatedinformation. Algorithmic trading which is popularly known as algo trading is trending now because of its data processing ability and executing trades at high speed.It is an automated trading system which usesa pre-defined set of rules for trading. Investors have to feed the trading instructions regarding the price, quantity, technical tools etc., The computer follows these instructions and executes the trade without any human Ms. Anusha S Sangondimath, Prof. S. B Kam The rising competition in the field of financial markets has made brokerage companies introduce and offer innovative services to their clients. Companies are providing technical, fundamental, and market-related information related to the particular company that is required to make informed investment decisions through websites and mobile-based trading platforms. Interactive technical chartsprovidereal-time access to the price changes and market depth of particular stocks and indices. Investors can analyze the stocks based on the combination of different technical tools to make investment decisions using interactive technical charts. With the advent of smartphones, companies are developing mobile-based applications which are making trading more convenient, effortless, and handy. The investors will be givena user id and password using which they can place orders by themselves directly through the broker’s website. These platforms alsonotify the investors with all the favourable and unfavourable activities of a company which affect their interest.Investors can now have the details of their portfolios like sector-wise investment, profit or loss, order execution etc., readily available to them.The Stop-loss Ms. Anusha S Sangondimath, Prof. S. B Kamashetty 83 IJAAR B Kamashetty 84 through automation and decentralisation and Figure 1: Graph showing the percentage of trades through different modes of trading in BSE g Figure 1: Graph showing the percentage of trades through different modes of trading in BSE Figure 1: Graph showing the percentage of trades through different modes of trading in BSE Ms. Anusha S Sangondimath, Prof. S. B Kamashetty Ms. Anusha S Sangondimath, Prof. S. B Kamashetty 84 B. Findings and Suggestions: NSDL and CDSL collectively added 22 Lakh new Demat Accounts in a single month of August 2022. Nevertheless, there is a wide gap between the number of Demat accounts and the active traders in the market.The ease of accessibility and simplified account opening process due to digitalization is another major reason for this increase. It is a good sign for the economy as it promotes capital formation. Conclusion: B. Findings and Suggestions: B. Findings and Suggestions: The concept of trading started in the 18th century in India. what was started by mere 22 stock brokers under a Banyan tree, is now a market worth of INR 2,61,81,064 crores. The establishment of SEBI as a regulatory body and its investor protection norms, rising financial literacy, technological innovation and emergence of technical tools haveaccelerated the market participation of individual investors in the Indian Stock Market. Further, these technological improvements have made the market more accessible and brought down transaction costs along with faster settlement cycles leading to the enhanced liquidity of the market. As a result, the share of individual investors has become 41.6% by the end of FY 2022 from 33% in FY 2016 (NSE Pulse). The study finds that the trades using Co-location services, Mobile platforms and Internet Based Trading are increasing year on year [fig(1)].Also, the number of Demat accounts in August 2022 crossed 10 Crores, which is a 5x increase from the year 2018. NSDL and CDSL collectively added 22 Lakh new Demat Accounts in a single month of August 2022. Nevertheless, there is a wide gap between the number of Demat accounts and the active traders in the market.The ease of accessibility and simplified account opening process due to digitalization is another major reason for this increase. It is a good sign for the economy as it promotes capital formation. Conclusion: The concept of trading started in the 18th century in India. what was started by mere 22 stock brokers under a Banyan tree, is now a market worth of INR 2,61,81,064 crores. The establishment of SEBI as a regulatory body and its investor protection norms, rising financial literacy, technological innovation and emergence of technical tools haveaccelerated the market participation of individual investors in the Indian Stock Market. Further, these technological improvements have made the market more accessible and brought down transaction costs along with faster settlement cycles leading to the enhanced liquidity of the market. As a result, the share of individual investors has become 41.6% by the end of FY 2022 from 33% in FY 2016 (NSE Pulse). The study finds that the trades using Co-location services, Mobile platforms and Internet Based Trading are increasing year on year [fig(1)].Also, the number of Demat accounts in August 2022 crossed 10 Crores, which is a 5x increase from the year 2018. IJAAR Source: BSE. (Compiled by the researcher) estions: g started in the 18th was started by mere Banyan tree, is now 2,61,81,064 crores. EBI as a regulatory tection norms, rising nological innovation technical tools regulatory bodies to some extent. Overall, digitalization has a positive impact on Indian Stock Market. Asset digitalization is the emerging concept in the stock market which is aimed at providing for fractional ownership and improved cross-border share transactions and security of the shares. This will lead to the metamorphosis of the stock market. by the researcher) regulatory bodies to some extent. Overall, digitalization has a positive impact on Indian Stock Market. Asset digitalization is the emerging concept in the stock market which is aimed at providing for fractional ownership and improved cross-border share transactions and security of the shares. This will lead to the metamorphosis of the stock market. by the researcher) regulatory bodies to some extent. Overall, digitalization has a positive impact on Indian Stock Market. Asset digitalization is the emerging concept in the stock market which is aimed at providing for fractional ownership and improved cross-border share transactions and security of the shares. This will lead to the metamorphosis of the stock market. References: 1. Agrika. (2022). India cross 10 crore Demat accounts in August 2022, 60% new Demat accounts opened in 29 months. Meel Bijendra. Retrieved from Meel Bijendra. 2. Angel One. (n.d.). What is Algo Trading? Retrieved from Angel One: https://www.angelone.in/knowledge- center/online-share-trading/what-is-algo- trading 2. Angel One. (n.d.). What is Algo Trading? Retrieved from Angel One: https://www.angelone.in/knowledge- center/online-share-trading/what-is-algo- trading g 3. Angel One. (n.d.). What is Dematerialisation. Retrieved from Angel One: https://www.angelone.in/knowledge- center/demat-account/what-is- dematerialisation 3. Angel One. (n.d.). What is Dematerialisation. Retrieved from Angel One: https://www.angelone.in/knowledge- center/demat-account/what-is- dematerialisation 4. BSE. (2023, April). Mode of Trading Data. Retrieved from BSE: https://www.bseindia.com/markets/equity /EQReports/Cmmode.aspx 5. CFI Team. (2023, January). Robo Advisors. Retrieved from Corporate Finance Institue: https://corporatefinanceinstitute.com/reso urces/wealth-management/robo-advisors/ 5. CFI Team. (2023, January). Robo Advisors. Retrieved from Corporate Finance Institue: https://corporatefinanceinstitute.com/reso urces/wealth-management/robo-advisors/ 6. Erik Feyen, Jon Frost, Leonardo Gambacorta, Harish Natarajan and Matthew Saal. (2021). Fintech and the digital transformation of financial services: implications for market structure and public policy . Bank for International Settlements. 6. Erik Feyen, Jon Frost, Leonardo Gambacorta, Harish Natarajan and Matthew Saal. (2021). Fintech and the digital transformation of financial services: implications for market structure and public policy . Bank for International Settlements. Ms. Anusha S Sangondimath, Prof. S. B Kam From the open outcry method to Algo trading, the Indian stock market and trading have come a long way. However, in a digitalised ecosystem, there is always a jeopardy of data infiltration, data stealing, data misutilization and technological glitches like connectivity and run-time issues. This will wake the need for the regulatory bodies to bring stringent regulations towards safeguarding investor interests in cyberspace as well. However, digitalisation has started just now and with the increasing number of internet users, individual investors are getting added day by day, this indicates the wider scope for growth of the stock markets.The adoption of the Blockchain, Smart Contracts and Distributed Ledger Technologies to their fullest extent would automate the trading and settlement procedures. This will modify the role of 7. Ervin, E. (2018, August). Blockchain Technology Set To Revolutionize Global Stock Trading. Forbes. Retrieved from https://www.forbes.com/sites/ericervin/20 18/08/16/blockchain-technology-set-to- revolutionize-global-stock- trading/?sh=ca987a74e568 8. ET Contributors. (2018, January). How blockchain will change the way you trade in stock markets. The Economic Times. Retrieved from https://economictimes.indiatimes.com/ma rkets/stocks/news/how-blockchain-will- Ms. Anusha S Sangondimath, Prof. S. B Kamashetty 85 Vol.4 No.20 Vol.4 No.20 Ms. Anusha S Sangondimath, Prof. S. B Kamashetty IJAAR 19. SAMCO. (2023, March). The Role of Artificial Intelligence in Stock Market Prediction. Retrieved from SAMCO: Stock Market Library: https://www.samco.in/knowledge- center/articles/artificial-intelligence-in- stock-market-prediction/ change-the-way-you-trade-in-stock- markets/articleshow/62161610.cms 9. Frankenfield, J. (2023, March). Distributed Ledger Technology (DLT): Definition and How It Works. Retrieved from Investopedia: https://www.investopedia.com/terms/d/dis tributed-ledger-technology-dlt.asp 20. Shobhit Seth. (2023, March). Automated Investing. Retrieved from Investopedia: https://www.investopedia.com/articles/act ive-trading/101014/basics-algorithmic- trading-concepts-and- examples.asp#:~:text=Algorithmic%20tra ding%20(also%20called%20automated,im possible%20for%20a%20human%20trade r. 10. FRANKENFIELD, J. (2023, February). What Are Smart Contracts on the Blockchain and How They Work. Retrieved from Investopedia: https://www.investopedia.com/terms/s/sm art-contracts.asp 11. GAA Advisory. (n.d.). Retail Investors in India. IGAA. 12. Gauri, D. P. (2023, February). Potential of smart contracts in blockchain. The Economic Times. 21. Siddharth Nair, D. G. (2020). A STUDY ON APPLICATION OF ARTIFICIAL INTELLIGENCE IN STOCK MARKET PREDICTION. International Journal of Creative Research Thoughts (IJCRT), 8(6), 1403-1414. 13. Jacqueline Prause. (2021). Digitization Vs Digitalization. Retrieved from sap.com: https://www.sap.com/products/erp/digitiza tion-vs-digitalization.html 22. The Associated Chambers of Commerce and Industry of India. (2023). Indian Capital Market: Driver of Growth During Amrit Kaal. 14. Jiya Goel, S. V. (2020). Artificial Intelligence in Stock Market: Concepts, Applications and Limitations. International Journal of Advances in Engineering and Management (IJAEM), 2(9), 578-583. doi:DOI: 10.35629/5252- 0209578583 23. The Future of Digital Transformation In India. (2023). Retrieved from Insight Success: https://www.insightssuccess.in/the- future-of-digital-transformation-in-india/ 15. Motilal Oswal. (n.d.). All You Need To Know About Dematerialization Of Shares. Retrieved from Motilal Oswal PHYGITAL: https://www.motilaloswal.com/blog- details/all-you-need-to-know-about- dematerialization-of-shares/19929 24. Vinith V Bhandarkar, A. A. (2019). DIGITAL STOCKS USING BLOCKCHAIN TECHNOLOGY THE POSSIBLE FUTURE OF STOCKS? International Journal of Management (IJM), 44-49. 25. Vonko, D. (2022, May). Neural Networks: Forecasting Profits. Retrieved from Investopedia: https://www.investopedia.com/articles/tra ding/06/neuralnetworks.asp 16. Motilal Oswal. (n.d.). The AI and Machine Learning Revolution in the Stock Trading Industry. Retrieved from Motilal Oswal PHYGITAL: https://www.motilaloswal.com/blog- details/the-ai-and-machine-learning- revolution-in-the-stock-trading- industry/20295 17. Prareek Rani, A. S. (2015). Digitalisation of Financial Markets: Impact and Future. International Journal of Research in Finance and Marketing, 29-33. 18. Priem, R. (2018). Distributed ledger technology for securities clearing and settlement: benefits, risks, and regulatory implications. Financial Innovation, 1-25. doi:https://doi.org/10.1186/s40854-019- 0169-6 Ms. Anusha S Sangondimath, Prof. S. B Kamashetty Ms. Anusha S Sangondimath, Prof. S. B Kamashetty 86
https://openalex.org/W4205944689	https://iris.uniroma1.it/bitstream/11573/1600892.2/1/D%e2%80%99Erme_Effect-fibrillated-cellulose_2022.pdf	English	null	Effect of Fibrillated Cellulose on Lime Pastes and Mortars	Materials	2,022	cc-by	8,347	Chiara D’Erme 1,2, Walter Remo Caseri 2,* and Maria Laura Santarelli 1,* Chiara D’Erme 1,2, Walter Remo Caseri 2,* and Maria Laura Santarelli 1,* 1 DICMA—Department of Chemical Engineering Materials and Environment, Sapienza Università di Roma, Via Eudossiana 18, 00184 Rome, Italy; chiara.derme@uniroma1.it 1 DICMA—Department of Chemical Engineering Materials and Environment, Sapienza U Via Eudossiana 18, 00184 Rome, Italy; chiara.derme@uniroma1.it 1 DICMA—Department of Chemical Engineering Materials and Environment, Sapienza Università di Roma, Via Eudossiana 18, 00184 Rome, Italy; chiara.derme@uniroma1.it y 2 Laboratory for Multifunctional Materials, Department of Materials, ETH Zürich, Vladimir-Prelog-Weg 5, 8049 Zürich, Switzerland Vladimir-Prelog-Weg 5, 8049 Zürich, Switzerland g g * Correspondence: walter.caseri@mat.ethz.ch (W.R.C.); marialaura.santarelli@uniroma1.it (M.L.S.) * Correspondence: walter.caseri@mat.ethz.ch (W.R.C.); marialaura.santarelli@uniroma1.it (M.L.S.) Abstract: The use of nanocellulose in traditional lime-based mortars is a promising solution for green buildings in the frame of limiting the CO2 emissions resulting from Portland Cement produc- tion. The influence of the fibrillated cellulose (FC) on lime pastes and lime-based mortars was stud- ied incorporating FC at dosages of 0%, 0.1%, 0.2% and 0.3wt% by weight of binder. The lime pastes were subjected to thermal and nitrogen gas sorption analyses to understand if FC affects the for- mation of hydraulic compounds and the mesoporosities volume and distribution. The setting and early hydration of the mortars were studied with isothermal calorimetry. The mechanical perfor- mances were investigated with compressive and three-point-bending tests. Furthermore, fragments resulting from the mechanical tests were microscopically studied to understand the reinforcement mechanism of the fibres. It was found that 0.3wt% of FC enhances the flexural and compressive strengths respectively by 57% and 44% while the crack propagation after the material failure is not affected. Keywords: lime; mortar; fibre reinforced concrete; natural fibres; cellulose Citation: D’Erme, C.; Caseri, W. R.; Santarelli, M.L. Effect of Fibrillated Cellulose on Lime Pastes and Mortars. Materials 2022, 15, 459. https://doi.org/10.3390/ma15020459 Academic Editor: Gabriele Milani Received: 24 November 2021 Accepted: 6 January 2022 Published: 8 January 2022 Citation: D’Erme, C.; Caseri, W. R.; Santarelli, M.L. Effect of Fibrillated Cellulose on Lime Pastes and Mortars. Materials 2022, 15, 459. https://doi.org/10.3390/ma15020459 Academic Editor: Gabriele Milani Received: 24 November 2021 Accepted: 6 January 2022 Published: 8 January 2022 www.mdpi.com/journal/materials Materials 2022, 15, 459. https://doi.org/10.3390/ma15020459 1. Introduction https://doi.org/10.3390/ma15020459 www.mdpi.com/journal/materials Materials 2022, 15, 459 2 of 19 fibres employed: the source (e.g., plant, waste materials, bacteria), their production pro- cess and the strategy of incorporation used. fibres employed: the source (e.g., plant, waste materials, bacteria), their production pro- cess and the strategy of incorporation used. The use of nanocellulose in traditional lime-based mortars is a promising solution for green buildings in the frame of limiting the CO2 emissions resulting from Portland Ce- ment production[16]. Unlike ordinary Portland cement, lime is fired at lower tempera- tures (below 1000 °C), causing less CO2 emissions. Additionally, slaked lime partially re- absorbs the carbon dioxide emitted during the production process as it recarbonates over its in-use phase, thus resulting in lower net carbon emissions [17,18]. Moreover, lime can as well be used for the retrofit of historical buildings [19], since it is well known that ce- ment-based mortars are not chemically and physically compatible with aged and porous materials [20]. In this context, the use of fibres can indeed help increasing the flexural strength, avoiding crack propagation in mortars and plasters, eventually delaying the need for maintenance interventions. However, to our knowledge, no extensive study of the influence of nano- and micro-fibrillated cellulose materials on the hydration and on the mechanical properties of lime-based mortars has been performed so far. This study aims at exploring the performance of cellulose nanomaterials in traditional lime-based mortars in order to understand the composites applicability and potential. 1. Introduction Cement is the most widely used building material and its production is responsible for a large share of greenhouse gas emissions. It has been estimated that cement produc- tion can be accounted for about 5% of the total anthropogenic CO2 emissions and aver- agely 12–15% of the industrial energy consumption of each country [1–3]. For this reason, several efforts have been directed on reducing the environmental impact of concrete e.g., replacing or blending ordinary Portland cement with alternative binders or using eco- friendly additives. https://doi.org/10.3390/ma15020459 The use of fibres in mortars and concrete can help to enhance the properties of the final composites. Natural fibres were commonly employed during ancient times to rein- force and to reduce the shrinkage in mortars and concrete; nowadays their application in modern composites is newly evaluated as they represent a renewable, economical and abundant resource [4] and can become an alternative to the use of other synthetic fibres. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Publisher’s Note: MDPI stays neu- tral with regard to jurisdictional claims in published maps and institu- tional affiliations. Nanocellulose materials can overall enhance the mechanical properties of modern concrete due to their high aspect ratio and high Young’s modulus [5–7]. Moreover, due to their high hygroscopicity they can act as an internal curing agent of cement, preventing self-desiccation and promoting hydration as well as autogenous healing phenomena [8– 10]. Their application as viscosity modifiers in self-compacting concrete (SCC) allows to stabilise the fresh concrete, inhibiting bleeding and segregation phenomena [11]. Cellu- lose fibres in concrete can also help tuning the composite porosity and changing its hy- grothermal behaviour [12–14]. On the other hand, some disadvantages must be consid- ered, e.g., the incorporation of this kind of fibres can highly decrease the workability of the fresh concrete, making the casting process more difficult. Moreover, the results in lit- erature are highly scattered [15] as they strongly depend on the nature of the cellulose Copyright: © 2022 by the authors. Li- censee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and con- ditions of the Creative Commons At- tribution (CC BY) license (https://cre- ativecommons.org/licenses/by/4.0/). Materials 2022, 15, 459. 2.1. Materials The samples used in the following analysis were prepared with natural hydraulic lime Hydradur NHL 5 (Otterbein, Großenlüder-Müs, Germany) as binder and a 0/4 sili- ceous quarry sand (Eberhard, Kloten, Switzerland) as aggregate. X-ray diffraction anal- yses show that the main phases present in NHL5 are portlandite, calcium silicates, calcium aluminates and calcite, while the sand is mainly constituted by quartz and carbonates (calcite and dolomite) and plagioclases (albite) in minor quantities (Figure 1). In thermo- gravimetric analysis (TGA) of NHL5 the presence of uncalcinated limestone is more evi- dent, the double peak in the derivative (DTG) between 500 °C and 700 °C corresponds to the loss in CO2 by decomposition of carbonates (Figure 2). (a) (b) Figure 1. X-ray diffraction patterns on the raw materials: NHL5 (a) and quarry sand (b). (a) (b) Figure 1. X-ray diffraction patterns on the raw materials: NHL5 (a) and quarry sand (b). 3 of 19 Materials 2022, 15, 459 Figure 2. TGA/DTG of NHL5 produced by Otterbein, Germany. 0 200 400 600 800 1000 0.00 -0.05 -0.10 -0.15 -0.20 -0.25 d[Weight(%)]/T(°C) T(°C) 80 85 90 95 100 Weight (%) Figure 2. TGA/DTG of NHL5 produced by Otterbein, Germany. 2.2. Methodology and Experiments 2.2. Methodology and Experiments Fibrillated Cellulose Celova, a type of fibrillated cellulose (FC) produced by Weidmann, (Weidmann, Rap- perswil-Jona, Switzerland), was applied for the following experiments. Celova was cho- sen after preliminary Vicat tests, to evaluate the setting time, and mechanical tests, as de- scribed previously [21]. The commercial batch, provided as gel, has an average solid con- tent of 3.3% w/w. The water retention value (WRV%, in percent) of cellulose was calculated to quantify the maximum amount of water the fibres can retain from a suspension. The as-received gel was diluted to 1% and 2% solid content (w/w) with distilled water and then sonicated in an ultrasonic bath for 10 min in order to improve the dispersion of the fibres. The suspension was then centrifugated at 4000 rpm (2701 RCF) for 20 min, the supernatant solution disposed and the cellulose pulp dried in an oven at 60 °C until constant mass was reached. The WRV% was calculated according to Equation (1) WRV% = 𝑚 − 𝑚ௗ௥௬ 𝑚ௗ௥௬ × 100 (1) (1) where m is the mass of the gel and 𝑚ௗ௥௬ the mass of the dry sample. To understand the fibrils morphology, the gel was diluted with deionized water to 0.001% (w/w) solid content, dropped on a silicon wafer and coated with carbon for scan- ning electron microscopy (SEM) investigations using a Zeiss Leo-1530 (Zeiss, Oberkochen, Germany). 2.2.1. Sample Preparation The lime pastes were prepared by manually mixing the water and the lime, with a ratio of 0.83, in a beaker for 5 min, until a homogeneous paste was obtained. Samples with 0%, 0.1%, 0.2% and a 0.3% of FC, dosed by weight of binder (w/w), were prepared by pre- dispersing the FC in water using an ultrasonic bath and then incorporated in the lime paste (Table 1). The water content was adjusted subtracting the water introduced with the gel Celova and already absorbed in the fibres. The mixtures were then transferred to a sealed box where a relative humidity (RH) of 65% was maintained with a saturated NaNO2 solution and kept at a temperature of 23 ± 1 °C until the designated sample ages for the tests. Materials 2022, 15, 459 4 of 19 Table 1. Sample codes of the lime pastes prepared with a water to binder ratio of 0.81 and relative weight content of fibrillated cellulose (FC) per weight of binder. Mix FC (%) n0 0 n01 0.1 n02 0.2 n03 0.3 Table 1. Sample codes of the lime pastes prepared with a water to binder ratio of 0.81 and relative weight content of fibrillated cellulose (FC) per weight of binder. A standard mixer (Hobart N50) was employed to prepare mortars with a binder:ag- gregate:water volume ratio of 1:1:0.6. Fractions of 0%, 0.1%, 0.2% and a 0.3% of FC dosed by weight of binder (w/w) were incorporated in the mixtures (Table 2). A polycarboxylate (PCE) superplasticiser , MasterGlenium ACE 30 (Master Builders Solution, Maharashtra, India), dosed by weight of binder (w/w), was used to increase the mortar workability and to obtain slumps of approximately 125 mm (Figure 3) keeping the same water to binder ratio (w/b). The slumps to evaluate the consistency of the mixes were obtained with the mean of a flow table following the procedure for mortars described in EN 1015-3[22]. The FC was pre-dispersed in the water and stirred at a speed of 281 rpm for 5 min, before proceeding to the mortar preparation. The process started by mixing the NHL, the water and the superplasticiser for 1 min at low speed (136 rpm). After that, the aggregate was added and blended for 1 min at a speed of 281 rpm. 2.2.3. Nitrogen Gas Sorption Nitrogen sorption experiments were performed on a Quantachrome Autosorb-iQ-C- XR (Quantachrome Instruments, Graz, Austria) at 77 K. Samples of lime pastes and mor- tars were crushed into pieces of 12 mm maximum diameter and were later outgassed at 60 °C for a minimum of 24 h, before the gas sorption analyses were performed. The spe- cific surface area of specimens was calculated with the Brunauer-Emmett-Teller (BET) multi-point method. The pore size and pore volume were determined by the density func- tional theory (DFT) analysis. 2.2.2. Hydration Study Thermogravimetric analyses were performed on the lime pastes to understand if the fibres can affect the hydration mechanisms. At each time a portion of the paste was sam- pled, crushed and analysed without any further treatment with a Mettler Toledo TGA/DSC 3 + Stare System (Mettler Toledo, Schwerzenbach, Switzerland) under air at- mosphere at a heating rate of 10 °C/min, from ambient temperature to 980 °C. It should be noted that in this work no sample processing or drying was made as it has been proved that this can affect the final results and render the hydrates instable [23]. The thermograms were divided in temperature ranges related to four phenomena assumed to occur in the sample (Figure 4) [23,24]. The first peak within the temperature range of 25 °C–105 °C was attributed to free water (Lw) and was not considered in the calculations. The phenomena within the temperature range of 105–370 °C was assumed to represent dehydration reac- tions (LdH), the third one between 400 °C and 600 °C to the dehydroxylation of portland- ite (LdX) and the last region to the decarbonation of calcite and dolomite impurities (LdC). Admittedly this division is somewhat arbitrary since certainly, for instance, the tempera- ture range of free water desorption and dehydration reactions is not sharply separated. The remaining paste was dried under vacuum (24 mbar) for a minimum of 24 h and then subjected to X-ray diffraction (XRD) measurements performed on a PANalytical Em- pyrean instrument (Malvern Panalytical, Malvern, UK) equipped with a Cu Kα X-ray tube (45 kV, 40 mA) and a monochromator. Figure 4. TGA/DTG plot of a lime paste and ranges of temperature related to the principal phenom- ena occurring in the sample (see text). Figure 4. TGA/DTG plot of a lime paste and ranges of temperature related to the principal phenom- ena occurring in the sample (see text). 2.2.1. Sample Preparation The mixer was stopped for 90 s to scrape the mortar from the walls and finally the mixture was blended at a speed of 281 rpm for 4 min. To prepare the samples for the mechanical tests, fresh mixtures were poured in 20 × 20 × 80 mm moulds. The moulds were filled to the half of their capacity and compacted for 30 s on a vibrating table. Afterwards, a second layer of mixture was poured, and the samples were compacted again. The samples were kept in climatic cham- ber at 99.9% RH and 20 °C for 5 days before demoulding; after being demoulded they were moved to a climate chamber at 65% RH and 20 °C and cured there until the age for testing was reached. Figure 3 Image of a lime mortar representative slump obtained in agreement with EN 1015-3 Figure 3. Image of a lime mortar representative slump obtained in agreement with EN 1015-3. Figure 3. Image of a lime mortar representative slump obtained in agreement with EN 1015-3. Table 2. Sample codes of the lime mortars prepared with w/b = 0.61 and relative fibrillated cellulose and superplasticiser (SP) dosage (w/w) per weight of binder. Mix FC (%) SP (%) N0 0 0.8 N01 0.1 1.0 N02 0.2 1.1 N03 0.3 1.2 Table 2. Sample codes of the lime mortars prepared with w/b = 0.61 and relative fibrillated cellulose and superplasticiser (SP) dosage (w/w) per weight of binder. 5 of 19 Materials 2022, 15, 459 2.2.4. Isothermal Calorimetry Calorimetric measurements were performed on about 70 g of mortar in an isothermal calorimeter TAM Air (Waters GmbH, UB TA Instruments, Eschborn, Germany) at 23 °C. The data were collected up to 30 h as at later stages the heat release was below the machine sensitivity. The calorimetric test was started just at the end of the mixing procedure and Materials 2022, 15, 459 6 of 19 6 of 19 after checking the target slump. The exact starting time of the measurement was consid- ered in the calculation. The results were normalised by the dry content of NHL and ex- pressed in terms of heat rate (mW/g) and cumulative heat (J/g). 2.2.5. Mechanical Tests Mechanical tests were performed using a ZwickRoell universal testing machine with a 20 kN load cell and the results were calculated according to the BS EN 12390 norm [25,26]. Three-point bending tests was carried out for each type of mix at 28 d and at 45 d of curing. The investigation at the longer time of curing was chosen because lime mortars develop their strength more slowly. The support span was set at 60 mm and the load was applied with a rate of 0.5 mm/min. After the flexural test, cubic portions (20 × 20 × 20 mm) of each specimen resulting halves were mechanically tested by compression applying the load at a rate of 1 mm/min. Furthermore, the curves resulting from the three-point-bending test were analysed to understand if the fibres also affect the brittle behaviour of the material. The experimental displacement data (mm) were transformed in strain ε (%) by the following equation: ε = 6𝛥𝑙 𝑑 𝑙ଶ × 100 (2) (2) where Δ𝑙 is the displacement (mm), d is the depth or thickness of tested beam (mm) and l is the support span (mm). The toughness of the material was estimated by integrating the total area (A) of the stress (MPa)–strain (%) curve, while the area (Ab) after the break point εb was calculated to have more information about the fibres possible effect on the crack propagation. The flexural modulus, E, was estimated according to Equation (3): E = 1 4 lଷ𝑠 𝑏𝑑ଷ (3) (3) where l is the support span (mm), b is the width of test beam (mm), d is the depth or thickness of tested beam (mm) and s is the slope of the initial straight-line portion of the load (N)—deflection (mm) curve. The results of the mechanical tests at 45 d were analysed according to Weibull statis- tics, which is often used to study the failure of brittle materials and that can also describe accurately the failure occurring in concrete [27]. To extrapolate the desired parameters the Weibull function was linearised: 𝑙𝑛ቜ𝑙𝑛ቆ 1 1 −𝑃௙ ቇቝ= 𝑚(𝑙𝑛𝜎௙ − 𝑙𝑛𝜎଴) (4) (4) where 𝑃௙ is the probability of failure, calculated according to Equation (5), m is the Weibull Modulus (also defined as the shape parameter), 𝜎௙ is the strength at failure, 𝜎଴ is the scale parameter or the characteristic strength for which 𝑃௙ is 63.2%. 2.2.5. Mechanical Tests 𝑙𝑛ቜ𝑙𝑛ቆ 1 1 −𝑃௙ ቇቝ= 𝑚(𝑙𝑛𝜎௙ − 𝑙𝑛𝜎଴) (5) (5) where i and n are respectively the rank and the number of data. 2.2.6. Microscopic Observations Fragments of mortars obtained from the mechanical test were embedded in epoxy resin to study the morphology of the samples. The samples were optically observed with a Keyence multiscan VHX 6000 microscope (Keyence, Osaka, Japan) and then coated with carbon and subsequently studied by scanning electron microscope (SEM) (Zeiss, Ober- kochen, Germany). The fractured surfaces obtained from the three-point-bending test were analysed us- ing the same Keyence multiscan VHX optical microscope. The instrument can produce a 3D scan image of the sample so that the morphology and the surface roughness can be Materials 2022, 15, 459 7 of 19 7 of 19 studied. Eleven samples for the reference and eleven samples with 0.3% FC were optically scanned at 100× magnification to understand if the fibres also affect the crack propagation and therefore the final roughness of the broken samples. The surface roughness parame- ters calculated with the Keyence built-in software are defined in the following way: • The arithmetical mean height (Sa) is the average value of the absolute value of height at each point, z(x,y), in the defined area (A). 𝑆𝑎= 1 𝐴ඵ\|𝑧(𝑥, 𝑦)\|𝑑𝑥𝑑𝑦 . ஺ (6) (6) • The root mean square height (Sq) is the root mean square of height at each point in the area; it is equivalent to the standard deviation of height. • The root mean square height (Sq) is the root mean square of height at each point in the area; it is equivalent to the standard deviation of height. 𝑆𝑞= ඨ1 𝐴ඵ𝑧ଶ(𝑥, 𝑦)𝑑𝑥𝑑𝑦 . ஺ (7) (7) • The maximum height (Sz) is the sum of the maximum peak height (Sp) and the max- imum valley (Sv) depth in the definition area. • The maximum height (Sz) is the sum of the maximum peak height (Sp) and the max- imum valley (Sv) depth in the definition area. • The maximum height (Sz) is the sum of the maximum peak height (Sp) and the max- imum valley (Sv) depth in the definition area. 𝑆𝑧= 𝑆𝑝 + 𝑆𝑣 (8) (8) • The skewness (Ssk) is the cubic average of height which is rendered dimensionless with the cube of Sq. It indicates the asymmetric property of height distribution which is centred on the reference surface. When Ssk equals 0, it means that the height dis- tribution is symmetric with respect to the reference plane. 2.2.6. Microscopic Observations When Ssk < 0, Ssk > 0, the height distribution is skewed either higher or lower, relative to the reference plane. 𝑆𝑠𝑘= 1 𝑆௤ଷቈ1 𝐴ඵ𝑧ଷ(𝑥, 𝑦)𝑑𝑥𝑑𝑦 . ஺ ቉ (9) (9) • The kurtosis (Sku) is the average fourth power of height which is rendered dimen- sionless with the fourth power of Sq. It indicates the sharpness of the height distribu- tion. When Sku equals 3, it means that the height distribution of a scale-limited sur- face in characterised by a normal distribution. When Sku < 3 or Sku > 3, the height distribution takes either a collapsed or sharpened shape. 𝑆𝑘𝑢= 1 𝑆௤ସቈ1 𝐴ඵ𝑧ସ(𝑥, 𝑦)𝑑𝑥𝑑𝑦 . ஺ ቉ (10) (10) 3.2. Hydration Study Hydraulic lime mainly sets by the reaction of dicalcium silicate (C2S) with water to form calcium silicate hydrates (CSH); some hydrated lime (free lime) is also formed in addition to the one already present in the powder before the reaction (Equation (11)) 𝐶ଶ𝑆+ HଶO → 𝐶𝑆𝐻 + Ca(OH)ଶ (11) (11) 𝐶ଶ𝑆+ HଶO → 𝐶𝑆𝐻 + Ca(OH)ଶ The lime paste is highly alkaline, but this condition changes progressively on aging because carbon dioxide starts penetrating into the system once the paste is set and trans- forms the hydrated lime into calcium carbonate. Carbon dioxide also reacts with the hy- drated calcium silicate forming calcium carbonate and amorphous silica (SH) (Equation (12)). (12) 𝐶𝑆𝐻+ Ca(OH)ଶ+ COଶ→ CaCOଷ+ 𝑆𝐻+ HଶO (12) As the introduced FC has a high WRV%, it was crucial to understand if the setting and hardening reactions in the lime pastes could be affected; more specifically emphasis was put in understanding if the formation of CSH, important for the final mechanical strength of the mortar, was inhibited or delayed. It has been discussed that in Portland cement-based mortars and concretes, natural fibres can slow down the hydration at early stages, but natural fibres can also act as an internal reservoir of water avoiding the desic- cation of composites and therefore at last promoting the hydration of mortars [9]. The XRD pattern (Figure 7) at 2 d and after 28 d show in all four samples a gradual decrease in the Portlandite content, while at the same time the peaks assigned to calcite increase in intensity. The signals associated to the formation of the hydrates (CSH and CAH in the form of katoite) slowly increase, too. To obtain a quantitative impression of the pastes composition, thermogravimetric analyses (TGA) were performed. The TGA curves show that the formation rate of the hy- drates is comparable in all the four samples examined. After 21 d of curing the mass loss between 105 °C and 370 °C fluctuates around 4.5% of the total mass up to 45 d. The frac- tions of portlandite and of calcite, on the other hand, are different in the samples with and without fibres. In samples with fibres the decrease in portlandite and the increase in the calcite content seem to be slower. 3.1. Fibrillated Cellulose The fibres used in this study are composed of a complicated network (Figure 5), as observed in the scanning electron microscope (SEM) images. The primary fibre diameters are below 30 nm, but due to the partial defibrillation bigger bundles of fibres were ob- served. The aspect ratio is high as the fibre length is in the micrometric scale: however, it was not possible to get an accurate average of the fibre lengths, due to the complicated network they form. 8 of 19 8 of 19 Materials 2022, 15, 459 (a) (b) Figure 5. SEM images at different magnifications of a sample of new Celova produced by Weidmann, Switzerland with SE InLens (a) and normal SE (b) detector; bundles of cellulose fibres above 100 nm arise all over the sample. (a) (b) (a) (b) Figure 5. SEM images at different magnifications of a sample of new Celova produced by Weidmann, Switzerland with SE InLens (a) and normal SE (b) detector; bundles of cellulose fibres above 100 nm arise all over the sample. The water retention value ranges between 1500% and 2000% per mass of dry cellulose (Figure 6). The loss in workability experienced in preparing the mortars and the required increase in the SP content to obtain similar slumps (see Section 2.2.1. Sample Preparation) are thus not only due to the complex 3D net of the FC but also to the high amount of water absorbed by the cellulose. However, since the mechanism of water release is still not clear, the absorbed water was not subtracted from the calculation of the total water to binder ratio. Figure 6. Water retention value (WRV%) of the fibrillated cellulose (FC) gel used to cast the samples diluted to 1% and 2% w/w. Figure 6. Water retention value (WRV%) of the fibrillated cellulose (FC) gel used to cast the samples diluted to 1% and 2% w/w. Materials 2022, 15, 459 9 of 19 9 of 19 3.3. Nitrogen Gas Sorption The calculated specific surface area for the lime pastes n0, n01, n02 and n03 ranges between 9 m2/g and 11 m2/g without a significant difference within the accuracy of the measurements. The pores volume distribution is also very similar among the series with the exception of n03 for which the mesoporosities are lower in the range of 20–70 nm (Figure 8). Moreover, the mortars show a similar trend, but the presence of aggregates and inhomo- geneities of the samples leads to a higher scatter in the results. These results could indicate that the fibres partially reduce the mesoporosities of the composite. Figure 8. Pore size distribution calculated by DFT method from N2 adsorption–desorption isotherms for lime pastes with 0% FC (n0) and 0.3% fc (n03). 0 10 20 30 40 50 60 70 80 0.00 0.02 0.04 0.06 0.08 0.10 dV(logd) (cm 3/g) Pore width (nm) n0 n03 Figure 8. Pore size distribution calculated by DFT method from N2 adsorption–desorption isotherms for lime pastes with 0% FC (n0) and 0.3% fc (n03). 3.2. Hydration Study At 45 d of curing the LdX for n0 is near 4% and between 5.3% and 6.5% for n01, n02 and n03, while LdC is over 22% for n0 and below 18% for n01, n02 and n03. Further studies at different relative humidities should be conducted to un- derstand if FC can play a role in CO2 penetration by affecting the paste porosity or if the water stored in the FC can indeed delay the carbonation by avoiding the self-desiccation. (a) (b) Figure 7. XRD pattern of the four pastes at 2 d (a) and after 28 d of curing (b). (b) ( ) (b) (a) Figure 7. XRD pattern of the four pastes at 2 d (a) and after 28 d of curing (b). Materials 2022, 15, 459 10 of 19 3.3. Nitrogen Gas Sorption 3.4. Isothermal Calorimetry The specific heat development per gram of binder is presented in Figure 9. A maxi- mum is observed after 1 h. The rapid increase in the heat release can be connected to the heat of wetting [28]. A retardant effect on the setting hydration of cement mortars con- nected to the use of PCE was often reported [29,30]; this phenomenon was not observed in our lime mixes, despite the difference in the superplasticiser content. On the contrary a slight shift on the left was observed for N02 and N03, the samples with higher superplas- ticiser content. The maximum heat rate value is higher in samples with FC and could be connected to the partial alkaline hydrolysis of the cellulose by peeling [31]. However, the curve associated to N0 is broader and the cumulative heat recorded is higher. 11 of 19 11 of 19 Materials 2022, 15, 459 Figure 9. Isothermal calorimetry results for the four different mortar mixtures expressed in heat rate (mW) and heat (J) normalised to the mass of binder. 0 10 20 30 0.0 0.5 1.0 0 2 4 6 8 10 Heat rate (mW/g) t (h) N0 N01 N02 N03 Heat (J/g) t (h) Figure 9. Isothermal calorimetry results for the four different mortar mixtures expressed in heat rate (mW) and heat (J) normalised to the mass of binder. 3.5. Mechanical Tests Preliminary tests showed that 0.1% and 0.2% FC affect less significantly the mechan- ical properties of the mortars, in particular the compression strength (Figure 10). There- fore, the study focused on the comparison between the 0% and 0.3% FC mixtures. Each mix was casted three times to increase the statistics, reduce the dispersion of data (typical of this kind of building materials) and understand the reproducibility of the results. For each mix, 10 and 15 samples were tested respectively at 28 d and at 45 d. The results from the mechanical tests in terms of flexural strength (ffl) and compression strength (fc) are respectively shown in Tables 3 and 4. The flexural and compression strength in samples with 0.3% FC are improved respectively by 47% and 41% at 28 d and by 50% and 42% at 45 d (Figure 11). (b) (a) (b) Figure 10. Flexural strength ffl (a) and compression strength fc (b) of samples with 0% (N0), 0.1% (N01), 0.2% (N02) and 0.3% FC (N03) at 28 d (red) and 45 d (blue). (a) (a) (b) Figure 10. Flexural strength ffl (a) and compression strength fc (b) of samples with 0% (N0), 0.1% (N01), 0.2% (N02) and 0.3% FC (N03) at 28 d (red) and 45 d (blue). 12 of 19 12 of 19 Materials 2022, 15, 459 (a) (b) Figure 11. Flexural strength ffl (a) and compression strength fc (b) of samples with 0% FC (N0) and 0.3% FC (N03) at 28 d (red) and 45 d (blue). (b) (b) (a) (a) Figure 11. Flexural strength ffl (a) and compression strength fc (b) of samples with 0% FC (N0) and 0.3% FC (N03) at 28 d (red) and 45 d (blue). Table 3. Flexural strength (ffl) of samples with 0% FC (N0) and 0.3% FC (N03). Mix Day28 Day45 N0 1.7 ± 0.4 2.2 ± 0.3 N03 2.5 ± 0.5 3.3 ± 0.3 Table 4. Compression strength (fc) of samples with 0% FC (N0) and 0.3% FC (N03). Mix Day28 Day45 N0 4.1 ± 0.3 4.8 ± 0.3 N03 5.8 ± 0.4 6.8 ± 0.5 Table 3. Flexural strength (ffl) of samples with 0% FC (N0) and 0.3% FC (N03). Representative experimental flexural stress–strain curves are shown in Figure 12 and were analysed to understand if the fibres also affect the brittle behaviour of the material. 3.5. Mechanical Tests The total area under the three-point-bending test curve is also improved in N03 (Table 5). On the other hand, Ab and εb are not significantly affected by the FC, so that the fibres seem to have little effect on the post-crack behaviour and deflection of the composite. The flexural elastic modulus E, on the contrary, is enhanced by 54% indicating the material is behaving in a more rigid way. Figure 12. Representative flexural stress–strain curves from the three-point-bending test for samples with 0% (N0) and 0.3% (N03) FC. Figure 12. Representative flexural stress–strain curves from the three-point-bending test for samples with 0% (N0) and 0.3% (N03) FC. Materials 2022, 15, 459 13 of 19 13 of 19 Table 5. Results of three-point bending test stress–strain curves: total area (A) of the stress (MPa)- strain (%) relation, area (Ab) after the break point (εb) and flexural modulus (E) of mortars with 0% FC (N0) and 0.3% FC (N03). Mix A (MPa%) Ab (MPa%) εb (%) E (MPa) N0 0.6 ± 0.1 0.22 ± 0.07 0.48 ± 0.04 641 ± 113 N03 0.8 ± 0.1 0.31 ± 0.07 0.42 ± 0.04 986 ± 104 The FC effect on the mechanical properties can be connected to the microstructural changes that the fibres can induce in composites and that were already observed in Port- land cement concrete. These changes can be due to the higher elastic modulus of the crys- talline portion of cellulose nanomaterials (usually in the range of 100–130 GPa) [32], their high specific surface area and the interaction of the surface –OH groups with CSH and portlandite that leads to a strong fibre–matrix bonding, which involves a better stress transfer from the matrix to the nanofibers, but in return can also cause an excessive em- brittlement of the composite [33,34]. The Weibull statistics was applied on the data of the mechanical strength at 45 d (Fig- ure 13), a time at which, in the frame of our work, the mortars reached the highest strength. The Weibull analysis confirms the overall enhancement of the mechanical properties (Ta- ble 6) determined by the fibres, and the characteristic strength σ0 is close to the normal distribution averages calculated. The flexural and compressive σ0 in the N03 series are enhanced respectively by 57% and 44%. 3.5. Mechanical Tests The Weibull modulus m of the flexural data is considerably higher in samples with 0.3% fibre content, indicating that the distribution is narrower and the results less scattered. A higher Weibull modulus is indeed usually more desirable as the material has a lower probability to fail at a stress much lower than σ0. (a) (b) Figure 13. Linear fit of the experimental data from the three-point bending (a) and compression (b) tests analysed with Weibull statistics. (a) (b) (b) (a) Figure 13. Linear fit of the experimental data from the three-point bending (a) and compression (b) tests analysed with Weibull statistics. Table 6. Average experimental strength values and parameters obtained after Weibull fitting. Mix ffl (MPa) Flexural σ0(MPa) m R2 N0 2.2 2.3 4.1 0.93 N03 3.3 3.6 7.0 0.94 fc (MPa) Compressive σ0(MPa) m R2 N0 4.8 5.2 6.3 0.97 N03 6.8 7.5 6.1 0.98 Table 6. Average experimental strength values and parameters obtained after Weibull fitting. Materials 2022, 15, 459 14 of 19 3.6. Microscopic Observation Optical microscope observations (Figure 14) showed that both N0 and N03 are ho- mogeneous, and the aggregate is well dispersed in the binder. On the other hand, the samples with FC seem to have less voids and cracks as the embedding resin did not pen- etrate as much as in N0. (b) (a) (b) Figure 14. Embedded samples with 0% FC (a) and 0.3% FC (b) prior to the carbon coating observed with an optical microscope. (a) (b) (a) Figure 14. Embedded samples with 0% FC (a) and 0.3% FC (b) prior to the carbon coating observed with an optical microscope. SEM images confirm that a content of 0.3% fibrillated cellulose increases the com- pactness of the mortar, by reducing the presence of voids. Additionally, the aggregate to binder interphase, the interfacial transition zone (ITZ), appears to be also improved (Fig- ure 15). The ITZ is considered the weakest element of mortars and concretes, as the first cracks leading to material failure generally originate there. Therefore, a better interaction between binder and aggregate can lead to an enhancement of mechanical properties. Moreover, the general reduction of cracks and voids may improve the durability of the material, as cracks can be the main point of entry for water or aggressive chemical ions [35]. The FC effect in strengthening the ITZ and reducing the crack formation has already been observed in Portland cement concrete [12]. For the materials described in this work these aspects may as well be connected to the tendency that the filaments have to form a 3D network (Figure 5). This effect was also described elsewhere upon application of sim- ilar cellulose materials [11]. In this proposed reinforcement mechanism, the key to have a significant effect of the fibres is the formation of a continuous network that connects the particles in the mortar. This mechanism would also explain why lower loads of fibres do not significantly improve the mechanical strength of the resulting materials. On the other hand, no clear evidence of a bridging effect by the FC was found. It must be noted, though, that given the fibre morphology and size, it is extremely complicated to confidently dis- tinguish them from the hydraulic compounds formed during lime hydration (Figure 16). 15 of 19 Materials 2022, 15, 459 (a) (b) (a) (b) Figure 15. Backscattered electron (BSE) SEM images of mortars with 0% FC (a) and 0.3% FC (b). 3.6. Microscopic Observation (a) (b) (a) Figure 15. Backscattered electron (BSE) SEM images of mortars with 0% FC (a) and 0.3% FC Figure 15. Backscattered electron (BSE) SEM images of mortars with 0% FC (a) and 0.3% FC (b). Figure 16. Secondary electron (SE) SEM image of a possible filament of cellulose detected in a crack and immersed in the forming gel. Figure 16. Secondary electron (SE) SEM image of a possible filament of cellulose detected in a crack and immersed in the forming gel. The 3D topographies of the fractured surfaces of four representative samples are shown in Figure 17. The roughness parameters describing the two series are similar (Table 7). The calculated skewness shows that the peaks and valleys are normally distributed as the values are close to 0. The Sku for N0 defines that the distribution has a more collapsed shape. Overall, though, the 3D topographic images and the surface roughness parameters calculated did not underline any remarkable difference among the two series. 16 of 19 Materials 2022, 15, 459 Figure 17. Representative 3D topographic images (20 mm × 20 mm ca) of the mortars fractured sur- faces with 0% FC (N0) and 0.3% FC (N03). N03 N0 N03 Figure 17. Representative 3D topographic images (20 mm × 20 mm ca) of the mortars fractured sur- faces with 0% FC (N0) and 0.3% FC (N03). Table 7. Average surface roughness values obtained from 3D scans of fractured surfaces after three- point-bending tests. Table 7. Average surface roughness values obtained from 3D scans of fractured surfaces after three- point-bending tests. Mix Sa (µm) Sz (µm) Sq (µm) Ssk (µm) Sku (µm) N0 1110 ± 322 6450 ± 1445 1341 ± 381 0.1 ± 0.2 2.4 ± 0.3 N03 1157 ± 352 6472 ± 1219 1324 ± 405 0.1 ± 0.4 2.7 ± 0.5 4. Conclusions Fibrillated cellulose (FC) was incorporated in lime-based pastes and mortars from 0% up to 0.3% content by weight of binder (w/w). The fibres do not affect the hydration of the mortars appreciably, as the thermal stud- ies on the lime pastes underlined that the levels of hydraulic compounds formed are sim- ilar in all the samples. Nitrogen gas sorption analyses showed that 0.3% FC content deter- mines a decrease in the pores volume in the range of 20–70 nm. The different quantities of superplasticiser used for casting the mortars do not influ- ence the setting and the early hydration in a significant way. The mechanical performance of mortars with 0.3% FC (N03) was indeed positively affected: the flexural and compres- sive σ0 were enhanced respectively by 57% and 44%. On the other hand, the study of the three-point bending test curves did not show any significant FC effect on the post-crack Materials 2022, 15, 459 17 of 19 behaviour and deflection of the composite. The flexural elastic modulus E, on the contrary, was enhanced by 54% indicating the material behaves in a more rigid way. The FC effect on the mechanical properties can be connected to the microstructural changes that the fibres can induce in composites and that were already observed in Portland-cement-based concrete. These changes can be due to the higher elastic modulus of the crystalline portion of cellulose nanomaterials and their high specific surface area. Additionally, the interac- tion of the surface –OH groups with CSH and portlandite led to a stronger fibre–matrix bonding, which involves a better stress transfer from the matrix to the nanofibers, but in return can also cause an excessive embrittlement of the composite. The optical microscopy observations and the 3D topographies confirmed that FC does not affect the post-cracking behaviour of the mortars, while the scanning electron microscope (SEM) images showed that FC may promote a better adhesion between the binder and the aggregate strengthen- ing the interfacial transition zone and therefore helping to get a more compact and homo- geneous composite. Furthermore, the reinforcement mechanism may as well be connected to the continuous 3D network that the fibres form when incorporated in the fresh mortar. In conclusion, FC is a good option for strengthening lime-based mortars to be used in green-buildings. 4. Conclusions Related composites are also of high interest for application in the field of historical building conservation, as natural hydraulic lime is compatible with aged and porous materials and improving the flexural strength eventually reduces the need for maintenance interventions. On the other hand, with the type of fibres used in the current study, it was not possible to act against crack propagation, delaying it with bridging mech- anisms. Different kinds of cellulose fibres may be evaluated for this scope. Moreover, sur- face modification of fibres may be tested to improve their performance in the composite. Author Contributions: Conceptualization, C.D., W.R.C. and M.L.S.; methodology, C.D., W.R.C. and M.L.S.; investigation, C.D.; writing—original draft preparation, C.D.; review and editing, W.R.C.; supervision, W.R.C. and M.L.S. All authors have read and agreed to the published version of the manu- script. Funding: This research received no external funding. Institutional Review Board Statement: Not Applicable. Institutional Review Board Statement: Not Applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgements: The authors would like to thank cordially Robert Flatt and all the Physical Chemistry of Building Materials group, in particular Sara Mantellato, for their support in the use of their facilities for the sample preparation and for the isothermal calorimetry. We would also like to thank Theo A. Tervoort for providing the instrument for the mechanical tests and Elena Tervoort- Gorokhova for her support in the use of the scanning electron microscope and for the fruitful discussions. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. p g j y 4. Ma, W.; Qin, Y.; Li, Y.; Chai, J.; Zhang, X.; Ma, Y.; Liu, H. Mechanical properties and engineering appl reinforced concrete. Mater. Today Commun. 2020, 22, 100818. https://doi.org/10.1016/j.mtcomm.2019.10 6. Hisseine, O.A.; Omran, A.F.; Tagnit-Hamou, A. Influence of cellulose filaments on cement paste and concrete. J. Mater. Civ. Eng. 2018, 30, 04018109. https://doi.org/10.1061/(ASCE)MT.1943-5533.0002287. References Effect of cellulose nanofibrils and nanocrystals on physical properties of concrete. Constr. Build. Mater. 2019, 223, 1–11. https://doi.org/10.1016/j.conbuildmat.2019.06.145. 12. Barnat-Hunek, D.; Szymańska-Chargot, M.; Jarosz-Hadam, M.; Łagód, G. Effect of cellulose nanofibrils and nanocrystals on physical properties of concrete. Constr. Build. Mater. 2019, 223, 1–11. https://doi.org/10.1016/j.conbuildmat.2019.06.145. p y p p p g j 13. Collet, F.; Chamoin, J.; Pretot, S.; Lanos, C. Comparison of the hygric behaviour of three hemp concretes. Energy Build. 2013, 62, 294–303. https://doi.org/10.1016/j.enbuild.2013.03.010. 13. Collet, F.; Chamoin, J.; Pretot, S.; Lanos, C. Comparison of the hygric behaviour of three hemp concretes. Energy Build. 2013, 62, 294–303. https://doi.org/10.1016/j.enbuild.2013.03.010. 14. Mejdoub, R.; Hammi, H.; Suñol, J.J.; Khitouni, M.; M‘nif, A.; Boufi, S. Nanofibrillated cellulose as nanoreinforcement in Portland cement: Thermal, mechanical and microstructural properties. J. Compos. Mater. 2017, 51, 2491–2503. https://doi.org/10.1177/0021998316672090. 14. Mejdoub, R.; Hammi, H.; Suñol, J.J.; Khitouni, M.; M‘nif, A.; Boufi, S. Nanofibrillated cellulose as nanoreinforcement in Portland cement: Thermal, mechanical and microstructural properties. J. Compos. Mater. 2017, 51, 2491–2503. https://doi.org/10.1177/0021998316672090. p g 15. Balea, A.; Fuente, E.; Blanco, A.; Negro, C. Nanocelluloses: Natural-based materials for fiber- reinforced cement composites. A critical review. Polymers 2019, 11, 518. https://doi.org/10.3390/polym11030518. p g 15. Balea, A.; Fuente, E.; Blanco, A.; Negro, C. Nanocelluloses: Natural-based materials for fiber- reinforced cement composites. A critical review. Polymers 2019, 11, 518. https://doi.org/10.3390/polym11030518. y p g p y 16. Ip, K.; Miller, A. Life cycle greenhouse gas emissions of hemp-lime wall constructions in the UK. Resour. Conserv. Recycl. 2012, 69, 1–9. https://doi.org/10.1016/j.resconrec.2012.09.001. y p g p y 16. Ip, K.; Miller, A. Life cycle greenhouse gas emissions of hemp-lime wall constructions in the UK. Resour. Conserv. Recycl. 2012, 69, 1–9. https://doi.org/10.1016/j.resconrec.2012.09.001. p g j 17. Kang, S.-H.; Kwon, Y.-H.; Moon, J. Quantitative Analysis of CO2 Uptake and Mechanical Properties of Air Lime-Based Materials. Energies 2019, 12, 2903. https://doi.org/10.3390/en12152903. 18. SAINT-ASTIER Preservation of Our Built Heritage: St. Astier NHL Mortars. Available online: http://www.stastier.co.uk/articles/preserving-heritage.htm (accessed on 23 December 2021). rks on the green retrofitting of historic buildings in Italy. Energy Build. 2015, 95, 15–22 16/j.enbuild.2014.11.001. 19. Filippi, M. Remarks on the green retrofitting of historic buildings in Italy. Energy https://doi.org/10.1016/j.enbuild.2014.11.001. p g j 20. Kang, S.H.; Lee, S.O.; Hong, S.G.; Kwon, Y.H. Historical and scientific investigations into the use of hydraulic lime in Korea and preventive conservation of historic masonry structures. Sustainability 2019, 11, 5169. https://doi.org/10.3390/su11195169. 21. References 2. Summerbell, D.L.; Barlow, C.Y.; Cullen, J.M. Potential reduction of carbon emissions by performance improvement: A cement industry case study. J. Clean. Prod. 2016, 135, 1327–1339. https://doi.org/10.1016/j.jclepro.2016.06.155. y y p g j j p 3. Thwe, E.; Khatiwada, D.; Gasparatos, A. Life cycle assessment of a cement plant in Naypyitaw, Myanmar. Clean. Environ. Syst. 2021, 2, 100007. https://doi.org/10.1016/j.cesys.2020.100007. p g j y 4. Ma, W.; Qin, Y.; Li, Y.; Chai, J.; Zhang, X.; Ma, Y.; Liu, H. Mechanical properties and engineering application of cellulose fiber- reinforced concrete. Mater. Today Commun. 2020, 22, 100818. https://doi.org/10.1016/j.mtcomm.2019.100818. 5. Fu, T.; Moon, R.J.; Zavattieri, P.; Youngblood, J.; Weiss, W.J. Cellulose nanomaterials as additives for cementitious materials. In Cellulose-Reinforced Nanofibre Composites; Elsevier Ltd.: Amsterdam, The Netherlands, 2017; Volume C, pp. 455–482, ISBN 9780081009659. 6. Hisseine, O.A.; Omran, A.F.; Tagnit-Hamou, A. Influence of cellulose filaments on cement paste and concrete. J. Mater. Civ. Eng. 2018, 30, 04018109. https://doi.org/10.1061/(ASCE)MT.1943-5533.0002287. Materials 2022, 15, 459 18 of 19 18 of 19 7. Cengiz, A.; Kaya, M.; Pekel Bayramgil, N. Flexural stress enhancement of concrete by incorporation of algal cellulose nanofibers. Constr. Build. Mater. 2017, 149, 289–295. https://doi.org/10.1016/j.conbuildmat.2017.05.104. p g j 8. Fu, T.; Montes, F.; Suraneni, P.; Youngblood, J.; Weiss, J. The influence of cellulose nanocrystals on the hydration and flexural strength of Portland cement pastes. Polymers 2017, 9, 424. https://doi.org/10.3390/polym9090424. p g j 8. Fu, T.; Montes, F.; Suraneni, P.; Youngblood, J.; Weiss, J. The influence of cellulose nanocrystals on the hydration and flexural strength of Portland cement pastes. Polymers 2017, 9, 424. https://doi.org/10.3390/polym9090424. 9. Onuaguluchi, O.; Panesar, D.K.; Sain, M. Properties of nanofibre reinforced cement composites. Constr. Build. Mater. 2014, 63, 119–124. https://doi.org/10.1016/j.conbuildmat.2014.04.072. 9. Onuaguluchi, O.; Panesar, D.K.; Sain, M. Properties of nanofibre reinforced cement composites. Constr. Build. Mater. 2014, 63, 119–124. https://doi.org/10.1016/j.conbuildmat.2014.04.072. p g j 10. Snoeck, D.; Smetryns, P.A.; De Belie, N. Improved multiple cracking and autogenous healing in cementitious materials by means of chemically-treated natural fibres. Biosyst. Eng. 2015, 139, 87–99. https://doi.org/10.1016/j.biosystemseng.2015.08.007. 10. Snoeck, D.; Smetryns, P.A.; De Belie, N. Improved multiple cracking and autogenous healing in cementitious materials by means of chemically-treated natural fibres. Biosyst. Eng. 2015, 139, 87–99. https://doi.org/10.1016/j.biosystemseng.2015.08.007. 11. Hisseine, O.A.; Basic, N.; Omran, A.F.; Tagnit-Hamou, A. Feasibility of using cellulose filaments as a v in self-consolidating concrete. Cem. Concr. Compos. 2018, 94, 327–340. https://doi.org/10.1016/j.cemconc 12. Barnat-Hunek, D.; Szymańska-Chargot, M.; Jarosz-Hadam, M.; Łagód, G. p g j 8. Fu, T.; Montes, F.; Suraneni, P.; Youngblood, J.; Weiss, J. The influence of cellulose nanocrystals on th strength of Portland cement pastes. Polymers 2017, 9, 424. https://doi.org/10.3390/polym9090424. 34. Ardanuy, M.; Claramunt, J.; Arévalo, R.; Parés, F.; Aracri, E.; Vidal, T. Nanofibrillated cellulose (Nfc) as a potential reinforcement for high performance cement mortar composites. BioResources 2012, 7, 3883–3894. https://doi.org/10.15376/biores.7.3.3883-3894. p // g/ / 35. Kockal, N.U.; Ozturan, T. Effects of lightweight fly ash aggregate properties on the behavior of lightweight concretes. J. Hazard. Mater. 2010, 179, 954–965. https://doi.org/10.1016/J.JHAZMAT.2010.03.098. References D’Erme, C.; Verticchio, E.; Frasca, F.; Caseri, W.; Cornaro, C.; Siani, A.M.; Santarelli, M.L. Preliminary Study of the Mechanical and Hygrothermal Performance of Concrete Reinforced with Nanofibrillated Cellulose; AIP Publishing: 2021; Melville, NY, Volume 2416. 22. European Standard: EN 1015-3:1999 “Methods of Test for Mortar for Masonry—Part 3: Determination of consistence of Fresh Mortar (by Flow Table)”, Brussels. 1999. ( y ) 23. Deboucha, W.; Leklou, N.; Khelidj, A.; Oudjit, M.N. Hydration development of mineral additives blended cement using thermogravimetric analysis ( TGA ): Methodology of calculating the degree of hydration. Constr. Build. Mater. 2017, 146, 687– 701. https://doi.org/10.1016/j.conbuildmat.2017.04.132. p g j 24. Monteagudo, S.M.; Moragues, A.; Gálvez, J.C.; Casati, M.J.; Reyes, E. Thermochimica Acta The degree of hydration assessment of blended cement pastes by differential thermal and thermogravimetric analysis . Morphological evolution of the solid phases. Thermochim. Acta 2014, 592, 37–51. https://doi.org/10.1016/j.tca.2014.08.008. p g j 25. British Standards Institute. BS EN 12390-5:2009 “Testing Hardened Concrete—Part 5: Flexural Strength of Test Specimens”; British Standards Institute: 2009, London. 26. British Standards Institute. BS EN 12390-3:2003 “Testing Hardened Concrete—Part 3: Compressive Strength of Test Specimens”; British Standards Institute: 2003, London. 27. Li, L.; Guan, J.; Yuan, P.; Yin, Y.; Li, Y. A Weibull distribution-based method for the analysis of concrete fracture. Eng. Fract. Mech. 2021, 256, 107964. https://doi.org/10.1016/J.ENGFRACMECH.2021.107964. p g 28. Jerman, M.; Tydlitát, V.; Keppert, M.; Čáchová, M.; Černý, R. Characterization of early-age hydration processes in lime-ceramic binders using isothermal calorimetry, X-ray diffraction and scanning electron microscopy. Thermochim. Acta 2016, 633, 108–115. https://doi.org/10.1016/J.TCA.2016.04.005. p g 29. Flatt, R.; Schober, I. Superplasticizers and the Rheology of Concrete; Woodhead Publishing Limited: 2012. 30. Marchon, D.; Flatt, R.J. Impact of Chemical Admixtures on Cement Hydration; Woodhead Publishing, Elsevier Ltd.: Amsterdam, The Netherlands, 2016; ISBN 9780081006962. 31. Knill, C.J.; Kennedy, J.F. Degradation of cellulose under alkaline conditions. Carbohydr. Polym. 2003, 51, 281–300. https://doi.org/10.1016/S0144-8617(02)00183-2. 32. Dufresne, A. Nanocellulose: A new ageless bionanomaterial. Mater. Today 2013, 16, 220–227. https://doi.org/10.1016/j.mattod.2013.06.004. p g j 33. Hisseine, O.A.; Wilson, W.; Sorelli, L.; Tolnai, B.; Tagnit-Hamou, A. Nanocellulose for improved concrete performance: A macro-to-micro investigation for disclosing the effects of cellulose filaments on strength of cement systems. Constr. Build. Mater. 2019, 206, 84–96. https://doi.org/10.1016/j.conbuildmat.2019.02.042. 19 of 19 19 of 19 Materials 2022, 15, 459
https://openalex.org/W4321152359	https://zenodo.org/record/7644449/files/108-112.pdf	Russian	null	MIRZO SALIMBEKNING "TARIXI SALIMIY" ASARI	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	1,692	АННОТАЦИЯ В данной статье описывается научная значимость “Истории Салимы” Мирзо Салимбека, события, описываемые в произведении, и мнения автора. Ключевые слова: “История Салимии”, рукопись, эмират, “младобухарцы” мангиты кушбеги арк деванбеги манифест парваначи В данной статье описывается научная значимость “Истории Салимы” Мирзо Салимбека, события, описываемые в произведении, и мнения автора. В данной статье описывается научная значимость Истории Салимы Мирзо Салимбека, события, описываемые в произведении, и мнения автора. Ключевые слова: “История Салимии”, рукопись, эмират, “младобухарцы”, мангиты, кушбеги, арк, деванбеги, манифест, парваначи, посольство, верхние кошбеги, нижние кошбеги. Ключевые слова: “История Салимии”, рукопись, эмират, “младобухарцы”, мангиты, кушбеги, арк, деванбеги, манифест, парваначи, посольство, верхние кошбеги, нижние кошбеги. ANNOTATION In this article, the scientific importance of Mirzo Salimbek’s “History of Salimiy”, the level of study of the work, the events described in the work, the opinions of the author and the level of study of the work are reflected. Key words: “History of Salimi”, manuscript, emirate, “young Bukharans”, mangits, koshbegi, Ark, devonbegi, manifest, parvanachi, embassy, high koshbegi, lower koshbegi. ANNOTATSIYA ANNOTATSIYA Ushbu maqolada Mirzo Salimbekning “Tarixi Salimiy” asarining ilmiy ahamiyati, asarda tasvirlangan voqealar, muallifning fikr-mulohazalari bayon etiladi. Kalit so‘zlar: “Tarixi Salimiy”, qo‘lyozma, amirlik, “yosh buxoroliklar”, mang‘itlar, qushbegi, Ark, devonbegi, manifest, parvonachi, elchilik, yuqori qo‘shbegi, quyi qo‘shbegi. Ushbu maqolada Mirzo Salimbekning “Tarixi Salimiy” asarining ilmiy ahamiyati, asarda tasvirlangan voqealar, muallifning fikr-mulohazalari bayon etiladi. Kalit so‘zlar: “Tarixi Salimiy”, qo‘lyozma, amirlik, “yosh buxoroliklar”, Ushbu maqolada Mirzo Salimbekning “Tarixi Salimiy” asarining ilmiy ahamiyati, asarda tasvirlangan voqealar, muallifning fikr-mulohazalari bayon etiladi. Kalit so‘zlar: “Tarixi Salimiy”, qo‘lyozma, amirlik, “yosh buxoroliklar”, mang‘itlar, qushbegi, Ark, devonbegi, manifest, parvonachi, elchilik, yuqori qo‘shbegi, quyi qo‘shbegi. Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 \| ISSUE 3 \| 2023 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 \| ISSUE 3 \| 2023 MIRZO SALIMBEKNING “TARIXI SALIMIY” ASARI Jaxongirov Islomxon Faxriddin o‘g‘li Farg‘ona davlat universiteti Tarix fakulteti 3-kurs talabasi islomxonjahongirov7@gmail.com Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 \| ISSUE 3 \| 2023 Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Sovet davri tarixshunosligida Buxoro amirligi tarixiga berilgan baho asosan salbiy edi. Bu davr turg‘unlik va tanazzul davri sifatida qaraldi. Bu borada Buxoroning mang‘it amirlari hukmronligi davri voqealarini o‘rganishda biryoqlamalik hukm surdi va doimo qoralanib kelindi. Mirzo Salimbek ushbu asari bizga bu davr haqida atroflicha ma’lumot beradi. KIRISH Mirzo Salimbekning “Tarixi Salimiy” qoʻlyozma asari, asosan, Buxoro amirligi tarixining Mangʻitlar sulolasining soʻnggi vakillari hukmronligi davridagi voqealarni oʻz ichiga oladi. Ushbu qoʻlyozmaning maʼlum bir qismlarining tarjimasini 1966-yilda N. Norqulov yakunlangan. Bu asar uning nomzodlik dissertatsiyasi uchun asosiy manba boʻlib xizmat qilgan va nomzod uni mashhur sharqshunos olim A. K. Arends rahbarligida muvaffaqiyatli himoya qilgan. 108 https://t.me/openidea_uz Multidisciplinary Scientific Journal February, 2023 108 ASOSIY QISM Mirzo Salimbek hayoti davomida turli xil mansabda bo‘lib, har bir badavlat musulmonga yarasha madrasa qurdirib, yer va mol-mulkining bir qismini vaqfga bergan. Qabr va masjidlarni taʼmirlagan, ariqlar qurgan, adabiy faoliyat bilan shugʻullangan va hokazo. Mirzo Salimbekning adabiy merosi ma’lum bir qadriyatga ega. U qator tarixiy, adabiy va diniy asarlar muallifi. Tarixchi olimning asosiy asari “Tarixi Salimiy” – uning xotiralaridir. O‘zi ta’kidlanganidek, qo‘lyozma faqat bir nusxada mavjud. U Buxoro amirligining keyingi davr tarixiga bag‘ishlangan. Mirzo Salimbek o‘z asarini 1917-yilda yozishni boshlagan va 1920-yilda tugatgan, buni qo‘lyozmaning bir necha joyidagi xronologik sanalar tasdiqlaydi[3;4-b]. Mirzo Salimbekning “Tarixi Salimiy” asari muhim tarixiy manba bo‘lib, Buxoro amirligida hukmronlik qilgan so‘nggi amirlar faoliyati, Buxoro amirligidagi shaharlarning hayoti, moddiy madaniyati, hunarmandchiligi, amirlikning boy tarixi haqida ma’lumotlar berilgan. “Tarixi Salimiy” muallifi Buxoroning so‘nggi amirlari faoliyatiga juda ehtiyotkorona baho beradi. U oʻsha davr ruhida Buxoro hukmdorlarini “adolatli va gʻolib hukmdor”, “xoʻjayin”, “barcha dindorlar hukmdori” va boshqa ta’riflarni beradi. Salimbek Amir Muzaffarni Chingizxon bilan solishtiradi. U shunday yozadi: “Amir Muzaffar qishda, kuchli ayozda, katta qoʻshin bilan Hisorga yoʻl oldi. U yerga yetib kelganida shunday qirg‘in qildiki, u haqida eshitganlarni hayratda qoldirdi va Chingizxonning to‘kkan qonlarini eslatdi, dovdirab qolgan raqiblar hayratdan barmoqlarini tishladilar”. Shu bilan birga, Salimbek Rossiya imperiyasi qoʻshinlarining Buxoro amirligiga hujumini tasvirlab, amir Muzaffarning “rus qoʻshinlari bilan uchrashib, jang qilmay, qochib, Samarqandga qaytib kelganini ham sovuqqonlik bilan qayd etadi. Mirzo Salimbek amirlik muxoliflarini, Shahrisabz va Kitob hukmdorlarining bo‘lginchiligini qoralab, ularni “qo‘zg‘olonchilar” deb ataydi. Salimbek turli yillarda Yakkabogʻ, Nurota, Boysun, Sherobod, Shahrisabz hokimi boʻlgan. Bu hududlar etnografiyasi, tarixi, qadimiy inshootlari va manzillari haqida muhim ma’lumotlar qoldirdi, bu esa XX asr o‘rtalarida olib borilgan arxeologik tadqiqotlar uchun asos bo‘lib xizmat qildi. 109 ps://t.me/openidea_uz Multidisciplinary Scientific Journal February, 2023 109 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 \| ISSUE 3 \| 2023 Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 \| ISSUE 3 \| 2023 Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 “Tarixi Salimiy” da Mirzo Salimbek zamondoshlari ijodida uchramaydigan ko‘plab ma’lumotlar mavjud. Bu faqat Buxoro amirligining ijtimoiy-iqtisodiy va siyosiy tarixiga oid ma’lumotlar emas, balki tarixiy-geografiya, etnografiya va me’morchilik tarixi hamda alohida beklar tarixiga oid ma’lumotlardir. Qo‘lyozma muallifi o‘zi guvohi bo‘lgan davr va voqealarni juda jonli, tushunarli tilda tasvirlab bergan. “Tarixi Salimiy” asarida juda ko‘p toponim va gidronimlar yozib qoldirilgan [2;43-b]. Amir Said Olimxon o‘z xotiralarida bir vaqtlar Salimbek (Mirza Salimbek parvonachi)ni inglizlar bilan muzokaralar olib borish uchun Chorjo‘yga jo‘natganligi ta’kidlagan [1;16-b]. “Tarixi Salimiy” qo‘lyozmasida o‘z aksini topgan turli ma’lumotlar va faktlarga tanqidiy yondashish zarurligi shubhasiz. Uning muallifi nafaqat o‘zi ko‘rganlarini, balki boshqalardan eshitganlarini ham qog‘ozga tushirgan (masalan, Nurotani Iskandar Zulqarnayn obod qilgani haqidagi afsona va boshqalar). Asar kundalik xarakteriga ega, uning koʻp qismi amir Olimxon saltanati tarixini koʻrsatishga bagʻishlangan. Mirza Salimbek u yoki bu voqeani tasvirlab, unga o‘ziga xos tarzda baho beradi, unga munosabat bildiradi. U amir Muzaffar qo‘l ostidagi oddiy mirzolikdan tortib, amir Olimxon davrida devonbegi darajasidagi zakotchi boshlig‘i martabasigacha bo‘lgan xizmat yo‘li haqida batafsil hikoya qiladi. Manbani o‘rganar ekanmiz, buxorolik amaldorlar vakillaridan birining hayot yo‘lini ham bilib olamiz. Shuningdek, amirlikning hukmron qatlamlari hayoti, Buxoro davlat amaldorlarining iyerarxik mansab pog‘onasidan ko‘tarilishining turli yo‘llari va usullarini qo‘llaganligi tasvirini ko‘ramiz. Bu esa Buxoro amirligida sodir bo‘lgan voqealarni aniq taqdim etish va baholash imkonini beradi. Asar buyurtma asosida, biron bir amirni maqtash maqsadida yozilmagani uchun Salimbekning xotiralarida tarixiy voqealarni ataylab soxtalashtirish alomatlari sezilmaydi. Lekin, davr hukmron mafaatlari yuzasidan yondashilgan jihatlar ham bor. Salimbekning xotiralari Buxoro amirligining XIX asr oxiri XX asr boshlari tarixiga oid toʻliq boʻlmagan maʼlumotlarni asosan faktik materiallar bilan toʻldirishi bilan qimmatlidir. Bu manba O‘zbekiston Fanlar akademiyasi qo‘lyozma fondida paydo bo‘lgach, olimlarning e’tiborini tortdi. Uning birinchi ilmiy tavsifini Mirzo Salimbekning tarjimai holi va ijodiy merosiga bagʻishlangan maqola yozgan A. A. Semenov qilgan. Hozirda ushbu nashr etilmagan maqola A. A. Semenovning Dushanbe shahridagi shaxsiy arxivida “So‘nggi buxorolik tarixchi (Mirza Salimbek)” nomi ostida saqlanmoqda. Salimbekning xotiralarida Buxoro amirligining maʼmuriy tuzilishi haqida aniqroq tasavvur beriladi; qator mansab va martabalarning vazifa va vazifalari aniqlangan: qoʻshbegi, amlokdara, zakotchi, mirshab va boshqalar. Maʼlumki, 110 https://t.me/openidea_uz Multidisciplinary Scientific Journal February, 2023 February, 2023 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 \| ISSUE 3 \| 2023 Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 amirlikda ijro etuvchi hokimiyat oliy qoʻshbegi (“qoʻshbegii bolo”) va quyi qushbegiga tegishli edi. Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 \| ISSUE 3 \| 2023 Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Birinchisi Arkda - amirning qarorgohida yashagan, shuning uchun uni yuqori deb atashgan. Markaziy moliyaviy qismga quyi qo‘shbegilar rahbarlik qilgan va barcha viloyat zakotchilari unga bo‘ysungan. U va uning idorasi - Ark etagida joylashgan hovlida joylashgan edi. Shuning uchun uni quyi qo‘shbegi deb atashgan. Mirzo Salimbek Buxoro jadidlarining 1917-yil aprel namoyishlari paytidagi faoliyati haqida ham batafsil hikoya qiladi. Bu namoyishlar bostirilgach, Yosh buxoroliklar kuchlari yanada kuchaydi. Yosh buxorolikaliklar yangi kurashga tayyorlanib, qizg‘in tashkiliy ishlar boshladilar, shahar bozorlari va amirlik qishloqlarida varaqalar paydo bo‘ldi. Targ‘ibot ommaviy tus oldi, qo‘shinlarga alohida e’tibor qaratildi. Sarbozlar amir qoʻshini saflarida xizmat qilishdan bosh torta boshladi va raqiblari tomoniga oʻtdi. Yosh buxoroliklar faoliyati o‘z samarasini bera boshladi. Sarbozlar va qishloq ahlining qoʻzgʻolonlari tez-tez uchrab turadi [4]. “Tarixi Salimi”da shunday qoʻzgʻolonlardan biri Shahrisabz va Kitobda Yosh buxoroliklar boshchiligida boʻlib oʻtgan haqida batafsil hikoya qilinadi. Qoʻzgʻolonni bostirish uchun Salimbekning yozishicha, oʻzining shafqatsizligi bilan mashhur boʻlgan Nizomiddin Xoʻja Qoʻshbegi 50 askar bilan yuborilgan. Boshqa maʼlumotlarga koʻra, u sarbozlarning katta otryadi bilan kelgan. Qo‘zg‘olonchilar hokimni lavozimidan chetlashtirishni va mahalliy aholidan boshqasini tayinlashni talab qilishadi. Qo‘shbegi qo‘zg‘olonchilar talabiga dahshat bilan javob bergan va uning qo‘zg‘olonchilarga nisbatan ko‘rgan choralari go‘yoki o‘rta asrlardagi “Ispan inkvizitsiyasidan” oshib ketgan. Ammo shunga qaramay qoʻshbegi qoʻzgʻolonchilarning quyidagi talablarini qondirib, qoʻzgʻolonni bostirishga muvaffaq boʻldi: a) hokim Akramxon bilan birga kelgan oʻn bir amlokdorni oʻz lavozimlaridan chetlatish; shariat belgilagan soliqlardan tashqari, hokimlar oʻylab topgan barcha toʻlovlarni bekor qilish; d) aholiga taalluqli barcha masalalarni Shahrisabz viloyatining og‘alig‘i (xalqning saylangan vakili) Abdushukur biy bilan kelishilgan holda hal qilish va uning hayotiga tajovuz qilmaslik. XULOSA Republikamizda mustaqillik yillarida o‘zbek xalqining ma’naviy-madaniy merosini tiklash va o‘rganish eng muhim vazifa bo‘ldi. O‘tgan davr mobaynida O‘zbekiston tarixiga oid qator birlamchi manbalarni o‘rganish va ilmiy foydalanishga joriy etish borasida katta ishlar qilindi. Buxoro amirligining 1860-1920 –yillar tarixiga oid bir qancha tarixiy manbalar tadqiq qilinib, ilmiy muomalaga kiritildi. Shunga https://t.me/openidea_uz Multidisciplinary Scientific Journal February, 2023 11 111 February, 2023 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 \| ISSUE 3 \| 2023 Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 qaramay, ko‘rsatilgan davrdagi Buxoro amirligi tarixining alohida muammolari asosan o‘rganilmaganligicha qolmoqda. Natijada tarixshunoslikda XIX asr oxiri XX asr boshlaridagi Buxoro amirligi tarixining bir qator masalalari yuzasidan yuzaki xulosalar qayd etilgan. Xususan, Mirzo Salimbekning “Tarixi Salimiy” asari fors tilidan rus tiliga N.Norqulov tomonidan tarjima qilingan, asarni o‘rganish, uni keng kitobxonlar ommasiga taqdim qilish dolzarb masalalardan biri bo‘lib qolmoqda. Asar bizga XIX asr oxiri XX asr boshlarida yuz bergan ijtimoiy-siyosiy jarayonlarni kengroq o‘rganishimizda ulkan yordam beradi. Shuni ham alohida qayd etish kerakki, xorijda boy tariximiz bizga nisbatan ancha mufassal o‘rganilgan. Biz o‘z tariximizni yanada chuqurroq o‘rganishimiz zarur. Xususan, Mirzo Salimbekning “Tarixi Salimiy” asari ham yana chuqur tahlil qilinib, sinkovlik bilan o‘rganishni talab etmoqda. FOYDALANILGAN ADABIYOTLAR RO‘YHATI: (REFERENCES 1. Амир Саййид Олимхон. Бухоро халқининг хасрати тарихи.Т . : Фан, 1991, б.16. 1. Амир Саййид Олимхон. Бухоро халқининг хасрати тарихи.Т . : Фан, 1991, б.16. 2. Бухорий А.Р. Рахимов К. Мирзо Салимбек қолдирган мерос // Мозийдан садо,2006. №. Б.43. 2. Бухорий А.Р. Рахимов К. Мирзо Салимбек қолдирган мерос // Мозийдан садо,2006. №. Б.43. 3. Мирза Салимбек. Тарих-и Салими (Источник по истории Бухарского эмирата) / Перевод с персидского Н.К.Норкулов. — Тошкент. 2009.C.424. 4. Cадриддин Айний. Бухоро инқилобининг тарихи учун материаллар. Москва, 1926. https://t.me/openidea_uz Multidisciplinary Scientific Journal February, 2023 112 112
https://openalex.org/W2899292276	http://uhmj.org.ua/index.php/journal/article/download/2/1	Ukrainian	null	Air pollution by nitrogen dioxide in Kiev city	Ukraïnsʹkij gìdrometeorologìčnij žurnal	2,017	cc-by	8,261	ЗАБРУДНЕННЯ АТМОСФЕРНОГО ПОВІТРЯ МІСТА КИЄВА ДВООКИСОМ АЗОТУ В статті описані основні джерела надходження двоокису азоту в атмосферне повітря великих міст. Розглянуто часову динаміку середньорічних концентрацій двоокису азоту в атмосфері м. Києва, про- аналізовано рівень забруднення повітря цією домішкою в різних частинах міста, встановлено повто- рюваність випадків перевищення двоокисом азоту максимально-разової та середньодобової гранично допустимих концентрацій. Найвищі концентрації двоокису азоту спостерігалися в центральній части- ні міста в деякі місяці теплого періоду. Частота випадків перевищення ГДК в повітрі зазвичай досяга- ла 50%, а в теплий сезон 2012 р. концентрація NO2 в повітрі була дуже високою і перевищувала ГДК в 100% випадків на всіх станціях моніторингу. Дослідження показали, що в теплий сезон року в повітрі м. Києва регулярно спостерігаються підвищені концентрації двоокису азоту, які є прекурсорами фо- тохімічного смогу, що при сприятливих метеорологічних умовах може призвести до формування цьо- го негативного феномену в атмосфері міста. Ключові слова: забруднення атмосферного повітря, двоокис азоту, перевищення гранично допус- тимої концентрації, динаміка рівня забруднення. рівня забруднення окремих міст або регіонів, в яких серед інших домішок також розглядається вміст дво- окису азоту в атмосферному повітрі. Проте, окремі роботи українських вчених сфокусовані на до- слідженні лише цієї домішки. Наприклад, в [6] запро- поновано методику прогнозу забруднення повітря міста двоокисом азоту, а Маренко А.Н. з колегами [7−8] досліджували сполуки азоту в приземному шарі повітря біля автомагістралей Києва. І хоча останні дослідження проводилися якраз у столиці, проте їх результати відображають особливості зміни концент- рацій та перебіг фотохімічних перетворень сполук азоту лише у безпосередній близькості до автомобі- льних шляхів. В наших попередніх дослідженнях, що були присвячені забрудненню атмосферного повітря м. Києва [10], також розглядалися деякі особливості просторово-часового розподілу концентрацій двооки- су азоту в атмосферному повітрі міста в 2003−2006 рр., проте для вивчення двоокису азоту, як прекурсору фотохімічного смогу та встановлення ймовірності виникнення цього явища в атмосфері м. Києва необхідно використовувати інформацію про сучасний стан забруднення повітря міста даною до- мішкою та особливості її просторово-часового розпо- ділу в повітрі міста в останні роки, а результати таких досліджень на сьогоднішній день відсутні. 1. ВСТУП Забруднення атмосферного повітря урбанізованих територій останнім часом викликає все більше зане- покоєння, адже, з одного боку у великих містах його постійного негативного впливу зазнає величезна кі- лькість людей, з іншого боку – в структурі основних забруднювачів міст зростає частка пересувних дже- рел, зниження викидів від яких у міському середови- щі пов’язано з низкою труднощів. Проблема переви- щення нормативів вмісту двоокису азоту в атмосфер- ному повітрі характерна для багатьох великих міст світу. Як свідчать дані моніторингу Гідрометслужби, в останні роки більш ніж у половині міст України, у яких здійснюється моніторинг двоокису азоту в пові- трі, середньорічні концентрації даного забруднювача перевищують ГДК. Особлива небезпека високого вмісту цієї речовини у повітрі пов’язана з тим, що вона може здійснювати не лише безпосередній нега- тивний вплив на здоров’я людини, а також за сприят- ливих умов брати участь у фотохімічних реакціях та призводити до формування смогу. Саме тому в усьо- му світі активно досліджують джерела надходження двоокису азоту в повітря, особливості його просторо- во-часового розподілу у великих містах, перебіг фо- тохімічних перетворень за його участю [1, 4, 6−8, 10, 13−16, 18−21] та намагаються знайти шляхи вирі- шення цієї серйозної проблеми. Hydrometeorological Aspects of Environmental Monitoring ГІДРОМЕТЕОРОЛОГІЧНІ АСПЕКТИ МОНІТОРИНГУ НАВКОЛИШНЬОГО СЕРЕДОВИЩА PACS: 92.60.Sz Air quality and air pollution Укр. гідрометеорол. ж., 2015, №16 Укр. гідрометеорол. ж., 2015, №16 Hydrometeorological Aspects of Environmental Monitoring Hydrometeorological Aspects of Environmental Monitoring ГІДРОМЕТЕОРОЛОГІЧНІ АСПЕКТИ МОНІТОРИНГУ НАВКОЛИШНЬОГО СЕРЕДОВИЩА 3. ВИКЛАД ОСНОВНОГО МАТЕРІАЛУ Джерела надходження двоокису азоту в атмо- сферне повітря. Діоксид азоту за класифікацією за- бруднювальних речовин належить до групи основних – це речовини, що входять до складу викидів біль- шості стаціонарних джерел шкідливих домішок. Діоксид азоту є одним з пріоритетних забруднювачів повітря великих міст, належить до речовин третього класу небезпеки. Тривалість його максимального перебування в атмосфері становить близько 3 діб. Його характерне рудувато-коричневе забарвлення можна часто побачити над трубами підприємств, за що його називають «лисячий хвіст». 2NO+O2→2NO2+120 кДж. (1) (1) Цей процес є зворотним, проте за нормальних умов хімічна рівновага зміщена в сторону діоксиду. Також можливе перетворення за участю немета- нових вуглеводнів (NMHC) NMHC+NO+hv→NO2+R (інші продукти). (2) (2) Усі джерела надходження двоокису азоту в атмо- сферне повітря за різними ознаками можна поділити на кілька груп. Перш за все, виділяють антропогенні та природні джерела. Природними джерелами цього забруднювача є лісові, торфові та вугільні пожежі, вулканічна діяльність, мікроорганізми, незначна кіль- кість цього газу надходить в атмосферне повітря та- кож від розрядів блискавок. Якщо стосовно надход- ження двоокису азоту в планетарному масштабі, можна знайти різні дані про внесок кожного виду з цих джерел, то суттєве переважання антропогенних джерел у великих містах на сьогодні не викликає жодних сумнівів. На концентрацію NOx в повітрі великих міст сут- тєво впливають їх перетворення за участю озону. Під дією сонячної радіації та підвищених температур повітря відбувається фотоліз NO2 NO2+hv (λ≤410 нм) →NO+O. (3) (3) Атомарний кисень О, що утворився взаємодіє з молекулярним О2 – в результаті чого утворюється озон О+О2+М→О3+М. (4) (4) Далі молекула озону може бути зруйнована в ре- зультаті взаємодії з NO Усі антропогенні джерела надходження даної домішки в повітря можуть бути розподілені на ста- ціонарні та пересувні. Крім того, двоокис азоту може не лише надходити в атмосферу безпосередньо від джерел викидів, він також може утворюватися в самій атмосфері з речовин-попередників. Одним із основ- них джерел безпосереднього надходження NO2 в ат- мосферу є спалювання органічного палива за темпе- ратур, що не перевищують 600°С (за вищих темпера- тур відбувається руйнування двоокису азоту). Це NO+O3→NO2+O2. (5) (5) Реакції (3) – (5), власне, описують формування фотохімічного смогу за участі одного з видів з його прекурсорів – сполук азоту. Як видно з (3) – (5) – це замкнутий цикл, що може досягнути стаціонарного стану за сталих концентрацій реагентів та відповідних умов. Відповідно для зміни концентрацій однієї зі складових необхідним є додаткове джерело іншої. клас небезпеки змінено згідно постанови Головного державного санітарного лікаря України від 4.06.2010 р. № 18. 2. АНАЛІЗ ОСТАННІХ ДОСЛІДЖЕНЬ Для виконання даної роботи було використано ма- теріали строкових спостережень Державної гідроме- теорологічної служби України за вмістом двоокису азоту на 14 із 16 постів спостереження за забруднен- Вивченню якості атмосферного повітря міст Укра- їни останнім часом приділяється все більше уваги [3, 5, 10]. Більшість робіт присвячені комплексній оцінці Ukr. gìdrometeorol. ž., 2015, No16 6 Забруднення атмосферного повітря міста Києва двоокисом азоту ням (ПСЗ) м. Києва за період 2008−2012 рр. Дані ПСЗ № 10 (вул. Межигірська, 56/60 (Поділ, поблизу ст.м. Тараса Шевченка)) та ПСЗ № 20 (Московська площа) не використовувалися нами через відсутність вимірювань протягом більшої частини обраного для досліджень періоду. Багаторічна динаміка забруднен- ня атмосферного повітря міста двоокисом азоту аналізувалася за даними ЦГО − використана інфор- мація про середньорічні концентрації за період з 1985 по 2012 рр. може відбуватися в двигунах внутрішнього згорання, при спалюванні органічного палива на ТЕС та в різноманітних промислових процесах. Ці ж процеси є також і джерелами надходження речовин- попередників – сполук азоту, основним серед яких є оксид азоту. Надходження в атмосферне повітря ок- сиду чи двоокису азоту залежатиме, перш за все, від температури в камері згорання, а концентрація окси- дів азоту у викидах – ще й від вмісту в ній вільного кисню. може відбуватися в двигунах внутрішнього згорання, при спалюванні органічного палива на ТЕС та в різноманітних промислових процесах. Ці ж процеси є також і джерелами надходження речовин- попередників – сполук азоту, основним серед яких є оксид азоту. Надходження в атмосферне повітря ок- сиду чи двоокису азоту залежатиме, перш за все, від температури в камері згорання, а концентрація окси- дів азоту у викидах – ще й від вмісту в ній вільного кисню. Двоокис азоту (NO2) та оксид азоту (NO) в атмо- сферній хімії прийнято називати оксидами азоту (NOx). Джерелами оксидів азоту, що утворюються в процесі горіння, є молекулярний азот повітря, що використовується в якості окиснювача при горінні та азотовмісні компоненти пального. Моноксид азоту NO в середньому становить близько 95–99 % від за- гальних викидів NOx, в той час на токсичніший NO2 припадає не більше 1–5 %. Після викидів в атмосферу моноксид азоту легко реагує з киснем повітря з утво- ренням NO2 З метою вивчення основних джерел надходження двоокису азоту нами було проаналізовано вітчизняну та зарубіжну наукову літературу, присвячену даній проблемі. 3. ВИКЛАД ОСНОВНОГО МАТЕРІАЛУ Крім рекомбінації атомарного кисню О з моле- кулярним О2, джерелом озону в тропосфері можуть бути цикли окислення метану (CH4) та моноксиду Укр. гідрометеорол. ж., 2015, №16 7 7 О.Г.Шевченко, С.І. Сніжко, Н.О.Данілова легкових автомобілів – знаходиться в межах 7 %, в той час як для таких же дизельних автомобілів з ка- талізаторами окислення – в межах 30–60 %. легкових автомобілів – знаходиться в межах 7 %, в той час як для таких же дизельних автомобілів з ка- талізаторами окислення – в межах 30–60 %. вуглецю СО, а також надходження його в атмосферу безпосередньо від джерел забруднення [12]. Оскіль- ки, швидкість реакцій (4) та (5) залежить від темпера- тури, а перебіг реакції (3) перебуває у прямій залеж- ності від сонячної радіації, то для їх перебігу в атмо- сфері необхідні відповідні метеорологічні умови – достатня кількість сонячної радіації з довжиною хвиль менше 410 нм, температура повітря вище 18ºС (оптимально 25–35ºС), а також – низька відносна вологість повітря та штиль або слабкий вітер (до 2 м/с). Не зважаючи на введення більш жорстких норма- тивів вмісту оксидів азоту в атмосферному повітрі європейських міст у 2008 р., і навіть деяке зниження концентрацій цих забруднювачів, для багатьох вели- ких міст високі концентрації NO2 в атмосферному повітрі все ще лишаються серйозною проблемою. За даними [13] у великих містах концентрації двоокису азоту можуть досягати дуже високих значень. На рис.1 представлено дані про концентрацію двоокису азоту у стовпі повітря, що отримані за допомогою газоаналізатора SCIAMACHY, встановленого на бор- ту супутника ENVISAT [13]. На карті чітко видно підвищений вміст забруднювача над великими міста- ми (в тому числі, над Києвом), над потужними вугі- льними басейнами, де високий вміст двоокису азоту може бути спричинений спалюванням супутніх газів на місті видобутку сировини та над промисловими агломераціями (наприклад, над Запорізькою та Дніп- ропетровською), де суттєвим є внесок промислових джерел [13]. Зв’язки між оксидами азоту можуть бути виражені через коефіцієнт трансформації (КТ) оксиду азоту в діоксид азоту: КТ=[NO2]/[NOx]. у 2011 р.), пасажирських автобусів – більш ніж у три рази (з 5,3 тис. до 17,2 тис.), легкових автомобілів – також майже вдвічі (з 394,4 тис до 743,2 тис). В той же час кількість громадського електротранспорту (що не призводить до забруднення повітря міста) за цей же період помітно знизилася: тролейбусних машин з 583 одиниці у 2000 р. до 490 у 2013 р., трамвайних вагонів – з 582 до 403 одиниць [11]. спостерігалися перевищення нормативу Всесвітньої організації охорони здоров’я 200 мкг/м3 для значення усередненого за 1 годину [9]. Як вже згадувалося вище, кілька років тому на те- риторії Європи вступили в дію жорсткіші нормативи якості повітря – Directive 2008/50/EC of the European Parliament and of the Council on ambient air quality and cleaner air for Europe [17]. В більшості європейських країн (навіть тих, в яких активно запроваджуються природоохоронні технології) в окремих населених пунктах до цього часу періодично фіксуються пере- вищення цих нормативів. За даними Німецького Фе- дерального Агентства з довкілля [24] в 2011 р. при- близно на 57 % станцій спостереження за забруднен- ням, що розташовані у містах під безпосереднім впливом автомобільного транспорту, було зафіксова- но перевищення середньорічного нормативу по дво- окису азоту. Проте, в деяких країнах, ситуація з за- брудненням повітря двоокисом азоту, є дещо кра- щою. Наприклад, в Білорусі за період 1999–2010 рр. середньорічні концентрації двоокису азоту у всіх містах є нижчими 40 мкг/м3 (що є нормативом ВООЗ для такого часового усереднення). Виключення ста- новить лише Могилів, в якому найвища за цей період середньорічна концентрація даного забруднювача становила 61,4 мкг/м3 [4]. Хоча, незважаючи на дот- римання середньорічних нормативів, максимальні концентрації інколи можуть бути дуже високими, наприклад, за даними [2] максимальні концентрації двоокису азоту в центральній частині Мінська у го- дини пік інколи сягають навіть до 6 ГДК м.р. (яке у цій країні дорівнює 250 мкг/м3). У структурі викидів шкідливих речовин в атмо- сферне повітря стаціонарними джерелами забруднен- ня за видами економічної діяльності протягом остан- ніх кількох років близько 80 % припадає на викиди від підприємств, що займаються виробництвом та розподілом електроенергії, газу та води. Підприємств, що займаються такою діяльністю, в Києві 12. Найбільшими з них є Дарницька ТЕЦ (ЗАТ "Укр-Кан- Пауер"), Акціонерні енергогенеруючі компанії "Київенерго" ТЕЦ-5, ТЕЦ-6, "Теплові мережі", філіал "Житлотеплоенерго" та філіал заводу з термічної переробки побутових відходів "Енергія". Джерела забруднення атмосферного повітря м. Києва. Антропогенне забруднення атмосферного повітря м. Києва в основному формується за рахунок пересувних джерел. В структурі забруднення атмо- сферного повітря м. КТ=[NO2]/[NOx]. Розрахунок значень КТ в реальних умовах при концентраціях оксидів азоту підтвердив положення про те, що повної трансформації оксиду азоту в діок- сид фактично не спостерігається, тому в розрахунках розсіювання оксидів азоту в атмосферному повітрі слід враховувати частковий ступінь трансформації оксиду азоту в діоксид. За даними [1] однією з при- чин підвищеного вмісту двоокису азоту в літній пері- од є значно вищий ніж взимку ступінь трансформації оксидів азоту, що, наприклад, для міста Стерлітамак, становить 86 %. Рис. 1 – Середній вміст діоксиду азоту в стовпі атмосфери в 2007 р. (Вміст в 1013 молекул/см2) [13]. Стосовно первинних джерел надходження дво- окису азоту, то крім тих, про які вже згадувалося вище, до них ще належать підприємства хімічної промисловості, виробництво мінеральних добрив, вибухових речовин, нітратної кислоти, бактеріальний розклад силосу та ін., а у великих урбанізованих міс- тах де переважають викиди від автомобільного транспорту – дизельні автомобілі – викиди NO2 в результаті їхньої експлуатації значно вищі, порівняно з бензиновими автомобілями. Була проведена ціла низка досліджень з метою з’ясування надходження первинного двоокису азоту від пересувних джерел у міському середовищі у різних європейських країнах [14–16, 19–21] і в результаті було встановлено, що зростання частки двоокису азоту у співвідношенні NO2/NOx від пересувних джерел переважно зумовле- не зростанням використання дизельних сажевих фі- льтрів (diesel particulate filters) та каталізаторів окис- лення (oxidation catalysts), а також зростанням кілько- сті дизельних автомобілів у загальному автопарку міст. Деякі дослідження свідчать, що для бензинових автомобілів частка первинних викидів NO2 становить близько 5 %, в той час як для автомобілів з дизельни- ми двигунами, що не оснащені новітніми установка- ми для очищення вихлопних газів цей показник дорівнює 10–13 % [16, 18, 20]. Дані [19] є ще менш оптимістичними – згідно цього дослідження надход- ження первинного двоокису азоту від бензинових Рис. 1 – Середній вміст діоксиду азоту в стовпі атмосфери в 2007 р. (Вміст в 1013 молекул/см2) [13]. Дані World Atlas of Atmospheric Pollution [22], що отримані в результаті безпосередніх вимірювань у найбільших містах світу протягом кількох останніх років, також свідчать про екстремально високі конце- нтрації двоокису азоту на окремих урбанізованих територіях – Мехіко, Братислава, Пекін, Моква, Санкт-Петербург, Ліма, Лос-Анджелес, Гвадалахара, Мадрид, Рим, Шанхай, Сеул (табл. 1). За даними WHO/UNEP [23] в окремих з цих міст і раніше спо- стерігався підвищений рівень забруднення двоокисом азоту. Наприклад, у Лос-Анджелесі, Мехіко, Москві у 1992 р. забруднення повітря двоокисом азоту оцінювалося від «помірного до сильного» і регулярно Ukr. gìdrometeorol. КТ=[NO2]/[NOx]. ž., 2015, No16 8 Забруднення атмосферного повітря міста Києва двоокисом азоту Таблиця 1 - Рівень забруднення атмосферного повітря великих міст світу двоокисом азоту в 2000-х рр.(підготовано за даними [22]) Середньорічні концен- трації NO2 (мкг/м3) М і с т а ˂ 20,0 Буенос-Айрес, Мельбурн 20,1 – 40,0 Ванкувер, Оттава, Монреаль, Рейк’явік, Осло, Берлін, Прага, Йоганнес- бург, Мумбай, Делі, Калькутта, Бангкок 40,1 – 60,0 Чикаго, Нью-Йорк, Сантьяго, Сан-Паоло, Лондон, Париж, Афіни, Токіо 60,1 – 80,0 Лос-Анджелес, Гвадалахара, Мадрид, Рим, Шанхай, Сеул ˃ 80,0 Мехіко, Братислава, Пекін, Моква, Санкт-Петербург, Ліма реважно від автомобільного транспорту). Забруднен- ня атмосфери міста автотранспортом має тери- торіальну прив’язку до міських автомагістралей, перехресть та вуличних каньйонів і характеризується інтенсивністю руху на них. Найбільш завантаженими в м. Києві можна назвати: на Лівому березі − просп. Бажана, Броварський просп., просп. генерала Ватутіна, Харківське шосе, Ленінградську пл.; у Правобережній частині міста це вул. Хрещатик, вул. Саксаганського, Бесарабська пл., Набережне шосе, бульв. Тараса Шевченка, просп. та пл. Перемо- ги, Червонозоряний просп., бульв. Івана Лепсе, просп. Комарова, Московська пл. Варто відмітити, що за даними Державного комітету статистики України [11] у м. Києві за період 2000–2011 рр. кількість автомобільного транспорту суттєво зросла. Наприк- лад, кількість вантажних автомобілів збільшилася удвічі (з 29,2 тис. у 2000 р. до 60,4 тис. у 2011 р.), пасажирських автобусів – більш ніж у три рази (з 5,3 тис. до 17,2 тис.), легкових автомобілів – також майже вдвічі (з 394,4 тис до 743,2 тис). В той же час кількість громадського електротранспорту (що не призводить до забруднення повітря міста) за цей же період помітно знизилася: тролейбусних машин з 583 одиниці у 2000 р. до 490 у 2013 р., трамвайних вагонів – з 582 до 403 одиниць [11]. реважно від автомобільного транспорту). Забруднен- ня атмосфери міста автотранспортом має тери- торіальну прив’язку до міських автомагістралей, перехресть та вуличних каньйонів і характеризується інтенсивністю руху на них. Найбільш завантаженими в м. Києві можна назвати: на Лівому березі − просп. Бажана, Броварський просп., просп. генерала Ватутіна, Харківське шосе, Ленінградську пл.; у Правобережній частині міста це вул. Хрещатик, вул. Саксаганського, Бесарабська пл., Набережне шосе, бульв. Тараса Шевченка, просп. та пл. Перемо- ги, Червонозоряний просп., бульв. Івана Лепсе, просп. Комарова, Московська пл. Варто відмітити, що за даними Державного комітету статистики України [11] у м. Києві за період 2000–2011 рр. кількість автомобільного транспорту суттєво зросла. Наприк- лад, кількість вантажних автомобілів збільшилася удвічі (з 29,2 тис. у 2000 р. до 60,4 тис. КТ=[NO2]/[NOx]. Києва в 2012 р. на пересувні джерела припадало 85,5 % і 14,5 % на стаціонарні. За даними Держкомстату в повітряний басейн Києва в 2012 р. надійшло 32,2 тис. т. сполук азоту, з яких 0,19 тис. т – оксид азоту. Переважна більшість сполук азоту (66,8 %) надійшла від пересувних джерел (пе- Отже, джерела забруднення атмосферного повітря м. Києва двоокисом азоту розосереджені по території усього міста. 9 Укр. гідрометеорол. ж., 2015, №16 О.Г.Шевченко, С.І. Сніжко, Н.О.Данілова 1987 та 2001 рр. Найвищі концентрації – 0,011 мг/м3 спостерігалися в 2006 та 2012 рр. Варто відмітити, що рівень забруднення повітря міста даним полютантом за весь період спостережень перевищує ГДК с.д., а в окремі роки цей норматив перевищується більш ніж у 2 рази. Динаміка вмісту двоокису азоту в атмосферному повітрі Києва. З метою встановлення основних тен- денцій вмісту двоокису азоту в повітрі Києва, нами були проаналізовані часові ряди середньорічних кон- центрацій по місту за період 1985−2012 рр. (рис. 2). Рис. 2 – Динаміка середньорічних концентрацій двоокису азоту в повітрі м. Києва за 1985–2012 рр. Наші попередні дослідження [10] вмісту двоокису азоту в атмосферному повітрі м. Києва дали змогу встановити, що рівень забруднення атмосферного повітря даною домішкою не є однаковим протягом року і суттєво відрізняється в теплий та холодний періоди (табл. 2). Концентрації двоокису азоту харак- теризуються вищими значеннями в теплий період – більш ніж на 15 % по відношенню до холодного. З 16-ти постів на двох спостерігаються вищі середні концентрації в холодний період − на ПСЗ № 5 (просп. Науки) та № 15 (Гідропарк). Слід зазначити, що ПСЗ № 5, № 13 та № 15 розташовані в зеленій зоні міста, де структура викидів і умови розсіювання домішок суттєво відрізняються від цих же характери- стик на інших постах. Очевидно, саме з цим і пов’язано те, що на даних ПСЗ усереднені концен- трації в холодний період є вищими від концентрацій теплого періоду. В той же час на постах, що розташо- вані поблизу навантажених автошляхів (ПСЗ № 6, 7, 8 та 10) перевищення рівня концентрацій теплого періоду над холодним становить 20–30 %. Рис. 2 – Динаміка середньорічних концентрацій двоокису азоту в повітрі м. Києва за 1985–2012 рр. Аналізуючи динаміку зміни середньорічних кон- центрацій двоокису азоту, на фоні незначних коли- вань концентрацій чітко простежується тенденція до їх зростання. За досліджувані 27 років найнижчі кон- центрації були зафіксовані на рівні 0,05 мг/м3 у 1985, аблиця 2 - Вміст двоокису азоту в теплий та холодний період в атмосферному повітрі м. Ukr. gìdrometeorol. ž., 2015, No16 КТ=[NO2]/[NOx]. Аналіз усереднених концентрацій по постах спо- стереження за теплий період (в даному дослідженні під «теплим періодом» ми розуміємо період з травня по вересень включно, оскільки саме в цей період для в Києві можуть спостерігатися сприятливі метеороло- гічні умови для формування фотохімічного смогу) дав змогу встановити, що найвищі по місту концент- рації спостерігаються в районі Бесарабської площі (ПСЗ №7) та площі Перемоги (ПСЗ №6) – тобто в центральній частинні міста, де зосереджена значна кількість автотранспорту, часто спостерігаються до- рожні затори, а щільна висотна забудова перешко- джає розсіянню домішок (табл. 3). Найнижчі концен- трації відмічаються на ПСЗ №5 (проспект Науки) та в районі Виставкового центру (ПСЗ №13). Різниці між В результаті аналізу концентрацій двоокису азоту за обрані роки по місяцях (табл. 4), було встановлено, максимальні середньомісячні концентрації даної домішки за теплий період спостерігалися в травні (2009, 2012 рр.), липні (2011 р.) та серпні (2008, 2010 рр.), що, очевидно, спричинено перш за все по- годними чинниками (переважанням синоптичних ситуацій, що були сприятливими для формування та накопичення двоокису азоту в повітрі міста в ці місяці), адже структура викидів забруднювальних речовин та їх обсяги як правило не зазнають різких змін протягом травня–вересня. Таблиця 3 - Середні концентрації двоокису азоту за травень–вересень 2008–2012 рр. на різних ПСЗ Києва ПСЗ 1 2 3 4 5 6 7 8 9 11 13 15 17 21 2008 0,07 0,09 0,08 0,08 0,02 0,09 0,11 0,08 0,07 0,09 0,04 0,04 0,09 0,07 2009 0,09 0,09 0,08 0,11 0,04 0,08 0,13 0,08 0,08 0,09 0,06 0,07 0,08 0,09 2010 0,09 0,10 0,09 0,14 0,04 0,14 0,14 0,10 0,11 0,11 0,08 0,09 0,10 0,12 2011 0,09 0,09 0,09 0,11 0,03 0,11 0,13 0,09 0,08 0,10 0,05 0,06 0,09 0,11 2012 0,13 0,14 0,16 0,15 0,05 0,17 0,21 0,12 0,15 0,16 0,07 0,07 0,15 0,16 середнє 0,09 0,10 0,10 0,12 0,04 0,12 0,14 0,09 0,10 0,11 0,06 0,07 0,10 0,11 Таблиця 4 - Концентрації двоокису азоту у повітрі м. Києва в різні місяці за період 2008–2012 р. Таблиця 4 - Концентрації двоокису азоту у повітрі м. Києва в різні місяці за період 2008–2012 р. Для представлення інформації про забруднення атмосферного повітря слід обирати таку її форму, щоб вона була максимально компактною і водночас вплив суб’єктивного підходу до її сприйняття був мінімальним. Величини концентрацій не можуть розцінюватись як самостійна інформація, адже, вони характеризують не ступінь небезпечності забруднен- ня атмосферного повітря, а лише кількість тієї чи іншої домішки в одиниці об’єму. КТ=[NO2]/[NOx]. Києва (усереднення за 2003–2006 р ПСЗ Конц. хол. періоду Конц. тепл. періоду Перевищення, % 1 0,1050 0,1203 +12,7 % 2 0,1189 0,1368 +13,1 % 3 0,111 0,1217 +8,8 % 4 0,0995 0,1198 +16,9 % 5 0,0511 0,0487 - 4,7 % 6 0,1017 0,1272 +20,0 % 7 0,1097 0,1572 +30,2 % 8 0,1054 0,1344 +21,6 % 9 0,0943 0,1141 +17,4 % 10 0,1004 0,1307 +23,2 % 11 0,1047 0,1303 +19,6 % 13 0,0516 0,0519 +0,6 % 15 0,0698 0,063 - 9,7 % 17 0,1138 0,1332 +14,6 % 20 0,1078 0,1276 +15,5 % 21 0,1176 0,1247 +5,7 % сер. по місту 0,0976 0,1151 +15,2 % Вміст двоокису азоту в теплий та холодний період в атмосферному повітрі м. Києва (усереднення за 2003–2006 рр.) Ukr. gìdrometeorol. ž., 2015, No16 10 Забруднення атмосферного повітря міста Києва двоокисом азоту найвищими та найнижчими концентраціями по місту, як і варто було очікувати, досить суттєві – більш ніж у 3 рази за весь період в цілому, а в 2008 р. – більш ніж у 5 разів. Отримані результати просторо- вого розподілу концентрацій двоокису азоту по території міста Києва в 2008–2012 рр. цілком співпадають з результатами, отриманими раніше (з періодом усереднення 2002–2006 рр.), що є цілком очікуваним, адже, структура викидів за цей період у місті практично не змінилася. Оскільки, двоокис азоту є прекурсором тропосфе- рного озону, його високі концентрації в повітрі міст у теплий період сприяють формуванню фотохімічного смогу, а попередні дослідження дали змогу встанови- ти вищі концентрації цього забруднювача у повітрі Києва у теплий період, то на нашу думку доцільним є здійснити детальний аналіз просторово-часових зако- номірностей розподілу даної домішки у місті в сучас- ний період як перший етап дослідження особливостей формування фотохімічного смогу у повітрі Києва. найвищими та найнижчими концентраціями по місту, як і варто було очікувати, досить суттєві – більш ніж у 3 рази за весь період в цілому, а в 2008 р. – більш ніж у 5 разів. Отримані результати просторо- вого розподілу концентрацій двоокису азоту по території міста Києва в 2008–2012 рр. цілком співпадають з результатами, отриманими раніше (з періодом усереднення 2002–2006 рр.), що є цілком очікуваним, адже, структура викидів за цей період у місті практично не змінилася. * - вибірка даних за цей місяць містила дуже велику кількість пропусків, тому отримані результати можуть бути не репрезен- тативними і не наводяться у таблиці КТ=[NO2]/[NOx]. Для точнішої харак- теристики негативного впливу забруднення повітря, виміряні концентрації слід порівнювати з певними нормативами. Найчастіше для цього використовують величину відповідної гранично допустимої концен- трації. Усереднені концентрації порівнюють з серед- Місяць/рік 2008 2009 2010 2011 2012 Травень 0,067 0,097 0,089 -* 0,155 Червень 0,069 0,081 0,121 0,098 -* Липень 0,061 0,076 0,106 0,103 0,137 Серпень 0,086 0,078 0,125 0,073 0,119 Вересень 0,076 0,084 0,071 0,074 0,121 11 Укр. гідрометеорол. ж., 2015, №16 О.Г.Шевченко, С.І. Сніжко, Н.О.Данілова норматив ВООЗ. Подібними є також і нормативи максимального вмісту двоокису азоту для коротких періодів – вони дорівнюють 0,2 мг/м3 і згідно вимог ВООЗ, і нормативів ЄС, і українських ГДК м.р. – дещо відрізняється лише період осереднення: у випа- дку європейських нормативів та ВООЗ він становить 1 год, українська ж ГДКм.р. передбачає порівняння з безпосередньо виміряними неусередненими концент- раціями. Але, враховуючи методики відбору проб двоокису азоту на постах спостереження за забруд- ненням повітря в містах України, коли відбирається 20-хвилинна проба, можна сказати, що таким чином для українського ГДКм.р. період усереднення стано- вить 20 хвилин, для нормативів ВООЗ та ЄС – 1 го- дина. Тобто, українське ГДК м.р. для двоокису є дуже близьким до граничних нормативів для цієї ж домішки в європейських країнах. ньодобовими гранично допустимими концентраціями (ГДК с.д.), фактично виміряні – з максимально разо- вими гранично допустимими концентраціями (ГДК м.р.). Відповідно до документів, що регламен- тують вміст забруднюючих речовин в атмосферному повітрі населених місць України, для двоокису азоту ГДК с.д. становить 0,04 мг/м3, а ГДК м.р. – 0,20 мг/м3 (табл. 5). Аналіз нормативів якості повітря ЄС та Всесвітньої організації охорони здоров’я (ВООЗ) [9] показав, що ГДКс.д., з яким в Україні порівнюються всі усереднені концентрації забруднювальних речо- вин (від середньодобових і до середньорічних) відповідає нормативу ЄС та ВООЗ для середнь- орічних значень двоокису азоту. Отже, таким чином, наприклад, у 2011 р. у Києві (див. рис. 2) середнь- орічна концентрація двоокису азоту перевищувала не лише українську ГДКс.д. у 2,75 рази, але й Європей- ський норматив якості повітря за цією домішкою та Таблиця 5 - Нормативи якості повітря в Україні, країнах Європейського Союзі та Всесвітньої організації охорони здоров’я Граничні нормативи для коротких періодів Граничні нормативи для усереднених кон- центрацій ВООЗ 0,2 мг/м3 (усереднене значення за 1 год) 0,04 мг/м3 (середньорічне значення) ЄС 0,2 мг/м3 (усереднене значення за 1 год, перевищення якого допускається не більш ніж 18 разів за рік) 0,04 мг/м3 (середньорічне значення) Україна 0,2 мг/м3 (ГДК м.р. КТ=[NO2]/[NOx]. стосуються випадків відбоpу пpоб пpотягом 20 хвилин й з цими ГДК поpівнюються pазові кон- центpації домішок) 0,04 мг/м3 (ГДКс.д., належать до тpивалої дії забруднювальних домішок і з цими ГДК поpівнюються середньодобові, сере- дньомісячні та сеpедньоpічні концентра- ції.) Таблиця 5 - Нормативи якості повітря в Україні, країнах Європейського Союзі та Всесвітньої організації охорони здоров концентраціями двоокису азоту ГДК м.р. сягала 50 %, а в 2012 р. на ПСЗ № 6, 7, 11 та 21 повторюваність була в межах 58,3–65,4 %. Розрахунок середньодобових концентрацій та нормування їх на ГДК с.д. показав, що в Києві в 2010–2012 рр. в травні–вересні майже на всіх постах концентрації двоокису азоту щодня, або майже щодня перевищують даний гігієнічний норматив (повто- рюваність випадків перевищення 90–100 %) (табл. 7). Виключення становить лише пост № 5 (проспект Науки), де повторюваність перевищень значно нижча, а в окремі роки – взагалі рівна 0 % (вересень 2010 р.), а в окремі місяці також пости №13 (Виставковий Центр) та 15 (Гідропарк), №3 (вул. Попудренка). Аналіз повторюваності випадків перевищення ГДК м.р. двоокисом азоту у м. Києві за 2008–2012 рр. (табл. 6) показав, що з певною періодичністю концен- трації вище даного нормативу спостерігаються на усіх постах, за виключенням ПСЗ № 5 (проспект Нау- ки), навіть на ПСЗ № 13 (що також розташований поза зоною прямого впливу джерел забруднення, в межах зеленої зони, на території Виставкового цен- тру) в окремі місяці повторюваність перевищення ГДК м.р. сягає 4 %. Найвища за місяць повто- рюваність перевищення ГДК м.р. спостерігається на ПСЗ №7 (Бесарабська площа), ПСЗ № 6 (площа Пе- ремоги), в окремі роки на постах №4 (район ДВРЗ), № 11 (перетин просп. Перемоги та вул. акад. Ту- полєва, біля ст.м. Святошин) та №21 (вул. Скляpенка, 5 (Куренівка)). Варто також відмітити, що в 2010 р. на ПСЗ №6 повторюваність випадків перевищення концентраціями двоокису азоту ГДК м.р. сягала 50 %, а в 2012 р. на ПСЗ № 6, 7, 11 та 21 повторюваність була в межах 58,3–65,4 %. Розрахунок середньодобових концентрацій та нормування їх на ГДК с.д. показав, що в Києві в 2010–2012 рр. в травні–вересні майже на всіх постах концентрації двоокису азоту щодня, або майже щодня перевищують даний гігієнічний норматив (повто- рюваність випадків перевищення 90–100 %) (табл. 7). Виключення становить лише пост № 5 (проспект Науки), де повторюваність перевищень значно нижча, а в окремі роки – взагалі рівна 0 % (вересень 2010 р.), а в окремі місяці також пости №13 (Виставковий Центр) та 15 (Гідропарк), №3 (вул. Попудренка). 12 Ukr. КТ=[NO2]/[NOx]. gìdrometeorol. ž., 2015, No16 Забруднення атмосферного повітря міста Києва двоокисом азоту Таблиця 6 - Повторюваність випадків перевищення ГДК м.р. на ПСЗ м. Києва (для кожного поста зазначена найвища та найнижча повторюваність за місяць з травня по вересень), % ПСЗ / Рік 2008 2009 2010 2011 2012 1 0,0– 4,0 1,0– 6,5 1,0–13,3 0,0– 4,8 5,0– 17,3 2 0,0–7,7 0,0–4,2 0,0–16,0 0,0–11,5 12,0–24,0 3 0,0–8,0 0,0–2,0 0,0–9,3 0,0–8,3 9,6–47,9 4 0,0–7,7 0,0–22,9 0,0–16,7 0,0–14,8 5,3–41,7 5 0,0 0,0 0,0 0,0 0,0 6 0,0–6,5 0,0–4,2 2,3–50,0 0,0–20,8 12,0–62,5 7 0,0–24,0 10,9–13,5 1,9–40,0 0,0–22,9 25,0–65,4 8 0,0–3,9 0,0–4,4 0,0–12,3 0,0–7,7 0,0–13,5 9 0,0–4,0 0,0–7,9 0,0–18,0 0,0–3,9 9,6–31,3 11 1,0–6,5 0,0–10,4 0,0–24,0 0,0–9,6 6,7–58,3 13 0,0 0,0–1,9 0,0–4,0 0,0 0,0–1,9 15 0,0–1,0 0,0 0,0–2,7 0,0 0,0 17 0,0–11,5 0,0–8,7 1,9–14,0 0,0–7,7 12,5–36,5 21 0,0–4,0 0,0–14,6 3,7– 9,1 0,0–16,7 7,7–58,3 Таблиця 6 - Повторюваність випадків перевищення ГДК м.р. на ПСЗ м. Києва (для кожного поста зазначена найвища та найнижча повторюваність за місяць з травня по вересень), % Таблиця 6 - Повторюваність випадків перевищення ГДК м.р. на ПСЗ м. Києва (для кожного поста зазначена найвища та найнижча повторюваність за місяць з травня по вересень), % 7 Рік ПСЗ 1 2 3 4 5 6 7 8 9 11 13 15 17 21 5 100,0 100,0 100,0 100,0 41,7 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 100,0 6 100,0 100,0 100,0 100,0 - - - - - - - - - - 7 100,0 100,0 100,0 100,0 69,2 100,0 100,0 100,0 100,0 100,0 100,0 73,1 100,0 100,0 8 100,0 100,0 100,0 100,0 46,2 100,0 100,0 100,0 100,0 100,0 96,2 100,0 100,0 100,0 2012 9 100,0 100,0 100,0 96,0 76,0 100,0 100,0 100,0 100,0 100,0 92,0 96,0 100,0 100,0 Якщо значення відсутнє – це означає, що на даному ПСЗ в цей місяць не проводилися спостереження, або кількість пропусків даних Якщо значення відсутнє – це означає, що на даному ПСЗ в цей місяць не проводилися спостереження, або кількість пропусків даних була значною і тому повторюваність перевищення ГДК не розраховувалася. Таблиця 8 - Повторюваність випадків перевищення 5 ГДК с.д. на ПСЗ м. Києва у 2012 р. Таблиця 8 - Повторюваність випадків перевищення 5 ГДК с.д. на ПСЗ м. Києва у 2012 р. ПСЗ/ Місяць 1 2 3 4 5 6 7 8 9 11 13 15 17 21 5 8,3 8,3 33,3 45,8 0,0 66,7 75,0 4,2 29,2 62,5 0,0 0,0 33,3 66,7 6 0,0 8,3 41,7 29,2 - - - - - - - - - - 7 15,4 15,4 23,1 0,0 0,0 19,2 53,9 11,5 11,5 30,8 0,0 0,0 15,4 19,2 8 3,9 7,7 11,5 5,3 0,0 11,5 23,1 3,9 3,9 0,0 0,0 0,0 7,7 0,0 9 0,0 0,0 4,0 0,0 0,0 0,0 64,0 0,0 0,0 16,0 0,0 0,0 0,0 8,0 Нормування концентрацій двоокису азоту на ГДК с.д. показало, що досить часто вони не просто перевищують цей норматив, а перевищують його у кілька разів, тому нами була також розрахована по- вторюваність випадків перевищення 5 ГДК с.д. Най- нижча кількість випадків перевищення 5 ГДК с.д. спостерігалася у 2008 р. – такі концентрації були зафіксовані лише на двох ПСЗ – № 7 (Бесарабська площа) та №2 (вул. Довженка, поблизу ст.м. Шулявська) – 12,0 % та 7,7 % відповідно. Про- тягом 2009–2011 рр. практично на усіх постах (за виключенням ПСЗ № 5, 13 та 15) в окремі місяці спо- стерігалися концентрації на рівні 5 ГДК с.д., крім того повторюваність таких випадків також суттєво зросла – на ПСЗ № 6 та 7 (що розташовані в центрі міста) в серпні 2010 р. Таблиця 6 - Повторюваність випадків перевищення ГДК м.р. на ПСЗ м. Києва (для кожного поста зазначена найвища та найнижча повторюваність за місяць з травня по вересень), % Таблиця 7 - Повторюваність випадків перевищення ГДК с.д. на постах спостереження за забрудненням міста Києва Рік ПСЗ 1 2 3 4 5 6 7 8 9 11 13 15 17 21 5 100 100,0 100,0 100,0 0,0 100,0 100,0 87,5 100,0 95,8 20,8 57,1 100,0 100,0 6 91,7 100,0 100,0 95,7 4,4 100,0 100,0 95,7 95,0 100,0 34,8 34,8 91,3 - 7 - 100,0 85,2 74,1 0,0 96,3 100,0 100,0 81,5 100,0 37,0 33,3 - - 8 96,0 100,0 100,0 92,0 0,0 100,0 100,0 100,0 100,0 100,0 68,0 64,0 100,0 100,0 2008 9 84,6 84,6 84,6 96,2 11,5 - 96,2 92,3 65,4 96,2 65,4 69,2 96,2 76,9 5 100,0 100,0 100,0 100,0 13,0 - 100,0 100,0 100,0 100,0 21,7 46,2 100,0 100,0 6 95,8 100,0 79,2 87,5 29,2 91,7 - 77,8 70,8 91,7 70,8 87,5 47,4 91,7 7 100,0 51,0 85,2 96,3 96,3 92,6 - 96,3 100,0 100,0 96,3 100,0 100,0 92,6 8 100,0 96,0 92,0 96,0 68,0 80,0 - 80,0 84,0 96,0 84,0 100,0 96,0 88,0 2009 9 100,0 100,0 92,3 100,0 73,1 96,2 100,0 100,0 100,0 100,0 96,2 100,0 100,0 100,0 5 100,0 100,0 95,5 100,0 22,7 100,0 100,0 100,0 100,0 100,0 95,5 100,0 100,0 100,0 6 100,0 100,0 100,0 100,0 56,0 100,0 100,0 100,0 100,0 100,0 100,0 96,0 100,0 - 7 100,0 100,0 100,0 100,0 55,6 100,0 100,0 96,3 100,0 100,0 100,0 - 100,0 100,0 8 96,0 100,0 76,0 - 56,0 100,0 100,0 100,0 100,0 100,0 84,0 77,8 96,0 - 2010 9 92,3 100,0 61,5 100,0 0,0 100,0 100,0 100,0 100,0 95,2 92,3 84,6 92,3 - 5 100,0 100,0 100,0 100,0 17,4 100,0 - - - - - - - - 6 100,0 100,0 95,8 100,0 50,0 100,0 100,0 100,0 95,8 100,0 79,2 100,0 100,0 100,0 7 100,0 100,0 100,0 100,0 38,5 100,0 100,0 96,2 100,0 100,0 80,8 92,9 100,0 100,0 8 100,0 100,0 - 100,0 11,5 100,0 100,0 100,0 100,0 100,0 100,0 84,6 100,0 100,0 2011 9 100,0 100,0 - 100,0 7,7 100,0 100,0 100,0 100,0 100,0 - - 100,0 100,0 Таблиця 7 - Повторюваність випадків перевищення ГДК с.д. на постах спостереження за забрудненням міста Києва 13 Укр. гідрометеорол. ж., 2015, №16 О.Г.Шевченко, С.І. Сніжко, Н.О.Данілова Продовження табл. Таблиця 6 - Повторюваність випадків перевищення ГДК м.р. на ПСЗ м. Києва (для кожного поста зазначена найвища та найнижча повторюваність за місяць з травня по вересень), % вона становила 48 та 40 % відповідно, в червні 2011 р. – 25 % на цих же постах. В 2012 р. середньорічна концентрація двоокису азоту сягнула 0,11 мг/м3, що перевищує ГДК с.д. у 2,75 рази, відповідно і середньодобові концентрації також були значно вищими від цього нормативу – в теплий період на окремих ПСЗ повторюваність перевищення 5 ГДК с.д. становила понад 50 % (ПСЗ № 6, 7, 11 та 21) (табл. 8). перевищення середньорічними концентраціями дво- окису азоту ГДК с.д., на фоні незначних коливань концентрацій домішки за період 1985–2012 рр. чітко простежується тенденція до подальшого зростання її вмісту в повітрі Києва. Найвищі концентрації двооки- су азоту спостерігаються в центральній частині міста – на постах, розташованих в районі Бесарабської площі та площі Перемоги – в окремі місяці теплого періоду року повторюваність випадків перевищення ГДК м.р. на цих ПСЗ перевищує 50 %. Повто- рюваність перевищення ГДК с.д. середньодобовими концентраціями двоокису азоту в повітрі міста в останні роки була дуже високою, а в теплий період 2012 р. майже на усіх постах міста (за виключенням тих, що розташовані в межах зелених зон) сягнула 100 %. перевищення середньорічними концентраціями дво- окису азоту ГДК с.д., на фоні незначних коливань концентрацій домішки за період 1985–2012 рр. чітко простежується тенденція до подальшого зростання її вмісту в повітрі Києва. Найвищі концентрації двооки- су азоту спостерігаються в центральній частині міста – на постах, розташованих в районі Бесарабської площі та площі Перемоги – в окремі місяці теплого періоду року повторюваність випадків перевищення ГДК м.р. на цих ПСЗ перевищує 50 %. Повто- рюваність перевищення ГДК с.д. середньодобовими концентраціями двоокису азоту в повітрі міста в останні роки була дуже високою, а в теплий період 2012 р. майже на усіх постах міста (за виключенням тих, що розташовані в межах зелених зон) сягнула 100 %. Таким чином, у теплий період року в повітрі м. Києва регулярно спостерігаються аномально висо- кі концентрації двоокису азоту, що є прекурсором фотохімічного смогу, і за сприятливих метеорологіч- ними умов, буде призводити до формування цього негативного явища в атмосфері міста. СПИСОК ЛІТЕРАТУРИ 1. Бикбулатов И.Х. Анализ содержания оксидов азота в воздуш- ном басейне г. Стерлитамак РБ / И.Х. Бикбулатов, Л.Р. Асфандиярова, А.А. Панченко // Актуальные проблемы науки и техники. Сборник трудов ІІІ научной конференции молодых ученых. – Уфа: Нефтегазовое дело, 2011. – С. 46–47. 1. Бикбулатов И.Х. Анализ содержания оксидов азота в воздуш- ном басейне г. Стерлитамак РБ / И.Х. Бикбулатов, Л.Р. Асфандиярова, А.А. Панченко // Актуальные проблемы науки и техники. Сборник трудов ІІІ научной конференции молодых ученых. – Уфа: Нефтегазовое дело, 2011. – С. 46–47. REFERENCES 1. Bikbulatov I.H., Asfandijarova L.R., Panchenko A.A. Aktual'nye problemy nauki i tehniki. Sbornik trudov ІІІ nauchnoj konferencii molodyh uchenyh [Actual problems of science and technology. Proc. ІІІ Conference of Young Scientists ]. Ufa: «Neftegazovoye delo», 2011, pp. 46–47. 7. Маренко А.Н. Окислы азота в приземном слое воздуха на автомагистралях Киева / А.Н. Маренко, А.Н. Гриценко // Тру- ды УкрНИИ Госкомгидромета. − 1986. − Вып. 216. − С. 46−53. 2. Glazacheva G.I., Kurlovich T.A., Zalygina I.A. Novosti nauki i tehnologiy - Science and Technology News, 2011, no.1 (18), pp. 3–10. (In Russian) 8. Маренко А.Н. Исследование смогообразующих примесей в атмосфере вблизи автомагистралей / А.Н. Маренко, А.Н. Гриценко, В.С. Титов и др. // Труды УкрНИИ Госком- гидромета. − 1987. − Вып. 224. − С. 49−54. 3. Kiptenko Ye.M., Kozlenko T.V. Hidrolohiya, hidrokhimiya i hidroekolohiya - Hydrology, hydrochemistry and hydroecology, 2007, no.13, pp. 208−216. (In Ukrainian) 9. Рекомендации ВОЗ по качеству воздуха, касающиеся твердых частиц, озона, двуокиси азота и двуокиси серы. Глобальные обновленные данные 2005 год. [Електронний ресурс]. − Режим доступу: http://whqlibdoc.who.int/hq/2006/WHO_SDE_PHE_OEH_06.02_ rus.pdf 4. Krukovskaja O.Ju. Monitoring okruzhayushchey sredy: sb. materialov II mezhdunarodnoy nauch.-prakt. konf. Brest, 25–27 sentyabrya 2013 g.: v 2 ch. Brest. gos. un-t imeni A.S. Pushkina [Environmental Monitoring: Material collec. the III International scientific-practical. conf. Brest, 25-27 September 2013 : in 2 parts. The A.S. Pushkin Brest State University]. Брест: BSU, 2013, pp. 180–182. (In Russian) 10. Сніжко С.І. Урбометеорологічні аспекти забруднення атмо- сферного повітря великого міста / С.І. Сніжко, О.Г. Шевченко – К. : Обрії, 2011. – 297 с. 5. Loyeva I.D., Hrudyev P.Kh., Demchyshyna N.M. Meteorologiya, klimatologiya y gidrologiya - Meteorology, climatology and hy- drology , 2004, vol. 48, pp. 279−286. (In Ukrainian) 11. Статистичний щорічник міста Києва за 2013 рік [статистичний довідник]. − К.: ТОВ «Август Трейд», 2012. − 344 с. 12. Суркова Г.В. Химия атмосферы / Г.В. Суркова. – М.: Изд-во Московского университета, 2002. – 210 с. 6. Loeva I.D. Salim Rubajja Sand. Meteorologiya, klimatologiya y gidrologiya - Meteorology, climatology and hydrology, 1995, vol. 32, pp. 107–113. (In Russian) 13. Тронин А.А. Диоксид азота в воздушном бассейне России по спутниковым данным / А.А. Тронин, С.Г. Крицук, И.Ш. Латыпов // Современные проблемы дистанционного зондирования Земли из космоса. – 2009. – Т.2. – № 6. – С. 217–223. 7. Marenko A.N. Gricenko A.N. Trudy UkrNII Goskomgidrometa - Proc. USRI SCHM, 1986, vol. 216, pp. 46−53. (In Russian) 8. Marenko A.N. Gricenko A.N., Titov V.S. et al. 4. ВИСНОВКИ Отже, вміст двоокису азоту в атмосферному пові- трі м. Києва переважно визначається його надход- женням від джерел викидів, серед яких основними є двигуни внутрішнього згорання і спалювання органі- чного палива на ТЕС, та перебігом фотохімічних реакцій за його участю. Незважаючи на постійне 2. Глазачева Г.И. Состояние атмосферного воздуха г. Минска и прилегающего района / Г.И. Глазачева, Т.А. Курлович, И.А. Залыгина // Новости науки и технологий. – 2011. – № 1 (18). – С. 3–10. 3. Кіптенко Є.М. Вплив метеорологічних умов забруднення 14 Ukr. gìdrometeorol. ž., 2015, No16 Забруднення атмосферного повітря міста Києва двоокисом азоту 21. Kessler C., Niederau A., Scholz W. Estimation of NO2/NOx rela- tions of traffic emissions in Baden-Wurttemberg from 1995 to 2005. 2nd Conference on Environment & Transport, including 15th Conference on Transport and Air Pollution. Reims, France, 12–14 June 2006. Proceedings no. 107, vol. 2. France: Inrets, Ar- ceuil, pp. 101–105. повітря у промислових містах України / Є.М. Кіптенко, Т.В. Козленко // Гідрологія, гідрохімія і гідроекологія. −2007. − № 13. − С. 208−216. 4. Круковская О.Ю. Анализ динамики содержания диоксида азота в атмосферном воздухе в Беларуси / О.Ю. Круковская // Мониторинг окружающей среды: сб. материалов II междуна- родной науч.-практ. конф. Брест, 25–27 сентября 2013 г.: в 2 ч. Брест. гос. ун-т имени А.С. Пушкина. – Брест: БрГУ, 2013. – С. 180–182. 22. The World Atlas of Atmospheric Pollution. Anthem Press, 2008. 345 p. (Ed.: Sokhi R.S.) 23. The Quality of Air in World Megacities (based on WHO/UNEP data, 1992). Available at: http://www.air-quality.org.uk/11.php 5. Лоєва І.Д. Оцінка антропогенного навантаження на повітряний басейн м. Одеси / І.Д. Лоєва, П.Х. Грудєв, Н.М. Демчишина // Метеорология, климатология и гидрология. − 2004. − Вып. 48. − С. 279−286. data, 1992). Available at: http://www.air-quality.org.uk/11.php 24. Umwelt Bundes Amt. How good is air quality in Germany? Press Release No. 06/2012. Available at: 6. Лоева И.Д. Прогноз поля загрязнения воздушного бассейна города двуокисью азота / И.Д. Лоева, Салим Рубайя Санд // Метеорология, климатология и гидрология. − 1995. − Вып. 32. − С. 107–113. REFERENCES Trudy UkrNII Goskomgidrometa - Proc. USRI SCHM, 1987, vol. 224, vol. 49−54. (In Russian) 14. Carslaw D.C. Evidence of an increasing NO2/NOx emissions ratio from road traffic emissions. Atmospheric Environment, 2005, vol. 39, pp. 4793–4802. 9. Rekomendacii VOZ po kachestvu vozduha, kasajushhiesja tver-dyh chastic, ozona, dvuokisi azota i dvuokisi sery. Global'nye obnov- lennye dannye 2005 god [The WHO air quality guidelines for par- ticulate matter, ozone, nitrogen dioxide and sulfur dioxide. Global update 2005]. Available at: http://whqlibdoc.who.int/hq/2006/WHO_SDE_PHE_OEH_06.02_ df 15. Carslaw D.C., Beevers S.D. Investigating the potential importance of primary NO2 emissions in a street canyon. Atmospheric Envi- ronment, 2004, vol. 38, pp. 3585–3594. 16. Carslaw D.C., Beevers S.D. Development of an urban inventory for road transport emissions of NO2 and comparison with estimates derived from ambient measurements. Atmospheric Environment, 2005,. vol. 39, pp. 2049–2059. http://whqlibdoc.who.int/hq/2006/WHO_SDE_PHE_OEH_06.02_ rus.pdf 10. Snizhko S.I., Shevchenko O.H. Urbometeorolohichni aspekty zabrudnennya atmosfernoho povitrya velykoho mista [Urbo Mete- orological aspects of air pollution of the big city]. Kyiv: Obriyi, 2011. 297 p. 17. Directive 2008/50/EC of the European Parliament and of the Council of 21 May 2008 on ambient air quality and cleaner air for Europe. Official Journal of the European Union, L 152/1 – L 152/44. 11. Statystychnyy shchorichnyk mista Kyyeva za 2013 rik [Statistical Yearbook of Kyiv for 2013]. Kyiv: LLC «Avhust Treyd», 2012. 344 p. 18. Grice S., Stedman J., Kent A., Hobson M., Norris J., Abbott J., Cooke S. Recent trends and projections of primary NO2 emissions in Europe. Atmospheric Environment, 2009, vol. 43 (13), pp. 2154–2167. 12. Surkova G.V. Himija atmosfery [Chemistry atmosphere]. Moskow: The Moskow University Publ., 2002, 210 p. 13. Tronin A.A., Kricuk S.G., Latypov I.Sh. Sovremennye problemy distancionnogo zondirovanija Zemli iz kosmosa – Modern prob- lems of remote sensing of the Earth from space, 2009, vol. 2, no.6, pp. 217–223. (In Russian) 19. Hopfner U., Lambrecht U. Emissions and air quality in urban areas of Germany. An actual survey. Proceedings of the 14th Interna- tional Conference on Transport and Air Pollution. Graz, Austria, 1–3 June 2005. 14. Carslaw D.C. Evidence of an increasing NO2/Nox emissions ratio from road traffic emissions. Atmospheric Environment, 2005, vol. 39, pp. 4793–4802. 20. Jenkin M.E. Analysis of sources and partitioning of oxidant in the UK Part 2: contributions of nitrogen dioxide emissions and back- ground ozone at a kerbside location in London. Atmospheric Envi- ronment, 2004,.vol. 38, pp. 5131–5138. 15. Carslaw D.C., Beevers S.D. AIR POLLUTION BY NITROGEN DIOXIDE IN KIEV CITY AIR POLLUTION BY NITROGEN DIOXIDE IN KIEV CITY O. Shevchenko, Cand. Sci. (Geogr.), S. Snizhko, Dr. Sci. (Geogr.), N.Danilova Taras Shevchenko National University of Kyiv; 64/13, Volodymyrska Street, City of Kyiv, Ukraine, 01601, tempo2007@meta.ua Taras Shevchenko National University of Kyiv; Taras Shevchenko National University of Kyiv; 64/13, Volodymyrska Street, City of Kyiv, Ukraine, 01601, tempo2007@meta.ua 64/13, Volodymyrska Street, City of Kyiv, Ukraine, 01601, tempo2007@meta.ua 64/13, Volodymyrska Street, City of Kyiv, Ukraine, 01601, tempo2007@meta.ua In the article main nitrogen dioxide emissions sources in a big cities was analysed. Shown the temporal dynamics of average annual concentrations of nitrogen dioxide in Kiev city for time period 1985 – 2012, analyzed concentration of this pollutant in different part of the city and frequency of cases of maximum allowable concentration (MAC) exceeding. The highest concentrations of nitrogen dioxide observed in the central part of the city in some months the warm season. The frequency of cases exceeding of MAC in the air usually exceeds 50% and in the warm season 2012 average concentration of NO2 in the air has been very high and frequency of exceeding of MAC reached 100% in almost all monitoring stations. In the article main nitrogen dioxide emissions sources in a big cities was analysed. Shown the temporal dynamics of average annual concentrations of nitrogen dioxide in Kiev city for time period 1985 – 2012, analyzed concentration of this pollutant in different part of the city and frequency of cases of maximum allowable concentration (MAC) exceeding. The highest concentrations of nitrogen dioxide observed in the central part of the city in some months the warm season. The frequency of cases exceeding of MAC in the air usually exceeds 50% and in the warm season 2012 average concentration of NO2 in the air has been very high and frequency of exceeding of MAC reached 100% in almost all monitoring stations. Studies show that in the warm season in the air of Kyiv regularly observed abnormally high concentra- tions of nitrogen dioxide, which is a precursor of photochemical smog, and under favorable meteorological conditions will result in the formation of this negative phenomenon in the atmosphere of the city. Keywords: air pollution, nitrogen dioxide, exceeding of maximum allowable concentration, air pollution level dynamics. REFERENCES Investigating the potential importance 15 Укр. гідрометеорол. ж., 2015, №16 О.Г.Шевченко, С.І. Сніжко, Н.О.Данілова 20. Jenkin M.E. Analysis of sources and partitioning of oxidant in the UK Part 2: contributions of nitrogen dioxide emissions and back- ground ozone at a kerbside location in London. Atmospheric Envi- ronment, 2004,.vol. 38, pp. 5131–5138. of primary NO2 emissions in a street canyon. Atmospheric Envi- ronment, 2004, vol. 38, pp. 3585–3594. of primary NO2 emissions in a street canyon. Atmospheric Envi- ronment, 2004, vol. 38, pp. 3585–3594. 16. Carslaw D.C., Beevers S.D. Development of an urban inventory for road transport emissions of NO2 and comparison with estimates derived from ambient measurements. Atmospheric Environment, 2005,. Vol. 39, pp. 2049–2059. 21. Kessler C., Niederau A., Scholz W. Estimation of NO2/NOx rela- tions of traffic emissions in Baden-Wurttemberg from 1995 to 2005. 2nd Conference on Environment & Transport, including 15th Conference on Transport and Air Pollution. Reims, France, 12–14 June 2006. Proceedings no. 107, vol. 2. France: Inrets, Arceuil, pp. 101–105. 17. Directive 2008/50/EC of the European Parliament and of the Council of 21 May 2008 on ambient air quality and cleaner air for Europe. Official Journal of the European Union, L 152/1 – L 152/44. 22. The World Atlas of Atmospheric Pollution. Anthem Press, 2008. 345 p. (Ed.: Sokhi R.S.) 18. Grice S., Stedman J., Kent A., Hobson M., Norris J., Abbott J., Cooke S. Recent trends and projections of primary NO2 emissions in Europe. Atmospheric Environment, 2009, vol. 43 (13), pp. 2154–2167. 23. The Quality of Air in World Megacities (based on WHO/UNEP data, 1992). Available at: http://www.air-quality.org.uk/11.php 19. Hopfner U., Lambrecht U. Emissions and air quality in urban areas of Germany. An actual survey. Proceedings of the 14th Interna- tional Conference on Transport and Air Pollution. Graz, Austria, 1–3 June 2005. 24. Umwelt Bundes Amt. How good is air quality in Germany? Press Release No. 06/2012. Available at: 24. Umwelt Bundes Amt. How good is air quality in Germany? Press Release No. 06/2012. Available at: http://www.umweltbundesamt.de/uba-info-medien-e/4211.html Release No. 06/2012. Available at: http://www.umweltbundesamt.de/uba-info-medien-e/4211.html http://www.umweltbundesamt.de/uba-info-medien-e/4211.html О.Г. Шевченко, к. геогр. н., С.И. Снежко, д. геогр. н. Н.А. Данилова Киевский национальний университет имени Тараса Шевченка, Украина, 01601, город Киев, ул. Владимирская, 64/13, tempo2007@meta.ua Киевский национальний университет имени Тараса Шевченка, Украина, 01601, город Киев, ул. Владимирская, 64/13, tempo2007@meta.ua В статье описаны основные источники поступления двуокиси азота в атмосферный воздух боль- ших городов. Рассмотрена временная динамика среднегодовых концентраций двуокиси азота в атмо- сфере г. Киева, проанализирован уровень загрязнения данной примесью в разных частях города, уста- новлена повторяемость случаев превышения двуокисью азота максимально-разовой и среднесуточной предельно-допустимых концентраций (ПДК). Наивысшие концентрации двуокиси азота наблюдались в центральной части города в некоторые месяцы теплого периода.Частота случаев превышения ПДК в воздухе обычно достигала 50%, а в теплый сезон 2012 г. концентрация NO2 в воздухе была очень вы- сокой и превышала ПДК в 100% случаев на всех станциях мониторинга. Исследования показали, что в теплый сезон года в воздухе г.Киева регулярно наблюдаются повышенные концентрации двуокиси азота, которые являются прекурсорами фотохимического смога, что при благоприятных метеорологи- ческих условиях может привести к формированию этого этого негативного феномена в атмосфере го- рода. Ключевые слова: загрязнение атмосферного воздуха, двуокись азота, превышение предельно- допустимой концентрации, динамика уровня загрязнения. Дата першого подання.: 07.10.2015 Дата надходження остаточної версії :23.11.2015 Дата публікації статті :26.11.2015 16 Ukr. gìdrometeorol. ž., 2015, No16
https://openalex.org/W2589444358	https://europepmc.org/articles/pmc5392565?pdf=render	English	null	Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation	Journal of biological chemistry/The Journal of biological chemistry	2,017	cc-by	9,738	Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation Vladimir Baytshtok‡, Jiejin Chen‡1, Steven E. Glynn‡2, Andrew R. Nager‡3, Robert A. Grant‡, Tania A. Baker‡§, and Robert T. Sauer‡4 From the ‡Department of Biology and §Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Edited by George N. DeMartino (ii) sequential hydrolysis by individual subunits that occurs in an invariant kinetic pattern (4); and (iii) probabilistic hydrolysis in which, following a power stroke, any ATP-bound subunit has some chance of hydrolyzing ATP to drive the next power stroke (5). (ii) sequential hydrolysis by individual subunits that occurs in an invariant kinetic pattern (4); and (iii) probabilistic hydrolysis in which, following a power stroke, any ATP-bound subunit has some chance of hydrolyzing ATP to drive the next power stroke (5). The HslUV proteolytic machine consists of HslV, a double- ring self-compartmentalized peptidase, and one or two AAA HslU ring hexamers that hydrolyze ATP to power the unfolding of protein substrates and their translocation into the proteolytic chamber of HslV. Here, we use genetic tethering and disulfide bonding strategies to construct HslU pseudohexamers con- taining mixtures of ATPase active and inactive subunits at defined positions in the hexameric ring. Genetic tethering impairs HslV binding and degradation, even for pseudohex- amers with six active subunits, but disulfide-linked pseudo- hexamers do not have these defects, indicating that the pep- tide tether interferes with HslV interactions. Importantly, pseudohexamers containing different patterns of hydrolyti- cally active and inactive subunits retain the ability to unfold protein substrates and/or collaborate with HslV in their deg- radation, supporting a model in which ATP hydrolysis and linked mechanical function in the HslU ring operate by a probabilistic mechanism. ( ) The HslUV protease consists of one or two AAA HslU hexamers and the dodecameric HslV peptidase (Fig. 1A) (6–12). In ATP-dependent reactions, HslU hexamers recog- nize protein substrates, unfold any native structure that is pres- ent, and then translocate the unfolded polypeptide into the luminal chamber of HslV for degradation. In all crystal struc- tures of the Escherichia coli or Haemophilus influenzae HslUV complexes and some structures of HslU alone, the HslU hex- amer is highly symmetric and binds six ATP or ADP molecules, as might be expected for a concerted mechanism of hydrolysis (6–12). Covalently linked HslU hexamers support a probabilistic mechanism that links ATP hydrolysis to protein unfolding and translocation However, in other structures of HslU alone, only three or four nucleotides are bound to the hexameric HslU ring (6), and solution experiments show detectable binding of a max- imum of 3–4 nucleotides and the existence of at least two types of nucleotide-binding sites (13). These results suggest that the six subunits of HslU assume non-equivalent func- tional roles within the hexamer and are more consistent with sequential or probabilistic models. Here, we test different models by which ATP hydrolysis could power the mechani- cal functions of E. coli HslU by introducing hydrolytically inactive subunits at defined positions in its hexameric ring. Using subunit cross-linking strategies involving genetic tethering or disulfide bonding, we find that HslU pseudohex- amers with mixtures of hydrolytically active and inactive subunits retain protein unfolding activity and support HslV degradation. These studies support a probabilistic mecha- nism in which ATP hydrolysis powers mechanical function in the HslU ring and also reveal new information about inter- actions between HslU and HslV. Enzymes of the AAA ATPase superfamily play roles in pro- teolysis, protein remodeling and disaggregation, replication, transcription, membrane fusion, vesicle transport, and other cellular processes in all organisms (1, 2). These enzymes share conserved sequence and structural motifs and typically func- tion as homohexameric or heterohexameric rings. Fueled by the energy of ATP binding and hydrolysis, AAA enzymes act as molecular machines that disassemble, remodel, or denature macromolecule targets. There are three general models for how subunits in AAA hexamers hydrolyze ATP and generate the mechanical power strokes required for function: (i) concerted ATP hydrolysis that occurs simultaneously in all subunits (3); This work was supported by National Institutes of Health Grant AI-16892. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Theatomiccoordinatesandstructurefactors(code5TXV)havebeendepositedin the Protein Data Bank (http://wwpdb.org/). p p g 1 Present address: Takeda Pharmaceuticals, Cambridge, MA 02139. 2 This work was supported by National Institutes of Health Grant AI-16892. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Theatomiccoordinatesandstructurefactors(code5TXV)havebeendepositedin the Protein Data Bank (http://wwpdb.org/). 1 Present address: Takeda Pharmaceuticals, Cambridge, MA 02139. 2 Present address: Dept. of Biochemistry and Cell Biology, Stony Brook Uni- versity, Stony Brook, NY 11794. 3 Present address: Dept. of Molecular and Cellular Physiology, Stanford Uni- versity, Stanford, CA 94305. 4 To whom correspondence should be addressed E mail: bobsauer@mit edu y y 3 Present address: Dept. of Molecular and Cellular Physiology, Stanford Uni- versity, Stanford, CA 94305. 2 Present address: Dept. of Biochemistry and Cell Biology, Stony Brook Uni- versity, Stony Brook, NY 11794. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 14, pp. 5695–5704, April 7, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. crossmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 14, pp. 5695–5704, April 7, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. ssmark THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 14, pp. 5695–5704, April 7, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 14, pp. 5695–5704, April 7, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 292, NO. 14, pp. 5695–5704, April 7, 2017 © 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. HslU dimers with covalent peptide tethers peptide tethered HslU dimer B first HslU subunit C-tail large AAA+ small AAA+ large AAA+ small AAA+ C-tail peptide tether second HslU subunit B peptide tethered HslU dimer W-E3 hexamers C HslUV structure. A, an HslU hexamer (secondary-structure representation) bound to an HslV dodecamer (surface representation; Protein Data Bank . The large and small AAA domains of HslU and its C-terminal tails are colored blue, cyan, and red, respectively. B, tandem HslU subunits connected cally encoded peptide tether. C, three W-E3 hexamers in the asymmetric unit of structure 5TXV are shown in a secondary-structure representation; the amer is shown in a ribbon representation with electron density from a composite omit map contoured at 1 W-E3 hexamers C C Figure 1. HslUV structure. A, an HslU hexamer (secondary-structure representation) bound to an HslV dodecamer (surface representation; Protein Data Bank code 1G3I). The large and small AAA domains of HslU and its C-terminal tails are colored blue, cyan, and red, respectively. B, tandem HslU subunits connected by a genetically encoded peptide tether. C, three W-E3 hexamers in the asymmetric unit of structure 5TXV are shown in a secondary-structure representation; the fourth hexamer is shown in a ribbon representation with electron density from a composite omit map contoured at 1 . connected by a 20-residue peptide tether (Fig. 1B). One encoded dimer consisted of two wild-type subunits (W-W), another had a wild-type subunit followed by an E257Q subunit (W-E), and a third had an E257Q subunit followed by a wild- type subunit (E-W). These dimers behaved like wild-type HslU during purification, suggesting that they form W-W3, W-E3, and E-W3 pseudohexamers. Indeed, the asymmetric unit of a low-resolution W-E3 crystal structure contained four hexamers similar to wild-type HslU (Fig. 1C and Table 1), although elec- tron density for the C-terminal 8–10 residues was missing in alternating subunits of several hexamers or was generally poor throughout a hexamer, as expected if the tether disrupts normal C-terminal contacts. structed and expressed a W-W-W trimer, but this protein was insoluble. To assay protein unfolding, we constructed an I37AArc-cp6GFP-st11-ssrA fusion protein with a thrombin cleavage site located between -strands 5 and 6 (14). I37AArc is a denatured variant of Arc repressor that targets the substrate to HslU, and the C-terminal st11-ssrA sequence increases the substrate turnover rate 2-fold (15, 16). HslU dimers with covalent peptide tethers HslU homohexamers containing the Walker B E257Q muta- tion are defective in ATP hydrolysis and protein degradation but retain the ability to bind HslV and protein substrates (13). We engineered genes to encode tandem E. coli HslU subunits 2 Present address: Dept. of Biochemistry and Cell Biology, Stony Brook Uni- versity, Stony Brook, NY 11794. 3 Present address: Dept. of Molecular and Cellular Physiology, Stanford Uni- versity, Stanford, CA 94305. 3 Present address: Dept. of Molecular and Cellular Physiology, Stanford Uni- versity, Stanford, CA 94305. 4 To whom correspondence should be addressed. E-mail: bobsauer@mit.edu. JOURNAL OF BIOLOGICAL CHEMISTRY 56 5695 APRIL 7, 2017•VOLUME 292•NUMBER 14 APRIL 7, 2017•VOLUME 292•NUMBER 14 Disulfide-linked HslU pseudohexamers Disulfide-linked HslU pseudohexamers A large B large B arg arg large AAA+ domains small AAA+ domains C-tails HslV HslU A W-E3 hexamers C peptide tethered HslU dimer B first HslU subunit C-tail large AAA+ small AAA+ large AAA+ small AAA+ C-tail peptide tether second HslU subunit A large B large B arg arg large AAA+ domains small AAA+ domains C-tails HslV HslU . HslUV structure. A, an HslU hexamer (secondary-structure representation) bound to an HslV dodecamer (surface representation; Protein Data Bank I). The large and small AAA domains of HslU and its C-terminal tails are colored blue, cyan, and red, respectively. B, tandem HslU subunits connected etically encoded peptide tether. C, three W-E3 hexamers in the asymmetric unit of structure 5TXV are shown in a secondary-structure representation; the xamer is shown in a ribbon representation with electron density from a composite omit map contoured at 1 . W-E3 hexamers C peptide tethered HslU dimer B first HslU subunit C-tail large AAA+ small AAA+ large AAA+ small AAA+ C-tail peptide tether second HslU subunit large B large B arg arg small AAA+ domains C-tails HslV HslU Figure 1. HslUV structure. A, an HslU hexamer (secondary-structure representation) bound to an HslV dodecamer (surface representation; Protein Data Bank code 1G3I). The large and small AAA domains of HslU and its C-terminal tails are colored blue, cyan, and red, respectively. B, tandem HslU subunits connected by a genetically encoded peptide tether. C, three W-E3 hexamers in the asymmetric unit of structure 5TXV are shown in a secondary-structure representation; the fourth hexamer is shown in a ribbon representation with electron density from a composite omit map contoured at 1 . Disulfide-linked HslU pseudohexamers Relatively efficient formation of disulfide-linked HslU dimers or trimers was achieved by cytosolic coexpression of appropriate variants in the oxidizing SHuffle strain of E. coli 0 50 100 150 200 250 W-W3 W-E3 E-W3 ATP hydrolyzed (min-1 enz-1) A HslU W-W3 E-W3 W-E3 unfolding rate (min-1 enz-1) [protein substrate] (µM) B 0 0.1 0.2 0 10 20 30 40 –Arc-st11-ssrA HslU/HslV 0 5 10 30 60 90 0 5 10 30 60 90 degradation time (min) W-W3/HslV C Figure 2. Activity of genetically tethered pseudohexamers. A, rates of hydrolysis of 5 mM ATP by the genetically tethered W-W3, W-E3, and E-W3 pseudo- hexamers. Values are averages (n 5) S.D. (error bars). B, rates of unfolding of different concentrations of thrombin-split I37AArc-cp6GFP-5/6-st11-ssrA by wild-type HslU or genetically tethered variants. Lines, non-linear least squares fits to the Michaelis-Menten equation. Km values for all enzymes were 1–3 M but werenotwelldeterminedbecauseofthesmallnumberoflow-concentrationdatapoints.AverageVmaxvaluesS.D.calculatedfromthehighestfoursubstrate concentrations were 0.155 0.003 min1 enz1 (HslU), 0.143 0.008 min1 enz1 (W-W3), 0.0802 0.007 min1 enz1 (E-W3), and 0.0716 0.006 min1 enz1 (W-E3). Fitted Vmax values were 10–15% higher. C, the kinetics of degradation of Arc-st11-ssrA (10 M) by HslV (10 M) and HslU (0.3 M) or W-W3 (0.3 M) at 50 °C was monitored by SDS-PAGE. Reactions contained 5 mM ATP and a regeneration system. Table 1 Crystallographic statistics Values in parenthesis represent the highest resolution shell. Protein Data Bank code 5TXV Wavelength (Å) 0.979 Space group P 1 21 1 Unit-cell dimensions (Å) a 86.5; b 420.9; c 176.5 Unit-cell angles (degrees) 90; 98.6 Resolution range (Å) 49.2–7.1 (7.3–7.1) Unique reflections 18,630 (1784) Completeness (%) 98.3 (94.6) Redundancy 4.5 (4.5) Rmerge 0.105 (0.694) Rmeas 0.119 (0.782) Rpim 0.055 (0.315) Rwork 0.274 (0.356) Rfree 0.298 (0.347) MolProbity score (percentile) 100 Root mean square bonds (Å) 0.003 Root mean square angles (degrees) 0.61 Clash score 5.8 Favored rotamers (%) 99.0 Poor rotamers (%) 0.39 Ramachandran favored 98.0 Ramachandran outliers (%) 0 Bad bonds/angles 0/1 C deviations 0 Table 2 HslV binding Titration assays, monitored by changes in HslV peptidase activity, were performed at 25 °C in the presence of 5 mM ATP. Protein K1/2 nM Wild-type HslU 78 11 W-W3 1603 278 WSSW3 59 9 WSSWSSW2 56 5 WSSESSW2 21 7 WSSE3 23 11 WSSESSE3 28 19 HslU dimers with covalent peptide tethers Following thrombin cleavage, unfolding of the split substrate by wild-type HslU results in an irreversible loss of GFP fluorescence. In experi- ments performed at different concentrations of the split sub- strate, unfolding by wild-type HslU and W-W3 occurred with steady-state Vmax rates that were similar, whereas Vmax for unfolding by the W-E3 and E-W3 pseudohexamers was about half of the wild-type value (Fig. 2B). Thus, pseudohexamers with alternating ATPase active and inactive subunits retain substantial protein-unfolding activity. However, compared W-W3 hydrolyzed ATP at about twice the rate of the W-E3 and E-W3 enzymes (Fig. 2A), suggesting that ATP hydrolysis is largely restricted to the W subunits in these enzymes. We con- VOLUME 292•NUMBER 14•APRIL 7, 2017 VOLUME 292•NUMBER 14•APRIL 7, 2017 96 JOURNAL OF BIOLOGICAL CHEMISTRY 5696 Construction of disulfide-cross-linked HslU pseudohexamers HslU hexamers consist of rigid body units formed by the large and small domains of adjacent subunits (17). We used the Disulfide by Design algorithm (18) to identify Glu47/Ala349 and Disulfide-linked HslU pseudohexamers Gln39/Thr361 as sites for potential disulfide bonds across the rigid body interfaces of an HslU hexamer. Fig. 3A shows a model of an otherwise Cys-free HslU pseudohexamer in which red subunits contain Cys47 and blue subunits contain Cys349, potentially allowing formation of three disulfide-linked dimers. To make WSSW3 pseudohexamers, the Cys47 and Cys349 sub- units both had wild-type Walker B ATPase motifs. To make WSSE3 pseudohexamers, the Cys47 subunit had a wild-type Walker B sequence, and the Cys349 subunit contained the Walker B E257Q mutation to inactivate ATP hydrolysis. Fig. 3B shows a pseudohexamer in which red subunits contain Cys349, green subunits contain Cys47 and Cys361, and blue subunits con- tain Cys39. In this configuration, formation of disulfide-linked trimers is possible. We designed WSSWSSW trimers, WSSESSW trimers, and WSSESSE trimers by changing which subunits had wild-type or E257Q Walker B sites. with wild-type HslU, W-W3 supported HslV degradation very poorly (Fig. 2C) and bound HslV 20-fold more weakly (Table 2), probably because the peptide tether interferes with contacts between HslU and HslV (see “Discussion”). Thus, we explored a different method of constructing cova- lently linked HslU hexamers. y p y ( g ) y (Table 2), probably because the peptide tether interferes with contacts between HslU and HslV (see “Discussion”). Thus, we explored a different method of constructing cova- lently linked HslU hexamers. Construction of disulfide-cross-linked HslU pseudohexamers HslU hexamers consist of rigid body units formed by the large and small domains of adjacent subunits (17). We used the Disulfide by Design algorithm (18) to identify Glu47/Ala349 and g y g model of an otherwise Cys-free HslU pseudohexamer in which red subunits contain Cys47 and blue subunits contain Cys349, potentially allowing formation of three disulfide-linked dimers. To make WSSW3 pseudohexamers, the Cys47 and Cys349 sub- units both had wild-type Walker B ATPase motifs. To make WSSE3 pseudohexamers, the Cys47 subunit had a wild-type Walker B sequence, and the Cys349 subunit contained the Walker B E257Q mutation to inactivate ATP hydrolysis. Fig. 3B shows a pseudohexamer in which red subunits contain Cys349, green subunits contain Cys47 and Cys361, and blue subunits con- tain Cys39. In this configuration, formation of disulfide-linked trimers is possible. We designed WSSWSSW trimers, WSSESSW trimers, and WSSESSE trimers by changing which subunits had wild-type or E257Q Walker B sites. Crystallographic statistics Crystallographic statistics Values in parenthesis represent the highest resolution shell. Values in parenthesis represent the highest resolution shell. Values in parenthesis represent the highest resolution shell. Protein Data Bank code 5TXV Wavelength (Å) 0.979 Space group P 1 21 1 Unit-cell dimensions (Å) a 86.5; b 420.9; c 176.5 Unit-cell angles (degrees) 90; 98.6 Resolution range (Å) 49.2–7.1 (7.3–7.1) Unique reflections 18,630 (1784) Completeness (%) 98.3 (94.6) Redundancy 4.5 (4.5) Rmerge 0.105 (0.694) Rmeas 0.119 (0.782) Rpim 0.055 (0.315) Rwork 0.274 (0.356) Rfree 0.298 (0.347) MolProbity score (percentile) 100 Root mean square bonds (Å) 0.003 Root mean square angles (degrees) 0.61 Clash score 5.8 Favored rotamers (%) 99.0 Poor rotamers (%) 0.39 Ramachandran favored 98.0 Ramachandran outliers (%) 0 Bad bonds/angles 0/1 C deviations 0 Relatively efficient formation of disulfide-linked HslU dimers or trimers was achieved by cytosolic coexpression of appropriate variants in the oxidizing SHuffle strain of E. coli Table 2 HslV binding Titration assays, monitored by changes in HslV peptidase activity, were performed at 25 °C in the presence of 5 mM ATP. 0 50 100 150 200 250 W-W3 W-E3 E-W3 ATP hydrolyzed (min-1 enz-1) A HslU W-W3 E-W3 W-E3 unfolding rate (min-1 enz-1) [protein substrate] (µM) B 0 0.1 0.2 0 10 20 30 40 B A –Arc-st11-ssrA HslU/HslV 0 5 10 30 60 90 0 5 10 30 60 90 degradation time (min) W-W3/HslV C C W-W3/HslV W-W3/HslV Figure 2. Activity of genetically tethered pseudohexamers. A, rates of hydrolysis of 5 mM ATP by the genetically tethered W-W3, W-E3, and E-W3 pseudo- hexamers. Values are averages (n 5) S.D. (error bars). B, rates of unfolding of different concentrations of thrombin-split I37AArc-cp6GFP-5/6-st11-ssrA by wild-type HslU or genetically tethered variants. Lines, non-linear least squares fits to the Michaelis-Menten equation. Km values for all enzymes were 1–3 M but werenotwelldeterminedbecauseofthesmallnumberoflow-concentrationdatapoints.AverageVmaxvaluesS.D.calculatedfromthehighestfoursubstrate concentrations were 0.155 0.003 min1 enz1 (HslU), 0.143 0.008 min1 enz1 (W-W3), 0.0802 0.007 min1 enz1 (E-W3), and 0.0716 0.006 min1 enz1 (W-E3). Fitted Vmax values were 10–15% higher. C, the kinetics of degradation of Arc-st11-ssrA (10 M) by HslV (10 M) and HslU (0.3 M) or W-W3 (0.3 M) at 50 °C was monitored by SDS-PAGE. Reactions contained 5 mM ATP and a regeneration system. JOURNAL OF BIOLOGICAL CHEMISTRY 5697 APRIL 7, 2017•VOLUME 292•NUMBER 14 APRIL 7, 2017•VOLUME 292•NUMBER 14 Disulfide-linked HslU pseudohexamers C349-C47 C349-C47 C349-C47 A B C C349-C47 C349-C47 C361-C39 C361-C39 2 SS-linked trimers form pseudohexamer wild-type HslU WSSW dimer WSSE dimer WSSWSSW trimer WSSESSW trimer WSSESSE trimer 75 50 37 25 100 20 150 250 standards MW kDa 3 SS-linked dimers form pseudohexamer Figure 3. Design and purification of disulfide-cross-linked HslU variants. A, spheres show the positions of Cys47 (normally Glu) in red subunits and Cys349 (normally Ala) in blue subunits of an HslU hexamer, suggesting that Cys47-Cys349 disulfides would stabilize a pseudohexamer consisting of three linked dimers. B, spheres show the positions of Cys349 in red subunits, Cys47 and Cys361 (normally Thr) in green subunits, and Cys39 (normally Gln) in blue subunits. Disulfide bond formation in this configuration would stabilize a pseudohexamer consisting of two linked trimers. C, non-reducing SDS-PAGE of purified wild-type HslU, purified WSSW, purified WSSE, purified WSSWSSW, purified WSSESSW, and purified WSSESSE. 5 The abbreviations used are: NTA, nitrilotriacetic acid; BisTris, 2-[bis(2- hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; enz, enzyme concentration. Degradation supported by disulfide-linked pseudohexamers Arc repressor, a good substrate for HslUV degradation, is a metastable dimer that unfolds/dissociates with a half-life of 10 s but refolds in milliseconds to maintain a predominantly native structure (15, 22). We assayed the ability of different disulfide-linked pseudohexamers to support HslV degradation of Arc-CysA, where CysA designates a unique cysteine labeled with an Alexa-488 fluorophore (23), as autoquenching of the fluorophores in the native protein is relieved upon degradation. The WSSW3 and WSSWSSW2 pseudohexamers supported HslV degradation of a nearly saturating concentration of Arc-CysA at rates comparable with the wild-type HslU hexamer (Fig. 5A). Importantly, WSSESSW2 and WSSE3 also supported degrada- tion at 35–45% of the wild-type rate, demonstrating that pseu- dohexamers with only three or four hydrolytically active sub- units also have substantial degradation activity. Degradation supported by WSSESSE2 proceeded very slowly, at 3% of the wild-type rate. Table 2 HslV binding In each case, a 0.5 M concentration of the purified protein (in hexamer equivalents) was loaded on the gel. The first lane contains molecular weight standards. C349-C47 C349-C47 C349-C47 A 3 SS-linked dimers form pseudohexamer C C39 wild-type HslU WSSW dimer WSSE dimer WSSWSSW trimer WSSESSW trimer WSSESSE trimer 75 50 37 25 100 20 150 250 standards MW kDa C A B C349-C47 C349-C47 C361-C39 C361-C39 2 SS-linked trimers form pseudohexamer B C349-C47 Figure 3. Design and purification of disulfide-cross-linked HslU variants. A, spheres show the positions of Cys47 (normally Glu) in red subunits and Cys349 (normally Ala) in blue subunits of an HslU hexamer, suggesting that Cys47-Cys349 disulfides would stabilize a pseudohexamer consisting of three linked dimers. B, spheres show the positions of Cys349 in red subunits, Cys47 and Cys361 (normally Thr) in green subunits, and Cys39 (normally Gln) in blue subunits. Disulfide bond formation in this configuration would stabilize a pseudohexamer consisting of two linked trimers. C, non-reducing SDS-PAGE of purified wild-type HslU, purified WSSW, purified WSSE, purified WSSWSSW, purified WSSESSW, and purified WSSESSE. In each case, a 0.5 M concentration of the purified protein (in hexamer equivalents) was loaded on the gel. The first lane contains molecular weight standards. sis by WSSW3 and WSSWSSW2 was stimulated 2-fold, but ATP hydrolysis by WSSE3, WSSESSW2, or WSSESSE2 was not markedly stimulated (Fig. 4C). Thus, the presence of E subunits suppressed normal HslV stimulation of ATP hydrolysis by HslU pseudohexamers. (19). For example, following purification by Ni2-NTA5 affinity chromatography, non-reducing SDS-PAGE showed 50% for- mation of WSSW and WSSE and 33% formation of WSS- WSSW, WSSESSW, and WSSESSE (not shown). To further purify disulfide-linked pseudohexamers, we performed ion exchange chromatography and gel filtration chromatography once in the presence and once in the absence of urea, which destabilizes unlinked HslU hexamers more than linked hexamers. Follow- ing the final chromatography step, the WSSW, WSSE, WSS- WSSW, WSSESSW, and WSSESSE variants had purities of 95% (Fig. 3C). The disulfide-linked pseudohexamers bound HslV slightly more tightly than wild-type HslU (Table 2). Discussion Our studies show that E. coli HslU variants containing hydrolytically active and inactive subunits at specific positions in the hexameric AAA ring can hydrolyze ATP, unfold pro- teins, and degrade substrates in collaboration with HslV. As we discuss below, these results support a probabilistic model of ATP hydrolysis and provide insights into the multivalent inter- actions between HslU and HslV that are required for efficient protein degradation. 0 100 200 300 400 500 600 ATP hydrolyzed (min-1 enz-1) HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 WSSESSE2 HslV present 2.2 1.9 1.1 1.2 0.87 3.4 fold-stimutation C C g Genetic tethering allowed us to express and purify HslU pseudohexamers consisting of a trimer of linked dimers. How- ever, the W-W3 enzyme binds HslV poorly, suggesting that the tether interferes with HslV binding. Consistently, disulfide- linked pseudohexamers bind HslV well. In crystal structures of HslU hexamers alone, the C-terminal tails dock into a pocket, and the -carboxyl group forms a salt bridge with an arginine in the sensor-2 motif of the same subunit (6, 10). These tail inter- actions were disrupted in several subunits in our low-resolution structure of W-E3 pseudohexamers, as expected if the attached tether prevents proper packing of these residues. In the H. influenzae HslUV complex, the C-terminal tails are detached from HslU and pack into grooves on HslV, with the HslU -carboxylate forming a salt bridge with an HslV lysine side chain (7, 11, 12). In E. coli HslUV structures, by contrast, the tails remain docked into HslU (8, 9). Moreover, deletion of the five C-terminal residues of E. coli HslU does not prevent stimulation of HslV peptidase activity or degradation (20). Nev- ertheless, peptides corresponding to the C-terminal residues of HslU activate peptide cleavage by E. coli HslV, and mutations in HslV predicted to disrupt contacts with the C-terminal tails pre- vent HslU activation (20, 24). Our results support the importance of the C-terminal tails in high-affinity HslV binding and indicate that more than three tails of E. coli HslU must interact optimally with HslV to allow tight binding and efficient proteolysis. Figure 4. ATP hydrolysis by disulfide-cross-linked variants. A, basal rates of ATP hydrolysis by disulfide-cross-linked variants and wild-type HslU. Assays con- tained 0.3 M HslU or pseudohexamers and 5 mM ATP. Values are averages of at least three replicates 1 S.D. (error bars). B, basal ATP hydrolysis rates for disul- fide-cross-linkedpseudohexamersplottedasafunctionofthenumberofWsub- units. Disulfide-linked HslU pseudohexamers 0 50 100 150 200 250 300 HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 WSSESSE2 ATP hydrolyzed (min-1 enz-1) A For Arc-cp6GFP-st11-ssrA, by contrast, the major discontinuity was between four and six subunits. For Arc-cp6GFP-st11-ssrA, by contrast, the major discontinuity was between four and six subunits. A To determine the energetic efficiency of degradation of Arc- cp6GFP-st11-ssrA, we assayed the rate of ATP-hydrolysis for each disulfide-linked pseudohexamer in the presence of HslV and Arc-cp6GFP-st11-ssrA (Fig. 5E). We then divided the ATPase rate by the degradation rate to determine the average number of ATPs hydrolyzed during degradation of a single substrate (Fig. 5F). Notably, WSSW3, WSSE3, WSSWSSW2, and WSSESSW2 all had similar energetic efficiencies, hydrolyzing 500 100 ATPs for each substrate degraded. Assuming that power strokes are tightly coupled to ATP hydrolysis, this result suggests that the WSSESSW2 and WSSE3 pseudohexamers use approximately the same number of power strokes as hexamers with six wild-type subunits to unfold and translocate Arc- cp6GFP-st11-ssrA. Thus, the slower degradation activities of the WSSESSW2 and WSSE3 enzymes compared with pseudohex- amers with only wild-type subunits are principally a conse- quence of their slower rates of ATP hydrolysis. 0 50 100 150 200 250 0 1 2 3 4 5 6 number W subunits B ATP hydrolyzed (min-1 enz-1) B ATP hydrolysis by disulfide-linked pseudohexamers Like the basal ATP hydrolysis activities of the genetically tethered pseudohexamers, those of the disulfide-linked en- zymes were roughly proportional to the number of hydrolyti- cally active W subunits in each pseudohexamer (Fig. 4, A and B). Compared with wild-type HslU, however, the WSSW3 and WSSWSSW2 pseudohexamers were 3-fold more hydrolyti- cally active (Fig. 4A), possibly as a consequence of small confor- mational changes stabilized by the disulfide bonds. In the pres- ence of HslV, the ATPase rate of wild-type HslU was stimulated 3-fold, as observed previously (13, 20, 21), and ATP hydroly- The degradation defects caused by E subunits in pseudohex- amers were more severe for Arc-cp6GFP-st11-ssrA, a stable substrate that wild-type HslUV degrades 5-fold more slowly than Arc-CysA. WSSW3 and WSSWSSW2 supported HslV deg- VOLUME 292•NUMBER 14•APRIL 7, 2017 VOLUME 292•NUMBER 14•APRIL 7, 2017 JOURNAL OF BIOLOGICAL CHEMISTRY 5698 0 50 100 150 200 250 0 1 2 3 4 5 6 number W subunits B ATP hydrolyzed (min-1 enz-1) 0 50 100 150 200 250 300 HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 WSSESSE2 ATP hydrolyzed (min-1 enz-1) A 0 100 200 300 400 500 600 ATP hydrolyzed (min-1 enz-1) HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 WSSESSE2 HslV present 2.2 1.9 1.1 1.2 0.87 3.4 fold-stimutation C Figure 4. ATP hydrolysis by disulfide-cross-linked variants. A, basal ATP hydrolysis by disulfide-cross-linked variants and wild-type HslU. Assa tained 0.3 M HslU or pseudohexamers and 5 mM ATP. Values are averag least three replicates 1 S.D. (error bars). B, basal ATP hydrolysis rates fo fide-cross-linkedpseudohexamersplottedasafunctionofthenumberof units. C, rates of ATP hydrolysis determined in the presence of 0.9 dodecamer (other conditions as inA). The numbers aboveeach bar repres rate in the presence of HslV divided by the rate in the absence of HslV. Discussion Degradation rates and energetic efficiencies of disulfide-cross-linked variants. A, rates of degradation of Arc-CysA (30 M) assayed by increased fluorescence. B, rates of degradation of Arc-cp6GFP-st11-ssrA (20 M) assayed by decreased fluorescence. C, degradation rates for Arc-CysA from A were divided by the number of wild-type subunits in the HslU variant and plotted against this number. D, degradation rates for Arc-cp6GFP-st11-ssrA from B were divided by the number of wild-type subunits in the HslU variant and plotted against this number. E, rates of ATP hydrolysis in the presence of 20 M Arc-cp6GFP-st11-ssrA and 1 M HslV. F, energetic efficiency determined by dividing the ATPase rates by the degradation rates. For A–D, experiments were performed using 0.3 M HslU or variants and 0.9 M HslV. For E and F, experiments were performed using 0.5 M HslU or variants and 1 M HslV. All assays contained 5 mM ATP and were performed at 37 °C. Values in A–E are averages (n 3) 1 S.D. (error bars). Error bars in F, propagated errors. In C and D, values for the three variants with six wild-type subunits were offset slightly on the x axis to allow visualization of error bars. independently to basal ATPase activity, making concerted or strictly sequential models unlikely. For example, if ATP hydro- lysis in a specific subunit required prior hydrolysis in a neigh- boring subunit, as expected in a strictly sequential model, then a non-linear relationship between W subunits and ATP hydro- lysis would be expected. HslV stimulates ATP hydrolysis by WSSW3 and WSSWSSW2 but caused little change in hydrolysis by WSSESSW2, WSSE3, or WSSESSE2. Because the E subunits in these latter enzymes also increase pseudohexamer affinity for HslV, stronger interactions between E subunits and HslV might restrictHslV-inducedconformationalchangesrequiredforhigher ATPase activity in neighboring W subunits and thus explain the lack of ATPase stimulation. Alternatively, HslV binding might slow ATP dissociation from inactive E subunits, which becomes rate-limiting for hydrolysis in W subunits, especially if only a sub- set of subunits is nucleotide-bound at any given time. Our strategies for engineering HslU rings with defined mix- tures of active and inactive subunits were motivated by prior studies that applied these subunit-cross-linking methods to dif- ferent hexameric AAA unfoldases and remodeling machines (5, 25–27). Discussion C, rates of ATP hydrolysis determined in the presence of 0.9 M HslV dodecamer (other conditions as inA). The numbers aboveeach bar represent the rate in the presence of HslV divided by the rate in the absence of HslV. radation of a high concentration of Arc-cp6GFP-st11-ssrA at nearly wild-type rates, but WSSESSW2 had only 20% activity, WSSE3 had only 10% activity, and WSSESSE2 was inactive (Fig. 5B). Degradation rates were normalized by dividing by the number of wild-type subunits in HslU or different variants and are plotted in Fig. 5C for the Arc-CysA substrate and Fig. 5D for the Arc-cp6GFP-st11-ssrA substrate. For Arc-CysA, there was a sharp discontinuity between two and three wild-type subunits. The pseudohexamers that we studied have basal ATP hydro- lysis rates roughly proportional to their total number of hydro- lytically active W subunits. This result supports a model in which the W subunits in these pseudohexamers contribute APRIL 7, 2017•VOLUME 292•NUMBER 14 5699 JOURNAL OF BIOLOGICAL CHEMISTRY APRIL 7, 2017•VOLUME 292•NUMBER 14 Disulfide-linked HslU pseudohexamers C D 0 100 200 300 400 500 ATP hydrolyzed (min-1 enz-1) HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 0 100 200 300 400 500 600 700 ATP hydrolyzed per substrate degraded HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 Arc-cysA degraded (min-1 enz-1) A HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 WSSESSE2 0 1 2 3 4 5 6 Arc-cp6GFP-st11-ssrA degraded (min-1 enz-1) B HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 WSSESSE2 0 0.2 0.4 0.6 0.8 1.0 1.2 0 0.2 0.4 0.6 0.8 1.0 0 1 2 3 4 5 6 number W subunits degradation per W subunit (min-1 enz-1) Arc-cysA 0 0.1 0.2 0 1 2 3 4 5 6 number W subunits degradation per W subunit (min-1 enz-1) Arc-cp6GFP-st11-ssrA E F Figure 5. Degradation rates and energetic efficiencies of disulfide-cross-linked variants. A, rates of degradation of Arc-CysA (30 M) ass fluorescence. B, rates of degradation of Arc-cp6GFP-st11-ssrA (20 M) assayed by decreased fluorescence. C, degradation rates for Arc-CysA fr by the number of wild-type subunits in the HslU variant and plotted against this number. D, degradation rates for Arc-cp6GFP-st11-ssrA from the number of wild-type subunits in the HslU variant and plotted against this number. E, rates of ATP hydrolysis in the presence of 20 M Ar and 1 M HslV. F, energetic efficiency determined by dividing the ATPase rates by the degradation rates. For A–D, experiments were perfo HslU or variants and 0.9 M HslV. Discussion For E and F, experiments were performed using 0.5 M HslU or variants and 1 M HslV. All assays contained 5 performed at 37 °C. Values in A–E are averages (n 3) 1 S.D. (error bars). Error bars in F, propagated errors. In C and D, values for the thre wild-type subunits were offset slightly on the x axis to allow visualization of error bars. Disulfide-linked HslU pseudohexamers Arc-cysA degraded (min-1 enz-1) A HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 WSSESSE2 0 1 2 3 4 5 6 Arc-cp6GFP-st11-ssrA degraded (min-1 enz-1) B HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 WSSESSE2 0 0.2 0.4 0.6 0.8 1.0 1.2 B A C W W 0 0.2 0.4 0.6 0.8 1.0 0 1 2 3 4 5 6 number W subunits degradation per W subunit (min-1 enz-1) Arc-cysA C D W W 0 0.1 0.2 0 1 2 3 4 5 6 number W subunits degradation per W subunit (min-1 enz-1) Arc-cp6GFP-st11-ssrA D D 0 100 200 300 400 500 ATP hydrolyzed (min-1 enz-1) HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 E 0 100 200 300 400 500 600 700 ATP hydrolyzed per substrate degraded HslU WSSW3 WSSWSSW2 WSSESSW2 WSSE3 F F E Figure 5. Degradation rates and energetic efficiencies of disulfide-cross-linked variants. A, rates of degradation of Arc-CysA (30 M) assayed by increased fluorescence. B, rates of degradation of Arc-cp6GFP-st11-ssrA (20 M) assayed by decreased fluorescence. C, degradation rates for Arc-CysA from A were divided by the number of wild-type subunits in the HslU variant and plotted against this number. D, degradation rates for Arc-cp6GFP-st11-ssrA from B were divided by the number of wild-type subunits in the HslU variant and plotted against this number. E, rates of ATP hydrolysis in the presence of 20 M Arc-cp6GFP-st11-ssrA and 1 M HslV. F, energetic efficiency determined by dividing the ATPase rates by the degradation rates. For A–D, experiments were performed using 0.3 M HslU or variants and 0.9 M HslV. For E and F, experiments were performed using 0.5 M HslU or variants and 1 M HslV. All assays contained 5 mM ATP and were performed at 37 °C. Values in A–E are averages (n 3) 1 S.D. (error bars). Error bars in F, propagated errors. In C and D, values for the three variants with six wild-type subunits were offset slightly on the x axis to allow visualization of error bars. Figure 5. Cloning, expression, and protein purification Mutants were constructed and cloned by standard PCR tech- niques unless otherwise noted. To construct genetically linked HslU dimers, a gene encoding two HslU subunits separated by the 20-residue ASGAGGSEGGGSEGGTSGAT linker was cloned into the pet11a vector (Novagen). HslU mutants used to make disulfide-cross-linked pseudohexamers were constructed in the cysteine-free C262A/C288SHslU background with or with- out the E257Q mutation. Cysteine-free HslU supports robust ATP hydrolysis and substrate degradation (38–40). To make disulfide-cross-linked dimers, a gene encoding untagged E47CHslU was cloned into the first multiple cloning site (MCS1) of the pCOLADuet-1 (Novagen) vector between the NcoI and BamHI sites and a gene encoding His6-ENLYFQS-A349CHslU was cloned into MCS2 between the NdeI and XhoI sites, where His6 is the hexahistidine tag and ENLYFQS is the sequence recognized and cleaved by the tobacco etch virus protease. E47CHslU had a glycine residue inserted after the initiator methionine as a result of cloning. This pCOLADuet-1 vector was transformed into the of E. coli SHuffle T7 Express strain (New England Biolabs) for expression. To make disulfide- cross-linked trimers, a gene encoding untagged A349CHslU was cloned into MCS1 of pCOLADuet-1 between the NcoI and BamHI sites, and a gene encoding His6-ENLYFQS- E47CT361CHslU was cloned into MCS2 between the NdeI and XhoI sites. This vector was co-transformed with a pet12b (Novagen) vector encoding the untagged Q39CHslU variant into the SHuffle T7 Express strain. Both A349CHslU and Q39CHslU contained an additional glycine after the initiator methionine as a result of cloning. A gene encoding the Arc repressor from phage P22 followed by a cysteine residue and a hexahistidine tag (Arc-Cys-His6) was cloned and expressed in a pet21b vector (Novagen). Wild-type HslU and HslV were expressed from pet12b vectors. Arc-cp6GFP-st11-ssrA and I37AArc-cp6GFP-5/ 6-st11-ssrA (in which a GGTEGSLVPRGSGESGGS sequence between -stands 5 and 6 allows thrombin cleavage and gener- ation of a split substrate), and Arc-st11-ssrA were expressed from pet21b vectors as described (14, 15, 23). g The tethered W-E3 and E-W3 HslU pseudohexamers have 50% of the protein unfolding activity of the parental W-W3 enzyme, and the disulfide-linked WSSESSW2 and WSSE3 enzymes support HslV degradation of an easily degraded Arc- CysA substrate at 35–45% of the parental rates. Thus, protein unfolding and translocation by the AAA ring of HslU do not require ATP hydrolysis in adjacent subunits or in subunits immediately across the ring from each other. Discussion Indeed, genetic tethering experiments originally showed that ClpX also operates by a probabilistic mechanism, because ClpX pseudohexamers containing different combina- tions of hydrolytically active and inactive subunits unfold and degrade protein substrates in collaboration with the ClpP pro- tease (5). Before the current study, however, it was not obvious VOLUME 292•NUMBER 14•APRIL 7, 2017 5700 JOURNAL OF BIOLOGICAL CHEMISTRY Disulfide-linked HslU pseudohexamers that HslU and ClpX would operate using similar probabilistic mechanisms of ATP hydrolysis. First, HslU and ClpX contain unique family-specific auxiliary domains. The I domain of HslU emerges from the top of the AAA ring, between the Walker A and Walker B ATPase motifs, and regulates ATP hydrolysis, degradation, and autoinhibition (9, 20, 23). The N domain of ClpX, by contrast, serves as a docking site for some adaptors/ substrates, but its deletion has little effect on ATP hydrolysis or degradation of many ClpXP substrates (28). Second, in crystal structures, the large and small AAA domains of each HslU subunit assume orientations that create a potential nucleotide binding site, whereas the corresponding domains of ClpX adopt structures that allow nucleotide binding in some subunits but prevent binding in other subunits (6–12, 29). Third, HslU hex- amers make symmetric interactions with hexameric rings of HslV, whereas ClpX hexamers make asymmetric interactions with heptameric rings of ClpP (30). m-AAA protease, for example, mutating the Walker A motif of either the Yta10 or the Yta12 subunits does not affect hydrolysis in the remaining wild-type subunits, but Walker B mutations in Yta12 trap ATP and prevent robust hydrolysis in adjacent Yta10 subunits (36). Finally, in the six distinct subunits of the Rpt1–6 AAA ring of the 26S proteasome, Walker B mutations in individual subunits have a wide range of effects on ATP hy- drolysis and mechanical activity, requiring a model with some subunit-subunit coordination within the context of an inher- ently probabilistic mechanism of ATP hydrolysis (37). Disulfide-linked HslU pseudohexamers mM isopropyl 1-thio--D-galactopyranoside for 20 h. Cells were pelleted and resuspended in buffer A (50 mM Tris, pH 7.5, 300 mM NaCl, 20 mM imidazole, 0.5 mM EDTA), and 0.5 tablets of Complete Ultra EDTA-free protease inhibitor mixture (Roche Applied Science) and 1.5 l of benzonase (250 units/l; Sigma) per liter of the original culture were added. Cells were soni- cated, the lysate was cleared by centrifugation, and 0.1% (v/v) PEI was added to the supernatant. This precipitate was cleared by centrifugation, and the supernatant was loaded onto Ni2- NTA beads equilibrated in buffer B (50 mM Tris, pH 7.5, 300 mM NaCl, 20 mM imidazole). The Ni2-NTA beads were washed extensively with buffer B, and protein was eluted with buffer C (50 mM Tris, pH 7.5, 300 mM NaCl, 250 mM imidazole). The eluate was diluted 3-fold with buffer D (50 mM Tris, pH 7.5, 10% (v/v) glycerol, 1 mM EDTA), loaded onto a Mono Q 10/100 GL column (GE Healthcare), and eluted with a linear gradient from 150 to 500 mM NaCl in buffer D (120 ml total). Appropri- ate Mono Q fractions were pooled, concentrated using an Ami- con Ultra-15 centrifugal filter unit, and chromatographed on a Superdex 200 16/60 column (GE Healthcare) equilibrated in buffer E (50 mM Tris, pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, 1.5 M urea, 1 mM EDTA), and 1-ml fractions were collected. Fractions with the highest purity of the cross-linked dimer as judged by non-reducing SDS-PAGE were pooled, concen- trated, and run on another Superdex 200 16/60 column equili- brated in buffer F (50 mM Tris, pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, 1 mM EDTA). Fractions with the highest purity of cross- linked dimer were pooled and concentrated. The concentration of cross-linked dimer was determined in hexameric equivalents by measuring the absorbance at 280 nm using 148,545 M1 cm1 as the molar extinction coefficient. Concentrated protein was divided into small aliquots and was flash-frozen at 80 °C. HslU dimers. After Ni2-NTA purification, protein was con- centrated and injected onto a Superdex 75 16/60 column (GE Healthcare) equilibrated in buffer H (50 mM Tris, pH 7.5, 300 mM NaCl, 10% glycerol (v/v), 1 mM EDTA, 2 mM DTT). Frac- tions containing the Arc protein were concentrated and flash- frozen at 80 °C. Arc-Cys-His6 was labeled with the maleimide derivative of Alexa-488 (Thermo Fisher Scientific) to generate Arc-CysA (23). Biochemical assays Unless noted, assays were performed at 37 °C in PD buffer (25 mM HEPES, pH 7.5, 5 mM KCl, 10% glycerol (v/v), 20 mM MgCl2, 0.032% Igepal CA-630). Hydrolysis of 5 mM ATP was measured using an NADH-coupled assay (41) by monitoring the loss of absorbance at 340 nm on a Spectramax M5 plate reader (Molecular Devices). Cleavage of I37AArc-cp6GFP-5/ 6-st11-ssrA with thrombin was performed as described (14). Rates of unfolding of different concentrations of thrombin-split I37AArc-cp6GFP-5/6-st11-ssrA were determined by changes in cp6GFP fluorescence (excitation, 467 nm; emission, 511 nm) in assays that contained 0.5 M HslU or tethered variants and 5 mM ATP. Degradation of 30 M Arc-CysA (monomer equiva- lents) was measured on a Spectramax M5 plate reader by mon- itoring the increase in fluorescence (excitation, 480 nm; emis- sion, 520 nm). Degradation of 20 M Arc-cp6GFP-st11-ssrA (monomer equivalents) was measured by monitoring the decrease in GFP fluorescence on a Spectramax M5 plate reader (excitation, 467 nm; emission, 511 nm). Degradation reactions contained 5 mM ATP and a regeneration system consisting of 16 mM creatine phosphate and 10 g/ml creatine kinase. HslV activation assays were performed at 25 °C in the presence of ATP as described (13, 23), and K1⁄2 values were determined by fitting to a hyperbolic equation. q Disulfide-cross-linked HslU trimers were expressed and purified largely as described for cross-linked dimers. His6 tags were present on Cys349 subunits for dimers and Cys47/Cys361 subunits for trimers. After precipitation with PEI, the sample was cleared by centrifugation, and the supernatant was loaded onto a 5-ml HisTrap HP column (GE Healthcare) equilibrated in buffer B. The column was washed with 75 ml of buffer B, the sample was eluted with a gradient of 20–500 mM imidazole in buffer B (100 ml total), and 2-ml fractions were collected. Frac- tions were pooled and dialyzed overnight against buffer G (50 mM Tris, pH 7.5, 150 mM NaCl, 10% glycerol (v/v), 1 mM EDTA) at 4 °C. The dialyzed material was loaded onto a Mono Q 10/100 GL column, and purification then proceeded by the method described for cross-linked dimers. The concentration of the cross-linked trimer was determined in hexameric equiv- alents by measuring absorbance at 280 nm using 147,180 M1 cm1 as the molar extinction coefficient. Concentrated protein was aliquoted and flash-frozen at 80 °C. Cloning, expression, and protein purification Again, these results support a model in which probabilistic hydrolysis in the AAA ring powers unfolding and translocation. Slower rates of ATP hydrolysis in rings with mixtures of active and inactive subunits correlate with their reduced mechanical activities, because the ATP cost of degradation of the more stable Arc- cp6GFP-st11-ssrA substrate is similar for the WSSESSW2 and WSSE3 pseudohexamers and their disulfide-linked parental enzymes containing six W subunits. Although optimal rates of ATP hydrolysis, unfolding, and degradation require six hydro- lytically active HslU subunits, rings with only three or four active subunits use approximately the same number of power strokes to degrade this substrate. g We note that the WSSESSE2 pseudohexamer hydrolyzes ATP at 40–60% of the WSSE3 rate but supports no degradation of Arc-cp6GFP-st11-ssrA. These results suggest that some sub- unit-subunit communication is required for efficient unfolding and degradation by WSSESSE2 and/or that a minimum rate of ATP hydrolysis is required to unfold Arc-cp6GFP-st11-ssrA. Probabilistic models of ATP hydrolysis do not exclude coordi- nation between ring subunits for efficient ATP hydrolysis, sub- strate binding, or mechanical function. In fact, the rate of ATP hydrolysis and the degree of substrate binding by HslU change in a positively cooperative fashion with ATP concentration, as expected for functional linkage between different subunits (13). In the AAA ring of ClpX, communication between subunits is necessary to allow staged ATP binding to drive conformational changes needed for function (31–33). Moreover, optical trap- ping experiments reveal that kinetic bursts of power strokes in the ClpX ring result in random patterns of shorter and longer translocation steps, supporting a probabilistic but coordinated mechanism (34, 35). Similar themes of probabilistic hydrolysis but coordinated function are seen in AAA unfolding rings assembled from non-identical subunits. In the Yta10/Yta12 Genetically linked HslU dimers were expressed and purified as described previously for wild-type HslU (20). Disulfide- cross-linked HslU dimers were expressed and purified as fol- lows. E. coli SHuffle T7 Express cells carrying the pCOLA- Duet1 vector coding for the appropriate HslU mutants were grown at 30 °C until A600 of 0.6–0.8, the temperature was shifted to 18 °C, and protein expression was induced with 0.5 APRIL 7, 2017•VOLUME 292•NUMBER 14 5701 JOURNAL OF BIOLOGICAL CHEMISTRY APRIL 7, 2017•VOLUME 292•NUMBER 14 Disulfide-linked HslU pseudohexamers His6-tagged wild type HslU, His6-tagged HslV, I37AArc-cp6GFP-5/6-st11-ssrA, Arc-cp6GFP-st11-ssrA, and Arc-st11-ssrA were expressed and purified as described (23). References (2000) Crystal and solution structures of an HslUV protease- chaperone complex. Cell 103, 633–643 ing subunit arrangement. J. Biol. Chem. 290, 9789–9800 28. Baker, T. A., and Sauer, R. T. (2012) ClpXP, an ATP-powered unfolding and protein-degradation machine. Biochim. Biophys. Acta 1823, 15–28 8. Wang, J., Song, J. J., Franklin, M. C., Kamtekar, S., Im, Y. J., Rho, S. H., Seong, I. S., Lee, C. S., Chung, C. H., and Eom, S. H. (2001) Crystal struc- tures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism. Structure 9, 177–184 29. Glynn, S. E., Martin, A., Nager, A. R., Baker, T. A., and Sauer, R. T. (2009) Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA protein-unfolding machine. Cell 139, 744–756 9. Song, H. K., Hartmann, C., Ramachandran, R., Bochtler, M., Behrendt, R., Moroder, L., and Huber, R. (2000) Mutational studies on HslU and its docking mode with HslV. Proc. Natl. Acad. Sci. U.S.A. 97, 14103–14108 30. Grimaud, R., Kessel, M., Beuron, F., Steven, A. C., and Maurizi, M. R. (1998) Enzymatic and structural similarities between the Escherichia coli ATP-dependent proteases, ClpXP and ClpAP. J. Biol. Chem. 273, 12476–12481 10. Trame, C. B., and McKay, D. B. (2001) Structure of Haemophilus influen- zae HslU protein in crystals with one-dimensional disorder twinning. Acta Crystallogr. D Biol. Crystallogr. 57, 1079–1090 31. Stinson, B. M., Nager, A. R., Glynn, S. E., Schmitz, K. R., Baker, T. A., and Sauer, R. T. (2013) Nucleotide binding and conformational switching in the hexameric ring of a AAA machine. Cell 153, 628–639 11. Sousa, M. C., Kessler, B. M., Overkleeft, H. S., and McKay, D. B. (2002) Crystal structure of HslUV complexed with a vinyl sulfone inhibitor: cor- roboration of a proposed mechanism of allosteric activation of HslV by HslU. J. Mol. Biol. 318, 779–785 32. Hersch, G. L., Burton, R. E., Bolon, D. N., Baker, T. A., and Sauer, R. T. (2005) Asymmetric interactions of ATP with the AAA ClpX6 unfoldase: allosteric control of a protein machine. Cell 121, 1017–1027 33. Stinson, B. M., Baytshtok, V., Schmitz, K. R., Baker, T. A., and Sauer, R. T. (2015) Subunit asymmetry and roles of conformational switching in the hexameric AAA ring of ClpX. Nat. Struct. Mol. Biol. 22, 411–416 12. Kwon, A. R., Kessler, B. M., Overkleeft, H. S., and McKay, D. B. References 22. Milla, M. E., and Sauer, R. T. (1994) P22 Arc repressor: folding kinetics of a single-domain, dimeric protein. Biochemistry 33, 1125–1133 1. Ogura, T., and Wilkinson, A. J. (2001) AAA superfamily ATPases: com- mon structure–diverse function. Genes Cells 6, 575–597 23. Baytshtok, V., Fei, X., Grant, R. A., Baker, T. A., and Sauer, R. T. (2016) A structurally dynamic region of the HslU intermediate domain con- trols protein degradation and ATP hydrolysis. Structure 24, 1766–1777 2. Snider, J., Thibault, G., and Houry, W. A. (2008) The AAA superfamily f f ti ll di t i G Bi l 9 216 2. Snider, J., Thibault, G., and Houry, W. A. (2008) The AAA superfamily of functionally diverse proteins. Genome Biol. 9, 216 of functionally diverse proteins. Genome Biol. 9, 216 3. Gai, D., Zhao, R., Li, D., Finkielstein, C. V., and Chen, X. S. (2004) Mech- anisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen. Cell 119, 47–60 24. Ramachandran, R., Hartmann, C., Song, H. K., Huber, R., and Bochtler, M. (2002) Functional interactions of HslV (ClpQ) with the ATPase HslU (ClpY). Proc. Natl. Acad. Sci. U.S.A. 99, 7396–7401 4. Enemark, E. J., and Joshua-Tor, L. (2006) Mechanism of DNA transloca- tion in a replicative hexameric helicase. Nature 442, 270–275 25. Glynn, S. E., Nager, A. R., Baker, T. A., and Sauer, R. T. (2012) Dynamic and static components power unfolding in topologically closed rings of a AAA proteolytic machine. Nat. Struct. Mol. Biol. 19, 616–622 5. Martin, A., Baker, T. A., and Sauer, R. T. (2005) Rebuilt AAA motors reveal operating principles for ATP-fuelled machines. Nature 437, 1115–1120 26. Biter, A. B., Lee, S., Sung, N., and Tsai, F. T. F. (2012) Structural basis for intersubunit signaling in a protein disaggregating machine. Proc. Natl. Acad. Sci. U.S.A. 109, 12515–12520 6. Bochtler, M., Hartmann, C., Song, H. K., Bourenkov, G. P., Bartunik, H. D., and Huber, R. (2000) The structures of HsIU and the ATP-dependent protease HsIU-HsIV. Nature 403, 800–805 27. Yamasaki, T., Oohata, Y., Nakamura, T., and Watanabe, Y. H. (2015) Anal- ysis of the cooperative ATPase cycle of the AAA chaperone ClpB from Thermus thermophilus by using ordered heterohexamers with an alternat- ing subunit arrangement. J. Biol. Chem. 290, 9789–9800 7. Sousa, M. C., Trame, C. B., Tsuruta, H., Wilbanks, S. M., Reddy, V. S., and McKay, D. B. References (2003) Structure and reactivity of an asymmetric complex between HslV and I-domain deleted HslU, a prokaryotic homolog of the eukaryotic protea- some. J. Mol. Biol. 330, 185–195 34. Sen, M., Maillard, R. A., Nyquist, K., Rodriguez-Aliaga, P., Pressé, S., Mar- tin, A., and Bustamante, C. (2013) The ClpXP protease unfolds substrates using a constant rate of pulling but different gears. Cell 155, 636–646 13. Yakamavich, J. A., Baker, T. A., and Sauer, R. T. (2008) Asymmetric nu- cleotide transactions of the HslUV protease. J. Mol. Biol. 380, 946–957 35. Cordova, J. C., Olivares, A. O., Shin, Y., Stinson, B. M., Calmat, S., Schmitz, K. R., Aubin-Tam, M.-E. Baker, T. A., Lang, M. J., and Sauer, R. T. (2014) Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine. Cell 158, 647–658 14. Nager, A. R., Baker, T. A., and Sauer, R. T. (2011) Stepwise unfolding of a barrel protein by the AAA ClpXP protease. J. Mol. Biol. 413, 4–16 15. Burton, R. E., Baker, T. A., and Sauer, R. T. (2005) Nucleotide-dependent substrate recognition by the AAA HslUV protease. Nat. Struct. Mol. Biol. 12, 245–251 36. Augustin, S., Gerdes, F., Lee, S., Tsai, F. T., Langer, T., and Tatsuta, T. (2009) An intersubunit signaling network coordinates ATP hydrolysis by m-AAA proteases. Mol. Cell 35, 574–585 16. Sundar, S., McGinness, K. E., Baker, T. A., and Sauer, R. T. (2010) Multiple sequence signals direct recognition and degradation of protein substrates by the AAA protease HslUV. J. Mol. Biol. 403, 420–429 37. Beckwith, R., Estrin, E., Worden, E. J., and Martin, A. (2013) Reconstitu- tion of the 26S proteasome reveals functional asymmetries in its AAA unfoldase. Nat. Struct. Mol. Biol. 20, 1164–1172 17. Wang, J., Song, J. J., Seong, I. S., Franklin, M. C., Kamtekar, S., Eom, S. H., and Chung, C. H. (2001) Nucleotide-dependent conformational changes in a protease-associated ATPase HslU. Structure 9, 1107–1116 38. Yoo, S. J., Kim, H. H., Shin, D. H., Lee, C. S., Seong, I. S., Seol, J. H., Shimbara, N., Tanaka, K., and Chung, C. H. (1998) Effects of the Cys mutations on structure and function of the ATP-dependent HslVU pro- tease in Escherichia coli. The Cys287 to Val mutation in HslU uncouples the ATP-dependent proteolysis by HslVU from ATP hydrolysis. J. Biol. Chem. 273, 22929–22935 18. Dombkowski, A. A. Crystallography The W-E3 pseudohexamer was crystallized by the hanging drop method using 100 mM BisTris (pH 5.8), 26% (w/v) PEG 3350, and 260 mM ammonium sulfate as the well solution. Molecular replacement using Phaser (42) was initially used to solve the structure using a 1HQY hexamer (17) as the search model. We then replaced each 1HQY subunit with a 5JI3 sub- unit (23) to improve geometry and used rigid body refinement of individual domains, refinement of one B-factor and TLS group per subunit, and very tightly constrained positional refinement with torsional NCS constraints in Phenix (43). Coot (44) was used for model building, and MolProbity (45) was used to assess the geometry of the model. Author contributions—J. C. performed experiments with HslU sub- units linked by genetically encoded tethers. A. R. N. designed the split substrate for unfolding experiments. S. E. G., R. A. G., and R. T. S. performed crystallographic experiments. V. B. performed all remaining experiments. V. B. and R. T. S. wrote the manuscript. V. B., J. C., S. E. G., A. R. N., R. A. G., T. A. B, and R. T. S. contributed to the design and interpretation of experiments and approved the final manuscript. A pet21b vector carrying Arc with a C-terminal CHHHHHH tail (Arc-Cys-His6) was transformed into the E. coli X90 (DE3) slyD::kan hslUV::tet strain, and cells were grown to an A600 of 0.6–0.8 at 37 °C. Protein expression was induced by the addi- tion of 1 mM isopropyl 1-thio--D-galactopyranoside and con- tinued for 4 h at room temperature. Cells were resuspended and lysed, and Arc-Cys-His6 was purified by Ni2-NTA affinity chromatography as described for purification of cross-linked VOLUME 292•NUMBER 14•APRIL 7, 2017 5702 JOURNAL OF BIOLOGICAL CHEMISTRY APRIL 7, 2017•VOLUME 292•NUMBER 14 References (2003) Disulfide by DesignTM: a computational method for the rational design of disulfide bonds in proteins. Bioinformat- ics 19, 1852–1853 19. Lobstein, J., Emrich, C. A., Jeans, C., Faulkner, M., Riggs, P., and Berkmen, M. (2012) SHuffle, a novel Escherichia coli protein expression strain capa- ble of correctly folding disulfide bonded proteins in its cytoplasm. Microb. Cell Fact. 11, 56 39. Seong, I. S., Oh, J. Y., Yoo, S. J., Seol, J. H., and Chung, C. H. (1999) ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli. FEBS Lett. 456, 211–214 40. Yakamavich, J. A. (2008) Control of HslUV Protease Function by Nucleo- tide Binding and Hydrolysis, Ph.D. thesis, Massachusetts Institute of Technology, Cambridge, MA 20. Seong, I. S., Kang, M. S., Choi, M. K., Lee, J. W., Koh, O. J., Wang, J., Eom, S. H., and Chung, C. H. (2002) The C-terminal tails of HslU ATPase act as a molecular switch for activation of HslV peptidase. J. Biol. Chem. 277, 25976–25982 41. Nørby, J. G. (1988) Coupled assay of Na,K-ATPase activity. Methods Enzymol. 156, 116–119 21. Sundar, S., Baker, T. A., and Sauer, R. T. (2012) The I domain of the AAA HslUV protease coordinates substrate binding, ATP hydrolysis, and pro- tein degradation. Protein Sci. 21, 188–198 42. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Sto- roni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 APRIL 7, 2017•VOLUME 292•NUMBER 14 5703 JOURNAL OF BIOLOGICAL CHEMISTRY 5703 45. Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 44. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 Disulfide-linked HslU pseudohexamers 43. Adams, P. D., Afonine, P. V., Bunko´czi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richard- son, D. C., et al. (2010) PHENIX: a comprehensive Python-based sys- tem for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 44. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 45. Chen, V. B., Arendall, W. B., 3rd, Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S., and Richardson, D. C. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 VOLUME 292•NUMBER 14•APRIL 7, 2017 VOLUME 292•NUMBER 14•APRIL 7, 2017 VOLUME 292•NUMBER 14•APRIL 7, 2017 5704 JOURNAL OF BIOLOGICAL CHEMISTRY 5704
https://openalex.org/W3004332630	https://europepmc.org/articles/pmc7019154?pdf=render	English	null	The c-Myc/miR-27b-3p/ATG10 regulatory axis regulates chemoresistance in colorectal cancer	Theranostics	2,020	cc-by	11,150	The c-Myc/miR-27b-3p/ATG10 regulatory axis regulates chemoresistance in colorectal cancer Wu Sun1,#, Jialu Li2,#, Likun Zhou1,#, Jiayi Han1,#, Rui Liu1,#, Haiyang Zhang1, Tao Ning1, Zhiying Gao3, Baorui Liu4,, Xi Chen3,, Yi Ba1, 1. Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy Tianjin’s Clinical Research Center for Cancer, Tianjin, China. j j . State Key Laboratory for Oncogenes and Related Genes, Key Laboratory of Gastroenterology and Hepatology, Ministry of He Gastroenterology and Hepatology, Shanghai Institute of Digestive Disease, Renji Hospital, School of Medicine, Shanghai Jiao China. 2. State Key Laboratory for Oncogenes and Related Genes, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, Division of Gastroenterology and Hepatology, Shanghai Institute of Digestive Disease, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China 2. State Key Laboratory for Oncogenes and Related Genes, Key Laboratory of Gastroenterology and Hepatology, Ministry of Health, Division of Gastroenterology and Hepatology, Shanghai Institute of Digestive Disease, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China. . State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biot Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China. 3. State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China. 3. State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Nanjing, China. ( ) j g y j g Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University and Clinical Cancer Institute of Nanjing University #These authors contributed equally to this study. #These authors contributed equally to this study. Received: 2019.06.15; Accepted: 2019.12.15; Published: 2020.01.12 Received: 2019.06.15; Accepted: 2019.12.15; Published: 2020.01.12 Theranostics 2020, Vol. 10, Issue 5 Ivyspring International Publisher The c-Myc/miR-27b-3p/ATG10 regulatory axis regulates chemoresistance in colorectal cancer  Corresponding authors: Yi Ba,Ph.D., Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Huan hu xi Road 18, Tianjin, China; 300060; (Tel): 022-2334-0123; Email: bayi@tjmuch.com or Xi Chen, Ph.D., State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Engineering Research Center for MicroRNA Biology and Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, Xianlin Road 163, Nanjing, Jiangsu, China; 210046; (Tel): 86-25-89681323; Email: xichen@nju.edu.cn or Baorui Liu, Ph.D., The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University and Clinical Cancer Institute of Nanjing University, Zhognshan Road 321, Nanjing, Jiangsu, China; 210008; (Tel): 025-83304616; Email: baoruiliu07@163.com. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions. 1981 1981 Theranostics 2020, Vol. 10, Issue 5 Abstract Oxaliplatin (OXA) resistance is the major obstacle to the anticancer effects of chemotherapy in colorectal cancer (CRC) patients. MicroRNAs (miRNAs) play an important role in the chemoresistance of various tumors. Our objective is to clarify the underlying mechanism of miRNAs in chemoresistance and provide a potential strategy to improve the response of CRC patients to chemotherapeutics. Methods: MiRNA microarray and Real-time PCR were performed to compare changes in miRNA expression between oxaliplatin-resistant and the parental cells. CCK8, apoptosis assay, immunofluorescence and xenograft studies were used to elucidate the impact of miR-27b-3p on regulating chemoresistance. Luciferase reporter assay and western blot were carried to assess the regulatory role of miR-27b-3p in ATG10 expression. The effects of miR-27b-3p and ATG10 on autophagy were investigated by GFP-LC3 fluorescence microscopy, transmission electron microscopy, and western blot. ChIP assay and luciferase assay were performed to test the c-Myc’s occupancy on the miR-27B promoter. Results: We observed that miR-27b-3p expression was significantly downregulated in oxaliplatin-resistant cell lines (SW480-OxR and HCT116-OxR) compared to the corresponding parental cell lines and that miR-27b-3p expression was positively correlated with disease-free survival (DFS) time in colorectal cancer patients. MiR-27b-3p could sensitize colorectal cancer cells to oxaliplatin in vitro and in vivo. Under oxaliplatin treatment, chemoresistant cells showed a higher autophagy level than parental cells. Moreover, we also identified that miR-27b-3p inhibited the expression of ATG10 at the posttranscriptional level, thus inhibiting autophagy. Further study demonstrated that c-Myc can inhibit the expression of miR-27b-3p via binding to the promoter region of miR-27B gene. Conclusions: Our study identifies a novel c-Myc/miR-27b-3p/ATG10 signaling pathway that regulates colorectal cancer chemoresistance. These results suggest that miR-27b-3p is not only a potential indicator for evaluating efficiency of chemotherapy, but also a valuable therapeutic target for CRC, especially for patients with chemoresistance. Key words: miR-27b-3p; ATG10; chemoresistance; colorectal cancer; autophagy http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1982 plays a vital role in regulating colorectal cancer chemoresistance, blocking of which will be developed as a promising therapy strategy for colorectal cancer treatment [10]. By modulating key autophagy-related proteins expression, miRNA has an effect on regulating autophagy [10,20]. More generally, it has attracted substantial attention that the contribution of modulation of autophagy is mediated by miRNAs in cancer therapy and drug resistance. Materials and Methods g p MiRNAs are small noncoding RNAs that control genes expression at the posttranscriptional level [7]. As a vital regulator of numerous cell biological processes, numerous miRNAs have been shown to be involved in tumor progression and response to therapy [8]. Evidence is mounting that numerous miRNAs are involved in regulating drug resistance, especially in colorectal cancer [9,10]. In our study, a miRNA microarray array analysis was conducted to identify the aberrant miRNAs that can regulate the tolerance of CRC cells to oxaliplatin. We discovered a single miRNA, miR-27b-3p, which was greatly downregulated in both two oxaliplatin-resistant cell lines. Due to the different cellular contexts of tumors, miR-27b-3p has been reported to serve as an oncogene [11] or a tumor suppressor [12,13] in tumor progression. Interestingly, previous studies suggested that miR-27b-3p could improve the anticancer effects of chemotherapeutic drugs in multiple human cancers [14]. However, the mechanism of miR-27b-3p in regulating oxaliplatin resistance in CRC cells remains elusive. Tissue samples Colorectal cancer tissues and adjacent normal tissues were also obtained from Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). Written consent was provided by all the patients (or their guardians), and the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital approved all aspects of this study. IHC and H&E staining were performed using paraffin-embedded sections of biopsies as described previously [22]. Introduction Colorectal cancer (CRC) has one of the highest incidence rates among malignant neoplasia and is the main cause of cancer deaths worldwide [1]. According to statistics, over 1.8 million new cases of colorectal cancer and 881,000 deaths from this disease occurred in 2018 [2]. Metastasis is present at diagnosis in 1/4 of the cases, and another 1/4 of CRC patients will subsequently develop metastases within 5 years [3]. As a component of first- and second-line combination therapies, oxaliplatin is used to treat metastatic colorectal cancer (mCRC) and has significantly improved response rates to greater than 50% and led to a significant increase in median survival times [4,5]. However, the majority of CRC patients will eventually develop drug resistance, and the five-year survival rate for advanced CRC patients is lower than 10% [6]. Thus, it is important to illuminate the mechanism of chemoresistance because this knowledge may develop new strategies to overcome drug resistance in CRC patients. Here, we demonstrate the effects of miR-27b-3p on inhibiting autophagy and resensitizing chemoresistant cells to oxaliplatin. Moreover, we provided evidence showing that miR-27b-3p could target a key autophagy-related protein: ATG10, which is associated with tumorigenesis. A previous work has showed that miR-27b-3p is downregulated by c-Myc [21], and the relationship was confirmed in present study. In simple terms, we have demonstrated that the c-Myc/miR-27b-3p/ATG10 regulatory axis plays a vital role in regulating chemoresistance by activating the autophagy pathway in CRC. MiRNA expression microarray Total RNA extracted from SW480, HCT116, SW480-OxR and HCT116-OxR cells were used for Affymetrix miRNA microarray analysis (CapitalBio Corp, Beijing, China), and the process was described on the web site of CapitalBio (http:// www.capitalbio.com). Transfection MiRNA mimics, inhibitors, negative controls (NC or NC inhibitors), lentiviruses to overexpress or knowndown miR-27b-3p were purchased from GenePharma (Shanghai, China). To overexpress or knock down the expression level of proteins, gene-specific overexpression plasmids (FulenGen, Guangzhou, China) or siRNAs (GenePharma) were transfected into cells. The siRNA sequences are listed in Supplementary Table S1. RNA isolation and real-time quantitative PCR (qRT-PCR) TRIzol reagent (Sigma, St. Louis, USA) was used for extracting total RNA from CRC tissues and cells. Total RNA was extracted from paraffin-embedded of cancer tissues using an RNA pre Pure FFPE Kit (Tiangen, Beijing, China) following the manufacturer’s protocol. qRT-PCR for miRNAs and mRNAs were performed as described previously [22]. U6 snRNA or ACTB was used as the internal control for miRNAs or protein-coding genes, respectively. The sequences of the primers are listed in Supplementary Table S1. Cell culture Mounting evidence has demonstrated that anti-cancer therapies, including the cytotoxic chemotherapy, can induce cyto-protective autophagy in most cancer cells [15]. Briefly, autophagy is a highly conserved cellular process during evolution, which is induced by diverse pathologies and cellular stresses containing nutrient deprivation, endoplasmic reticulum stress and hypoxia [16]. Autophagy has also been involved in cancer resistance to multiple chemotherapeutic drugs, including cisplatin [17], doxorubicin [18], 5-Fu [19], and so on. Autophagy Six human colorectal cancer cell lines HCT116, SW480, HT29, SW620, Caco2 and LOVO were obtained from the Shanghai Institute of Cell Biology (Shanghai, China).The related oxaliplatin-resistant cell lines SW480-OxR and HCT116-OxR were generated by continuous exposure to increasing concentrations of oxaliplatin for a 10-month period as described previously [23]. We performed cytotoxicity testing to confirm that chemoresistance could be stable for about 4 weeks without oxaliplatin exposure. The oxaliplatin-resistant cell lines were used at no http://www.thno.org 1983 Theranostics 2020, Vol. 10, Issue 5 harboring the wild-type 3’UTR of ATG10, ATG4C, ATG2A or ATG2B were constructed. We also constructed a mutant 3’UTR of ATG10, which was mutated from ACUGUGA to TGACACT. For the miR-27B promoter activity assay, miR-27B promoter regions containing different c-Myc binding sites were inserted into pGL3-Basic reporter gene vectors from Genescript (Nanjing, China). We cotransfected SW480-OxR cells with luciferase vectors, small RNA oligos and a β-galactosidase expression plasmid (Ambion, Carlsbad, CA, USA). Twenty-four hours after transfection, Luciferase activity was measured using a luciferase assay kit (Promega, USA). harboring the wild-type 3’UTR of ATG10, ATG4C, ATG2A or ATG2B were constructed. We also constructed a mutant 3’UTR of ATG10, which was mutated from ACUGUGA to TGACACT. For the miR-27B promoter activity assay, miR-27B promoter regions containing different c-Myc binding sites were inserted into pGL3-Basic reporter gene vectors from Genescript (Nanjing, China). We cotransfected SW480-OxR cells with luciferase vectors, small RNA oligos and a β-galactosidase expression plasmid (Ambion, Carlsbad, CA, USA). Twenty-four hours after transfection, Luciferase activity was measured using a luciferase assay kit (Promega, USA). higher than 15 passages from creation. All cells were cultured in the appropriate medium (RPMI-1640 for HT29, SW620, SW480 and SW480-OxR cells; DMEM for Caco2, LOVO, HCT116 and HCT116-OxR cells) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) in a humidified atmosphere with 5% CO2 at 37 °C. Xenograft studies SW480, SW480-OxR, HCT116 and HCT116-OxR cells were infected with the miR-27b overexpression lentivirus, or negative control lentivirus, according to the manufacturer’s instructions. In addition, SW480 and HCT116 were also transfected with miR-27b inhibitor sponge lentivirus or negative control lentivirus. The cells were then collected for quantitative RT-PCR, or animal experiments. To explore the role of miR-27b-3p in CRC chemoresistance in vivo, we designed twelve groups (n = 5): SW480-OxR /control, SW480-OxR /control + OXA, SW480-OxR /miR-27b + OXA, SW480/control, SW480 /control + OXA, SW480/miR-27b inhibitor sponge + OXA. HCT116-OxR /control, HCT116-OxR /control + OXA, HCT116-OxR /miR-27b + OXA, HCT116/control, HCT116 /control + OXA, HCT116/miR-27b inhibitor sponge + OXA. Equal numbers of cells (5 × 106) were subcutaneously injected into each mouse to establish the CRC xenograft model. One week later, mice received an intraperitoneal injection of PBS or oxaliplatin (10 mg/kg) once per weekly for 3 weeks. On day 28, the animals were euthanized and tumors were removed. In the following experiments, SW480/control, SW480/miR-27b, HCT116/control, HCT116/miR-27b were subcutaneously injected into each mouse to establish the CRC xenograft, and 5-Fu (50 mg/kg) was used to treat the mice. Xenograft tumor tissues were removed for haematoxylin and eosin (H&E) staining or immunohistochemical (IHC) staining for Ki-67, ATG10 and cleaved-caspase 3.All the procedures were performed on the basis of the guidelines of the Laboratory Animal Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. Reagents and antibodies Oxaliplatin (S1224) and CQ (S4330) were purchased from Selleck Chemicals (Houston, TX, USA). The antibodies used for western blot were as follows: anti-c-Myc antibody (sc-40, 1:1500, Santa Cruz, CA, USA), anti-ACTB antibody (sc-10731, 1:2000, Santa Cruz), anti-p62 antibody (66184-1-Ig, 1:1000, Proteintech, IL, USA), anti-LC3 antibody (14600-1-AP, 1:1000, Proteintech), anti-cleaved PARP antibody (#5625, 1:1000, Cell Signaling Technology, MA, USA), anti-cleaved-caspase 3 antibody (#9664, 1:1000, Cell Signaling Technology), anti-ATG10 antibody (DF8366, 1:1000, Affinity, OH, USA), anti-γ-H2AX (ab2893; Abcam, Cambridge, MA, USA), anti-ATG2A antibody (23226-1-AP, 1:1000, Proteintech), anti-ATG2B antibody (251551-1-AP, Proteintech), anti-ATG4C antibody (20382-1-AP, 1:1000, Proteintech). Oxaliplatin-resistant colorectal cancer cells express decreased levels of miR-27b-3p CRC cells were transfected with GFP-LC3 vectors (HanBio Technology, Shanghai, China) and cotreated as indicated. GFP-expressing spots, which were indicated by green puncta, were imaged by a Nikon confocal microscope (Nikon, Tokyo, Japan) equipped with a 100× oil immersion objective. The number of spots per cell was determined by dividing the total number of spots by the number of nuclei in each field. To screen miRNAs that can participate in the response of colorectal cancer cells to oxaliplatin, we generated two drug resistant cell lines (SW480-OxR and HCT116-OxR) (Supplementary Figure S1A and B). Next, miRNA microarray technology showed that the expression levels of various miRNAs were changed between oxaliplatin-resistant and the parental cells (Figure 1A and Supplementary Table S2). Figure 1B lists all miRNAs with common aberrant expression in oxaliplatin-resistant cells compared to that in the corresponding parental cells. Furthermore, the qRT-PCR analysis results confirmed that the expression levels of eight miRNAs were different (Figure 1C). Then, we transfected the mimics or inhibitors of preselected miRNAs into oxaliplatin-resistant cells to detect the function of these miRNAs in regulating chemoresistance. Among all miRNAs identified to regulate drug resistance, the effect of miR-27b-3p on reversing chemoresistance was the most powerful (Supplementary Table S3). In addition, The Pearson correlation analysis showed a significant negative correlation between the miR-27b-3p level and drug resistance in eight CRC cell lines (Figure 1D). Statistical analysis The data are presented as the means ±S.E.M. of at least three independent experiments. GraphPad Prism Software (GraphPad) and Statistical Program for Social Sciences 20.0 software (SPSS) were used for statistical analyses. Differences between groups were analyzed using Student’s t-test or x2 test. The Pearson correlation test were calculated to estimate the correlations. The Kaplan-Meier survival function was calculated and compared with a log-rank test. Analysis of univariate or multivariate Cox proportional hazards regression was conducted with the hazard ratios and p values indicated. Statistically significance was defined: p<0.05, p<0.01 and p<0.001. Luciferase reporter assay The cell proliferation assay was performed as described before [24]. Briefly, CRC cells were transfected as indicated. After 12 h, 1 × 104 cells were Luciferase vectors were purchased from Genescript (Nanjing, China). Briefly, for miRNA binding site assays, luciferase reporter gene plasmids http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1984 seeded into 96-well plates, and medium containing oxaliplatin was added to each well. After 48h incubation, a CCK8 (Dojindo, Japan) assay was performed. The IC50 and the cell viability rate were calculated. Apoptosis analysis was performed using an Annexin V FITC/PI double staining assay (BD Biosciences, San Jose, CA) following the manufacturer’s protocol. seeded into 96-well plates, and medium containing oxaliplatin was added to each well. After 48h incubation, a CCK8 (Dojindo, Japan) assay was performed. The IC50 and the cell viability rate were calculated. Apoptosis analysis was performed using an Annexin V FITC/PI double staining assay (BD Biosciences, San Jose, CA) following the manufacturer’s protocol. amplification products were then separated on 2.5% agarose gels. The primers used for amplification are listed in Supplementary Table S1. Transmission electron microscopy (TEM) CRC cells were treated as indicated and harvested in a 1.5 ml microcentrifuge tube. For electron microscopy, cells were fixed with 2.5% glutaraldehyde diluted in phosphate buffer and stored at 4 °C until embedding, followed by staining with 1% OsO4. After dehydration in an increasing gradient alcohol series, thin sections were stained with 3% lead citrate–uranyl acetate and photographed with a JEM-1100 transmission electron microscope (JEOL, Tokyo, Japan). In situ hybridization and and immunofluorescence (IF). In situ hybridization (ISH) for miR-27b-3p was performed on fixed paraffin-embedded sections of biopsies from CRC samples by Roche Technology via standard protocols. The oligonucleotide probes complementary to miR-27b-3p were purchased from the GenePharma. Immunofluorescence was done as before [22]. Briefly, Cells were treated as indicated, then were fixed and incubated with primary, secondary antibodies, respectively, and DAPI for nuclear staining. Images were recorded using microscope. Chromatin immunoprecipitation (ChIP) assay The ChIP assay was performed with a commercial kit (Beyotime, Shanghai, China), following the manufacturer’s instructions. Briefly, after fragmentation of genomic DNA extracted from SW480-OxR cells, an anti-c-Myc antibody (Santa Cruz; sc-40) was used to immunoprecipitate c-Myc-chromatin complexes, and anti-IgG (Santa Cruz) was used as the negative control antibody. PCR was performed to amplify the ChIP products, and the Moreover, we found that the expression of miR-27b-3p was dramatically decreased in cancer tissues compared to noncancerous tissues (Figure 1E and F and Supplementary Table S4). In addition, we detected the expression of miR-27b-3p in 62 colorectal cancer patients who received oxaliplatin-based chemotherapy. The results showed that miR-27b-3p http://www.thno.org 1985 Theranostics 2020, Vol. 10, Issue 5 univariate and multivariate Cox regression analyses revealed that low levels of miR-27b-3p was an independent prognostic factor for poor prognosis of patients with colorectal cancer (Supplementary Figure S1D and E and Table S6).Our data indicate that miR-27b-3p is clinically associated with colorectal cancer recurrence and patient outcome. expression was significantly downregulated in patients with recurrence compared to that in patients without recurrence (Supplementary Figure S1C). Kaplan–Meier survival analysis suggested that low levels of miR-27b-3p was significantly associated with shorter disease-free survival (DFS) time (Figure 1G and Supplementary Table S5). Furthermore, orter disease-free survival (DFS) time (Figure 1G d Supplementary Table S5). Furthermore, miR-27b-3p is clinically associated with colorect cancer recurrence and patient outcome. re 1. Oxaliplatin-resistant colorectal cancer cells express decreased levels of miR-27b-3p. (A) Different miRNA expressions levels in parental cells (SW480 and HCT11 chemoresistant cells (SW480-OxR and HCT116-OxR) were determined by using the miRNA microarray. (B) Twenty-two miRNAs were dysregulated in oxaliplatin-resista relative to their expression in the corresponding parental cells. (C) The relative levels of miRNAs in SW480, HCT116, SW480-OxR and HCT116-OxR cell lines we rmined using qRT-PCR. (D) The correlation between the expression level of miR-27b-3p and IC50 for oxaliplatin in 8 CRC cell lines (SW480, HCT116, SW480-Ox T116-OxR, SW620, Caco2, HT-29 and LOVO) was shown. (E) MiR-27b-3p expression levels were decreased in human colorectal cancer samples compared with those in t ed noncancerous tissues (n=20). (F) Representative images of the expression of miR-27b-3p in paired tissues using ISH. Scale bars: 100 μm. (G) Kaplan–Meier plots stigating the correlation of miR-27b-3p expression level with disease-free survival (DFS). Patients were split into the high- and low-expression groups by the mean expressi of the miR-27b-3p (n=62; log-rank test). Chromatin immunoprecipitation (ChIP) assay (D) Cleaved-caspase 3 and PARP expression were observed by western blot in SW480 cells (left) and SW480-OxR cells(right).(E) Formation of γ-H2AX foci was observed in SW480 (left) and SW480-OxR (right) cells. Scale bars: 20 μm. (F) γ-H2AX expression was detected by western blot in SW480 and SW480-OxR cells. p < 0.01, *p < 0.001. Figure 2. MiR-27b-3p reverses the chemoresistance of colorectal cancer cells. (A) Growth curves of SW480 cells (left) and SW480-OxR cells (right) after transfection as indicated. (B) The CCK8 assay showed a change in cell viability in response to oxaliplatin after transfection of SW480 cells (left) and SW480-OxR cells (right). (C) Cell apoptotic rates of SW480 (left) and SW480-OxR (right) cells were detected by flow cytometry. (D) Cleaved-caspase 3 and PARP expression were observed by western blot in SW480 cells (left) and SW480-OxR cells(right).(E) Formation of γ-H2AX foci was observed in SW480 (left) and SW480-OxR (right) cells. Scale bars: 20 μm. (F) γ-H2AX expression was detected by western blot in SW480 and SW480-OxR cells. p < 0.01, **p < 0.001. while the miR-27b-3p inhibitor had contrasting effects (Figure 2B and Supplementary Figure S2D). Apoptosis is believed to be an important indicator of the antitumor effects of platinum-based chemotherapy [24]. To further investigate the role of miR-27b-3p in oxaliplatin-induced apoptosis, we measured the apoptosis rate in colorectal cancer cells. The results showed that the inhibition of miR-27b-3p could reduce apoptosis of oxaliplatin-sensitive cells, whereas overexpression of miR-27b-3p accelerated the apoptosis of oxaliplatin-resistant cells. (Figure 2C and Supplementary Figure S2E and F). Additionally, western blot confirmed that oxaliplatin increased the expression levels of cleaved-caspase 3 and PARP, and these effects were enhanced by miR-27b-3p overexpression, while inhibition of miR-27b-3p blocked the effects (Figure 2D and Supplementary Figure S2G). Oxaliplatin could cause DNA DSBs, are Chromatin immunoprecipitation (ChIP) assay p < 0.05, p < 0.01, p < 0.001. Figure 1. Oxaliplatin-resistant colorectal cancer cells express decreased levels of miR-27b-3p. (A) Different miRNA expressions levels in parental cells (SW480 and HCT116) and chemoresistant cells (SW480-OxR and HCT116-OxR) were determined by using the miRNA microarray. (B) Twenty-two miRNAs were dysregulated in oxaliplatin-resistant cells relative to their expression in the corresponding parental cells. (C) The relative levels of miRNAs in SW480, HCT116, SW480-OxR and HCT116-OxR cell lines were determined using qRT-PCR. (D) The correlation between the expression level of miR-27b-3p and IC50 for oxaliplatin in 8 CRC cell lines (SW480, HCT116, SW480-OxR, HCT116-OxR, SW620, Caco2, HT-29 and LOVO) was shown. (E) MiR-27b-3p expression levels were decreased in human colorectal cancer samples compared with those in the paired noncancerous tissues (n=20). (F) Representative images of the expression of miR-27b-3p in paired tissues using ISH. Scale bars: 100 μm. (G) Kaplan–Meier plots for investigating the correlation of miR-27b-3p expression level with disease-free survival (DFS). Patients were split into the high- and low-expression groups by the mean expression level of the miR-27b-3p (n=62; log-rank test). p < 0.05, p < 0.01, p < 0.001. http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1986 Figure 2. MiR-27b-3p reverses the chemoresistance of colorectal cancer cells. (A) Growth curves of SW480 cells (left) and SW480-OxR cells (right) after transfection as indicated. (B) The CCK8 assay showed a change in cell viability in response to oxaliplatin after transfection of SW480 cells (left) and SW480-OxR cells (right). (C) Cell apoptotic rates of SW480 (left) and SW480-OxR (right) cells were detected by flow cytometry. (D) Cleaved-caspase 3 and PARP expression were observed by western blot in SW480 cells (left) and SW480-OxR cells(right).(E) Formation of γ-H2AX foci was observed in SW480 (left) and SW480-OxR (right) cells. Scale bars: 20 μm. (F) γ-H2AX expression was detected by western blot in SW480 and SW480-OxR cells. p < 0.01, *p < 0.001. Figure 2. MiR-27b-3p reverses the chemoresistance of colorectal cancer cells. (A) Growth curves of SW480 cells (left) and SW480-OxR cells (right) after transfection as indicated. (B) The CCK8 assay showed a change in cell viability in response to oxaliplatin after transfection of SW480 cells (left) and SW480-OxR cells (right). (C) Cell apoptotic rates of SW480 (left) and SW480-OxR (right) cells were detected by flow cytometry. MiR-27b-3p suppresses tumor growth when combined with oxaliplatin in vivo Considering that autophagy can protect cancer cells from cytotoxic drugs, we explored whether autophagy participated in chemoresistance to oxaliplatin in colorectal cancer. Thus, we measured the LC3 and p62 levels, which are the widely used markers of autophagy [15]. Remarkably, oxaliplatin-resistant cells showed higher LC3-II protein levels and lower p62 protein levels than the corresponding parental cells after treatment with oxaliplatin, suggesting that autophagic flux was induced when chemoresistance occurred (Figure 4A and Supplementary Figure S6A).Consistent with this result, oxaliplatin treatment significantly induced the formation of LC3 puncta (Figure 4B and C) and autophagosomes (Figure 4D and E) in SW480-OxR cells. Next, we sought to explore whether chemotherapy-induced autophagy has an effect on the efficacy of chemotherapy. Thus, we co-treated SW480-OxR cells with chloroquine (CQ) and oxaliplatin, and found that CQ enhanced the antitumor activity of oxaliplatin, as evidenced by the decreased IC50 (Figure 4F and G). To assess the effect of miR-27b-3p combined with oxaliplatin on tumor growth in vivo, we stably transfected SW480-OxR and HCT116-OxR cells with lentivirus overexpressing miR-27b-3p or with lentivirus expressing miR-NC (Supplementary Figure S4A). In addition, we also stably transfected SW480 and HCT116 cells with lentivirus expressing a miR-27b-3p inhibitor sponge or miR-NC (Supplementary Figure S4A). Because miR-27b lentivirus could overexpress both miR-27b-3p and miR-27b-5p, we further examined the expression level of miR-27b-5p in CRC cell lines. The expression of miR-27b-5p was much lower than that of miR-27b-3p in miRNA microarray, and qRT-PCR further conformed the result (Supplementary Figure S4B and Table S2). We then overexpressed miR-27b-5p in SW480-OxR and HCT116-OxR cells, and miR-27b-5p could not significantly enhance the effect of oxaliplatin on inhibiting cell proliferation (Supplementary Figure S4C and D). In the CRC xenograft mouse models, SW480-OxR cells and SW480 cells were subcutaneously transplanted into nude mice, and then offer treatment with oxaliplatin, as shown in Figure 3A. The results showed that SW480-OxR and HCT116-OxR cells stably overexpressing miR-27b-3p were more sensitive to oxaliplatin therapy than control group (Figure 3B and C and Supplementary Figure S5A and B). Oppositely, inhibition of miR-27b-3p in SW480 and HCT116 cells weakened the effect of oxaliplatin on inhibiting tumor growth (Figure 3D and E and Supplementary Figure S5C and D). qRT-PCR showed that oxaliplatin We next investigated the role of miR-27b-3p in autophagic activity. Overexpression of miR-27b-3p resulted in reducing level of LC3-II and enhancing level of p62 in oxaliplatin-resistant cells (Figure 4H and Supplementary Figure S6B). MiR-27b-3p reverses the chemoresistance of colorectal cancer cells Based on the accumulated data, we sought to examine the effect of miR-27b-3p on CRC cells chemoresistance in vitro. We inhibited miR-27b-3p expression in oxaliplatin-sensitive cells and overexpressed miR-27b-3p in oxaliplatin-resistant cells, respectively (Supplementary Figure S2A and B). Subsequently, the growth curves showed that miR-27b-3p inhibitor increased the IC50 of oxaliplatin in oxaliplatin-sensitive cells. On contrary, the IC50 of oxaliplatin concomitantly decreased in miR-27b-3p-overexpressing cells (Figure 2A and Supplementary Figure S2C). Next, by adding oxaliplatin to the corresponding cells, we found that overexpression of miR-27b-3p could enhance the effect of oxaliplatin on inhibiting cell proliferation, http://www.thno.org http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1987 treatment decreased miR-27b-3p level, which were recovered by expression of miR-27b-3p in SW480-OxR and HCT116-OxR xenograft tumors. In contrast, miR-27b-3p expression was downregulated by miR-27b-3p sponge inhibitor in SW480 and HCT116 xenograft tumors (Supplementary Figure S5E). Additionally, the reduction of Ki67 and upregulation of cleaved-caspase 3 were detected in SW480-OxR and HCT116-OxR cells treated with the combination of miR-27b-3p-overexpressing lentivirus and oxaliplatin (Figure 3F and G and Supplementary Figure S5F and G), while SW480 and HCT116 cells stably expressing the miR-27b-3p inhibitor exhibited an increased Ki67 and reduced cleaved-caspase 3 level (Figure 3H and I and Supplementary Figure S5H and I). Altogether, these results strongly indicate that miR-27b-3p suppresses tumor growth and inhibits therapeutic resistance in vivo. associated with the formation of γ-H2AX [14]. Indeed, overexpression of miR-27b-3p resulted in the accumulation of γ-H2AX in oxaliplatin-resistant cells. Conversely, miR-27b-3p down-regulation reduced the level of oxaliplatin-induced foci formation of γ-H2AX in oxaliplatin-sensitive cells (Figure 2E and F and Supplementary Figure S2H and I). pp y g ) In addition to oxaliplatin, 5-Fu also serves as the backbone of systemic combination chemotherapy in CRC treatment [25]. Thus, we investigated whether miR-27b-3p could affect proliferation and apoptosis of CRC cells, when exposed to 5-Fu. Following treatment with 5-Fu, miR-27b-3p could inhibit proliferation (Supplementary Figure S3A and B) and enhance apoptosis (Supplementary Figure S3C-E). What’s more, miR-27b-3p could markedly increase the sensitivity of colorectal cancer cells to 5-Fu in vivo (Supplementary Figure S3F-I). Thus, miR-27b-3p may enhance the sensitivity of CRC cells to chemotherapeutic agents. MiR-27b-3p suppresses tumor growth when combined with oxaliplatin in vivo In contrast, in oxaliplatin-sensitive cells with miR-27b-3p suppression, the level of LC3-II was increased, while the level of p62 was decreased (Figure 4H and Supplementary Figure S6B). Overexpression of miR-27b-3p has diminished the numbers of LC3 puncta, whereas suppression of miR-27b-3p led to an increase in the number of LC3 puncta (Figure 4I and J http://www.thno.org 1988 Theranostics 2020, Vol. 10, Issue 5 chemoresistant cells show enhanced autophagy activity when compared to the corresponding parental cells, and that miR-27b-3p inhibits autophagic activity. and Supplementary Figure S6C and D). In addition, upregulation of miR-27b-3p blocked the formation of autophagosomes and that inhibition of miR-27b-3p enhanced the formation of autophagosomes (Figure 4K and L). Collectively, our data indicate that K and L). Collectively, our data indicate that ure 3. MiR-27b-3p suppresses tumor growth combined with oxaliplatin in vivo. (A) A schematic outline of the experimental design. (B) Representative images of tu e mice bearing SW480-OxR cells in different groups (n= 5 for each group). Scale bars: 1 cm. (C) Tumor weights were measured in different groups. (D) Representative umors in nude mice bearing SW480 cells in different groups (n= 5 for each group). Scale bars: 1 cm. (E) Tumor weights were measured in different groups, (F) Repres ges of tumor samples derived from SW480-OxR group that were stained with H&E (left) and immunohistochemistry of Ki67 (middle) and cleaved-caspase 3 (right). Sca μm; (insets) 25 μm. (G) Statistical analysis of Ki-67 and cleaved-caspase 3 protein levels in (F). (H) Representative images of tumor samples derived from SW480 gro re stained with H&E (left) and immunohistochemistry of Ki67 (middle) and cleaved-caspase 3 (right). Scale bars: 100 μm; (insets) 25 μm. (I) Statistical analysis of K ved-caspase 3 protein levels in (H). p < 0.05, p < 0.01, p < 0.001. Figure 3. MiR-27b-3p suppresses tumor growth combined with oxaliplatin in vivo. (A) A schematic outline of the experimental design. (B) Representative images of tumors in nude mice bearing SW480-OxR cells in different groups (n= 5 for each group). Scale bars: 1 cm. (C) Tumor weights were measured in different groups. (D) Representative images of tumors in nude mice bearing SW480 cells in different groups (n= 5 for each group). Scale bars: 1 cm. MiR-27b-3p suppresses tumor growth when combined with oxaliplatin in vivo Autophagosomes were observed by transmission electr microscopy (TEM) in SW480-OxR cells cultured with oxaliplatin. Representative images are shown in (D), and autophagosomes per cell were quantified in (E). Scale bar: 1 μ (insets) 250 nm. (F)Western blot was performed in SW480-OxR cells treated with oxaliplatin in the presence of CQ. (G) IC50 for oxaliplatin in SW480-OxR cells in the presen or absence of CQ. (H-L) SW480-OxR and SW480 cells were transfected with mimics or inhibitor of miR-27b-3p, respectively. After culturing with oxaliplatin, (H) autopha element expression levels were detected by western blot, (I) green fluorescent LC3 puncta were observed under confocal microscope, (K) autophagosomes were observed TEM, respectively. LC3 puncta per cell were quantified in (J). Scale bar: 10 μm. Autophagosomes per cell were quantified in (L). Scale bar: 1 μm; (insets) 250 nm. p < 0.01 Figure 4. MiR-27b-3p inhibits autophagic activity in chemoresistant CRC cells. (A) Autophagy element expression levels were detected by western blot in SW480 and SW480-OxR cells cultured with oxaliplatin. (B and C) Confocal microscopic analysis was performed to observe green fluorescent LC3 puncta in SW480-OxR cells cultured with oxaliplatin. Representative images are shown in (B), and LC3 puncta per cell were quantified in (C). Scale bar: 10 μm. Autophagosomes were observed by transmission electron microscopy (TEM) in SW480-OxR cells cultured with oxaliplatin. Representative images are shown in (D), and autophagosomes per cell were quantified in (E). Scale bar: 1 μm; (insets) 250 nm. (F)Western blot was performed in SW480-OxR cells treated with oxaliplatin in the presence of CQ. (G) IC50 for oxaliplatin in SW480-OxR cells in the presence or absence of CQ. (H-L) SW480-OxR and SW480 cells were transfected with mimics or inhibitor of miR-27b-3p, respectively. After culturing with oxaliplatin, (H) autophagy element expression levels were detected by western blot, (I) green fluorescent LC3 puncta were observed under confocal microscope, (K) autophagosomes were observed by TEM, respectively. LC3 puncta per cell were quantified in (J). Scale bar: 10 μm. Autophagosomes per cell were quantified in (L). Scale bar: 1 μm; (insets) 250 nm. p < 0.01. genes, namely, ATG10, ATG4C, ATG2A and ATG2B. Among these 4 genes, ATG10 exhibited the most significant reduction in luciferase activity when ectopic miR-27b-3p was expressed in SW480-OxR cells (Figure 5A). By blocking the conversion of LC3-I to LC3-II, ATG10 plays a vital role in regulating autophagy [29]. However, the function of ATG10 in regulating drug responses is unclear. MiR-27b-3p suppresses tumor growth when combined with oxaliplatin in vivo The predicted MiR-27b-3p suppresses tumor growth when combined with oxaliplatin in vivo (E) Tumor weights were measured in different groups, (F) Representative images of tumor samples derived from SW480-OxR group that were stained with H&E (left) and immunohistochemistry of Ki67 (middle) and cleaved-caspase 3 (right). Scale bars: 100 μm; (insets) 25 μm. (G) Statistical analysis of Ki-67 and cleaved-caspase 3 protein levels in (F). (H) Representative images of tumor samples derived from SW480 group that were stained with H&E (left) and immunohistochemistry of Ki67 (middle) and cleaved-caspase 3 (right). Scale bars: 100 μm; (insets) 25 μm. (I) Statistical analysis of Ki-67 and cleaved-caspase 3 protein levels in (H). p < 0.05, p < 0.01, p < 0.001. http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1989 , , . MiR-27b-3p inhibits autophagic activity in chemoresistant CRC cells. (A) Autophagy element expression levels were detected by western blot in SW OxR cells cultured with oxaliplatin. (B and C) Confocal microscopic analysis was performed to observe green fluorescent LC3 puncta in SW480-OxR cells cultu . Representative images are shown in (B), and LC3 puncta per cell were quantified in (C). Scale bar: 10 μm. Autophagosomes were observed by transmission py (TEM) in SW480-OxR cells cultured with oxaliplatin. Representative images are shown in (D), and autophagosomes per cell were quantified in (E). Scale b 50 nm. (F)Western blot was performed in SW480-OxR cells treated with oxaliplatin in the presence of CQ. (G) IC50 for oxaliplatin in SW480-OxR cells in the e of CQ. (H-L) SW480-OxR and SW480 cells were transfected with mimics or inhibitor of miR-27b-3p, respectively. After culturing with oxaliplatin, (H) a xpression levels were detected by western blot, (I) green fluorescent LC3 puncta were observed under confocal microscope, (K) autophagosomes were obs pectively. LC3 puncta per cell were quantified in (J). Scale bar: 10 μm. Autophagosomes per cell were quantified in (L). Scale bar: 1 μm; (insets) 250 nm. p ification of ATG10 as a direct target of genes, namely, ATG10, ATG4C, ATG2A and AT A h 4 ATG10 hibi d h Figure 4. MiR-27b-3p inhibits autophagic activity in chemoresistant CRC cells. (A) Autophagy element expression levels were detected by western blot in SW480 a SW480-OxR cells cultured with oxaliplatin. (B and C) Confocal microscopic analysis was performed to observe green fluorescent LC3 puncta in SW480-OxR cells cultured w oxaliplatin. Representative images are shown in (B), and LC3 puncta per cell were quantified in (C). Scale bar: 10 μm. Identification of ATG10 as a direct target of miR-27b-3p To investigate the target gene involved in mediating the effect of miR-27b-3p on modulating autophagy, we using a combination of three prediction softwares: TargetScan [26], miRanda [27] and miRPathDB [28], and we selected four candidate http://www.thno.org 1990 Theranostics 2020, Vol. 10, Issue 5 interactions between miR-27b-3p and the 3ʹ-UTR of ATG10 are shown in Figure 5B. To further verify that ATG10 is a direct target of miR-27b-3p, we constructed a luciferase reporter vector containing wild-type or mutant 3' UTR fragments harboring the miR-27b-3p binding site of ATG10. The results showed that ectopic expression of miR-27b-3p significantly reduced the fluorescence intensity, whereas downregulation of miR-27b-3p enhanced the interactions between miR-27b-3p and the 3ʹ-UTR of ATG10 are shown in Figure 5B. To further verify that ATG10 is a direct target of miR-27b-3p, we constructed a luciferase reporter vector containing wild-type or mutant 3' UTR fragments harboring the miR-27b-3p binding site of ATG10. The results showed that ectopic expression of miR-27b-3p significantly reduced the fluorescence intensity, whereas downregulation of miR-27b-3p enhanced the luciferase activity. When the binding site of miR-27b-3p was mutated, miR-27b-3p had no influence on the luciferase activity (Figure 5C). Furthermore, western blot showed that ATG10 was the most significantly upregulated protein in oxaliplatin-resistant colorectal cancer cells (Figure 5D and Supplementary Figure S7A). Overexpression of miR-27b-3p inhibited the expression of ATG10, whereas inhibition of miR-27b-3p increased the 5. Identification of ATG10 as a direct target of miR-27b-3p. (A)Luciferase assays showing suppression of luciferase activity of candidate genes by miR-27b-3p -OxR cells. (B) Schematic of the hypothetical duplexes formed by miR-27b-3p and the 3’-UTR of ATG10. (C) Relative luciferase activity in SW480-OxR cells transfect e miR-27b-3p mimic or inhibitor. (D) Western blot showing the expression levels of ATG10 in four CRC cell lines. (E and F) Western blot analysis was performed e the expression level of ATG10 in oxaliplatin-resistant cells transfected with the miR-27b-3p mimic and the corresponding parental cells transfected with the miR-27b- r. (G) Protein levels of ATG10 were measured in 20 pairs of samples using western blot as previously described. (H) The levels of ATG10 protein expression we ed. (I) Representative images of tumor samples that were stained for ATG10 by IHC. Scale bar: 100 μm; (insets) 25 μm. (J) The correction between the fold changes ression of miR-27b-3p and the ATG10 protein in human CRC tissue pairs (n=20). p<0.05, p<0.01, p<0.001. Figure 5. Identification of ATG10 as a direct target of miR-27b-3p Identification of ATG10 as a direct target of miR-27b-3p. (A)Luciferase assays showing suppression of luciferase activity of candidate genes by miR-27b-3p in SW480-OxR cells. (B) Schematic of the hypothetical duplexes formed by miR-27b-3p and the 3’-UTR of ATG10. (C) Relative luciferase activity in SW480-OxR cells transfected with the miR-27b-3p mimic or inhibitor. (D) Western blot showing the expression levels of ATG10 in four CRC cell lines. (E and F) Western blot analysis was performed to measure the expression level of ATG10 in oxaliplatin-resistant cells transfected with the miR-27b-3p mimic and the corresponding parental cells transfected with the miR-27b-3p inhibitor. (G) Protein levels of ATG10 were measured in 20 pairs of samples using western blot as previously described. (H) The levels of ATG10 protein expression were measured. (I) Representative images of tumor samples that were stained for ATG10 by IHC. Scale bar: 100 μm; (insets) 25 μm. (J) The correction between the fold changes in the expression of miR-27b-3p and the ATG10 protein in human CRC tissue pairs (n=20). p<0.05, p<0.01, p<0.001. Figure 5. Identification of ATG10 as a direct target of miR-27b-3p. (A)Luciferase assays showing suppression of luciferase activity of candidate genes by miR-27b-3p in SW480-OxR cells. (B) Schematic of the hypothetical duplexes formed by miR-27b-3p and the 3’-UTR of ATG10. (C) Relative luciferase activity in SW480-OxR cells transfected with the miR-27b-3p mimic or inhibitor. (D) Western blot showing the expression levels of ATG10 in four CRC cell lines. (E and F) Western blot analysis was performed to measure the expression level of ATG10 in oxaliplatin-resistant cells transfected with the miR-27b-3p mimic and the corresponding parental cells transfected with the miR-27b-3p inhibitor. (G) Protein levels of ATG10 were measured in 20 pairs of samples using western blot as previously described. (H) The levels of ATG10 protein expression were measured. (I) Representative images of tumor samples that were stained for ATG10 by IHC. Scale bar: 100 μm; (insets) 25 μm. (J) The correction between the fold changes in the expression of miR-27b-3p and the ATG10 protein in human CRC tissue pairs (n=20). p<0.05, p<0.01, p<0.001. http://www.thno.org 1991 Theranostics 2020, Vol. 10, Issue 5 cleaved-caspase 3 and PARP (Figure 6H) in SW480-OxR cells. Moreover, the increase in the oxaliplatin-induced cell apoptosis rate by the miR-27b-3p mimic was significantly reversed by ATG10 overexpression (Figure 6F and H and Supplementary Figure S8). ATG10 protein level, respectively (Figure 5E and F). Expression of miR-27b-3p is inhibited by c-Myc Expression of miR-27b-3p is inhibited by c-Myc To investigate the mechanism underlying miR-27b-3p inhibition in chemoresistant CRC cells, we first measured the levels of pri-miR-27b in CRC cells and observed that they were also significantly lower in chemoresistant cells than in the corresponding parental cells, suggesting that miR-27b-3p is transcriptionally inhibited in chemoresistant cells (Supplementary Figure S9A). It has been widely reported that dysregulation of transcription factors (TFs), which can also regulate the expression of miRNA, is commonly involved in tumorigenesis [30]. Interestingly, our previous work showed that c-Myc can transcriptionally downregulate miR-27b-3p [21]. To examine the effect of c-Myc on regulating the miR-27b-3p, we overexpressed or knocked down c-Myc in SW480 cells or SW480-OxR cells, respectively (Supplementary Figure S9B). As shown in Figure 7A and Supplementary Figure S9C, the levels of mature miR-27b-3p and pri-miR-27b were markedly decreased or increased after transfection with the c-Myc overexpression plasmid or siRNA, respectively. Identification of ATG10 as a direct target of miR-27b-3p However, the mRNA levels of ATG10 in CRC cells have not been changed (Supplementary Figure S7B and C), which indicated that miR-27b-3p could regulate ATG10 expression at the post-transcriptional level. Furthermore, we measured the ATG10 levels in the abovementioned 20 pairs of CRC tissues and found that the ATG10 levels were significantly upregulated in CRC tissues than in the paired normal colorectal tissues (Figure 5G-I). The Pearson correlation analysis revealed that the expression level of ATG10 was significantly negatively related to the level of miR-27b-3p (Figure 5J). In addition, the expression of ATG10 was decreased after overexpression of miR-27b-3p (Supplementary Figure S7D and F) and increased after inhibition of miR-27b-3p (Supplementary Figure S7E and G) in the subcutaneous colorectal tumors. In sum, these results demonstrate that miR-27b-3p posttranscriptionally regulates the expression of ATG10. cleaved-caspase 3 and PARP (Figure 6H) in SW480-OxR cells. Moreover, the increase in the oxaliplatin-induced cell apoptosis rate by the miR-27b-3p mimic was significantly reversed by ATG10 overexpression (Figure 6F and H and Supplementary Figure S8). cleaved-caspase 3 and PARP (Figure 6H) in SW480-OxR cells. Moreover, the increase in the oxaliplatin-induced cell apoptosis rate by the miR-27b-3p mimic was significantly reversed by ATG10 overexpression (Figure 6F and H and Supplementary Figure S8). Next, we investigated the effect of ATG10 on autophagic activity in colorectal cancer cells. In SW480 cells, silencing of ATG10 attenuated the effect of the miR-27b-3p inhibitor on the protein levels of ATG10, p62 and LC3-II (Figure 6I) and the formation of LC3 puncta (Figure 6J and K). Conversely, in SW480-OxR cells, ATG10 overexpression diminished the inhibitory effect of miR-27b-3p on the protein levels of ATG10, p62 and LC3-II (Figure 6L) and the formation of LC3 puncta formation (Figure 6M and N). Collectively, these results suggest that miR-27b-3p inhibits CRC cell chemoresistance and autophagy by suppressing ATG10. MiR-27b-3p enhances the sensitivity of CRC cells to oxaliplatin by inhibiting ATG10 and thereby inhibiting autophagy Given the effect of autophagy on regulating drug resistance as described before, we hypothesized that miR-27b-3p reverses chemoresistance by attenuating autophagic activity through inhibiting ATG10. To further evaluate whether ATG10 mediates the function of miR-27b-3p in the autophagic process and chemoresistance, we performed a series of rescue experiments. The results showed that inhibition of ATG10 can suppress the proliferation and attenuate the drug resistance of SW480 cells (Figure 6A and C). In contrast, overexpression of ATG10 promoted cell proliferation and drug resistance of SW480-OxR cells (Figure 6B and D). Suppression of ATG10 abolished the enhancement of cell proliferation and drug resistance in SW480 cells induced by the miR-27b-3p inhibitor (Figure 6A and C). Moreover, the miR-27b-3p-induced inhibition of SW480-OxR cell proliferation and chemoresistance was reduced by the ATG10 overexpression vector (Figure 6B and D). In combination with oxaliplatin, downregulation of ATG10 increased oxaliplatin-induced apoptosis (Figure 6E and Supplementary Figure S8) and the protein levels of cleaved-caspase 3 and PARP (Figure 6G) in SW480 cells. More importantly, the attenuation of SW480 cell apoptosis in response to oxaliplatin by the miR-27b-3p inhibitor was significantly reversed by ATG10 downregulation (Figure 6E and G and Supplementary Figure S8). In addition, we showed that ATG10 overexpression significantly reduced oxaliplatin-induced cell apoptosis (Figure 6F and Supplementary Figure S8) and the expression levels of By binding to the E-box sequence CACGTG or CATGTG, c-Myc can downregulate the expression of miRNA at the transcriptional level [31]. After analyzing the potential promoter region, we identified three putative c-Myc binding sites (Figure 7B). Chromatin immunoprecipitation technique showed that c-Myc was significantly recruited to the region around the binding sites 1 and 2 in SW480-OxR cells (Figure 7C and Supplementary Figure S9D). Subsequently, we cloned the binding sites 1 and 2 into the upstream region of a firefly luciferase reporter http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1992 whereas luciferase activity was unaffected when the binding sites were mutated (Figure 7D). gene and then performed luciferase reporter assays in SW480-OxR cells. Silencing c-Myc enhanced luciferase activity in binding sites 1 and 2-containing plasmids, Figure 6. MiR-27b-3p enhances the sensitivity of CRC cells to oxaliplatin by inhibiting ATG10 and thereby inhibiting autophagy. (A and B) Growth curves of SW480 cells (A) and SW480-OxR cells (B) after transfection as indicated. MiR-27b-3p enhances the sensitivity of CRC cells to oxaliplatin by inhibiting ATG10 and thereby inhibiting autophagy (C and D) The CCK8 assay showed a change in cell viability in response to oxaliplatin after transfection of SW480 cells (C) and SW480-OxR cells (D). (E and F) Apoptosis was detected by flow cytometry in SW480 cells (E) and SW480-OxR cells (F) with the indicated modifications and then were incubated with oxaliplatin, respectively.(G and H) The protein levels of cleaved-caspase 3 and PARP in SW480 cells (G) and SW480-OxR cells (H) after transfection and then were stimulated with oxaliplatin. (I)Western blot analysis for autophagy element expression levels in SW480 cells, after treated as in (G). (J and K) Representative photographs of LC3 puncta (green) in SW480 cells with the indicated modifications (J). Quantification of LC3 puncta in the indicated SW480 cells (K). Scale bar: 10 μm. (L) Western blot analysis for autophagy elements expression levels in SW480-OxR cells, after treated as in (H). (M and N) Representative photographs of LC3 puncta (green) in SW480-OxR cells with the indicated modifications (M). Quantification of LC3 puncta in the indicated SW480-OxR cells (N). Scale bar: 10 μm. p<0.05, p<0.01, p<0.001. http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1993 igure 7. Expression of miR-27b-3p is inhibited by c-Myc. (A) The influence of c-Myc on the expression of miR-27b-3p. (B) Schematic showing the three putative c-Myc-binding motifs in the miR-27B promoter region. (C) ChIP assay for c-Myc occupancy on the miR-27B promoter region. (D) Luciferase reporter assays were performed to confirm the uppression of miR-27B promoter by c-Myc. (E and F) Western blot analysis of the c-Myc and ATG10 protein levels in SW480 cells (E) and SW480-OxR cells (F). (G and H) Western blot analysis of the c-Myc expression level in 20 pairs of CRC tissues and NATs. G: representative images; H: quantitative analysis (n=20). (I) The correction between he fold changes in the expression of miR-27b-3p and the c-Myc protein in CRC tissues as mentioned previously (n = 20). (J) Schematic of the c-Myc/miR-27b-3p/ATG10 axis in CRC. p<0.01, p<0.001. Subsequently, we sought to investigate whether -Myc could regulate ATG10 expression by inhibiting c-Myc expression significantly upregulated miR-27b-3p, and this effect was attenuated by Figure 7. Expression of miR-27b-3p is inhibited by c-Myc. (A) The influence of c-Myc on the expression of miR-27b-3p. (B) Schematic showing the three putative c-Myc-binding motifs in the miR-27B promoter region. (C) ChIP assay for c-Myc occupancy on the miR-27B promoter region. MiR-27b-3p enhances the sensitivity of CRC cells to oxaliplatin by inhibiting ATG10 and thereby inhibiting autophagy (D) Luciferase reporter assays were performed to confirm the suppression of miR-27B promoter by c-Myc. (E and F) Western blot analysis of the c-Myc and ATG10 protein levels in SW480 cells (E) and SW480-OxR cells (F). (G and H) Western blot analysis of the c-Myc expression level in 20 pairs of CRC tissues and NATs. G: representative images; H: quantitative analysis (n=20). (I) The correction between the fold changes in the expression of miR-27b-3p and the c-Myc protein in CRC tissues as mentioned previously (n = 20). (J) Schematic of the c-Myc/miR-27b-3p/ATG10 axis in CRC p<0 01 ***p<0 001 Subsequently, we sought to investigate whether c-Myc could regulate ATG10 expression by inhibiting miR-27b-3p. As shown, transfection of the c-Myc vector increased the protein level of ATG10, which was attenuated by cotreatment with the miR-27b-3p mimic (Figure 7E). In contrast, inhibition of c-Myc expression significantly downregulated ATG10, and this effect was rescued following miR-27b-3p inhibitor transfection (Figure 7F). Meanwhile, we also found that transfection of the c-Myc vector decreased the level of miR-27b-3p, which was abolished by cotreatment with the miR-27b-3p mimic (Supplementary Figure S9E). In contrast, inhibition of c-Myc expression significantly upregulated miR-27b-3p, and this effect was attenuated by transfecting with miR-27b-3p inhibitor (Supplementary Figure S9F).Taken together, these results reveal that c-Myc indirectly upregulates the expression of ATG10 through inhibiting miR-27b-3p. We then examined the c-Myc protein levels in the 20 abovementioned pairs of CRC tissues, and the results showed that the expression level of c-Myc was markedly elevated in the CRC tissues (Figure 7G and H). Notably, The Person correlation test showed a significant inverse relation between the levels of the c-Myc protein and miR-27b-3p in CRC tissues (Figure http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1994 7I). These results indicate that the decreased levels of the miR-27b-3p are, at least in part, attributed to the overexpression of c-Myc in CRC. Thus, we concluded that c-Myc specifically regulates miR-27b-3p expression transcriptionally and indirectly promotes ATG10 expression (Figure 7J). in CRC cells. In summary, we introduced miR-27b-3p as a vital autophagy-regulating miRNA that acts as a tumor suppressor in CRC cells, by blocking autophagy to promote cell sensitivity to oxaliplatin. ATG10 is an autophagic E2-like enzyme that interacts with ATG7 to recruit ATG12 and modulates the conversion of LC3-I to LC3-II [29]. Thus, ATG10 plays a critical role in autophagosome formation. Discussion Due to the target genes diversity and tissue type specificity of miRNAs, the specific regulatory functions of miRNAs have yet to be fully delineated [7]. In our study, we have identified miR-27b-3p could significantly reduce cell chemoresistance and act as a promising marker for predicting prognosis in colorectal cancer patients receiving oxaliplatin-based chemotherapy. The role of miR-27b-3p in tumorigenesis remains to be elucidated. Previous reports showed that miR-27b-3p expression was elevated in certain human malignancies and that miR-27b-3p thus served as an oncogenic miRNA [11]. However, it has also been reported that in colorectal cancer [32], gastric cancer [33], breast cancer [34], miR-27b-3p acts as a tumor suppressor. These conflicting conclusions from different studies may be due to the use of different cellular models. In terms of regulating drug-resistance, studies have shown that miR-27b-3p may increase drug resistance in anaplastic thyroid cancer [35]. Conversely, in gastric cancer [36], breast cancer [34], and nasopharyngeal cancer [37], miR-27b-3p was reported to enhance the response to anticancer drugs such as doxorubicin and paclitaxel. However, the mechanism of miR-27b-3p regulating CRC chemoresistance requires further clarification. In present study, we found decreased expression of miR-27b-3p in the oxaliplatin-resistant cells, but the mechanism requires further study. As a transcription factor, c-Myc is equipped with the ability to regulate tumor development in many types of human cancer by orchestrating gene expression [30]. It has been shown that aberrant expression of c-Myc is a key driver of CRC progression [43]. Numerous studies have revealed that c-Myc acts as either a transcriptional activator or inhibitor that modulates miRNA expression and contributes to cancer progression [30]. Interestingly, our previous work showed that c-Myc could reduce the expression of miR-27b-3p, and we demonstrated this effect in CRC cells. MiR-27B is located within the 14th intron of its host gene, and far away from the host gene’s transcriptional start sites (TSSs). It has been reported that intronic miRNAs located far away from their host TSSs may rely on independent novel TSSs to increase the speed and efficiency of transcription [44]. Thus, the potential promoter region (approximately 2 kb upstream of the TSS) of miR-27B was analyzed, and the ChIP assay results suggested that c-Myc can bind to the promoter region of the miR-27B gene. MiR-27b-3p enhances the sensitivity of CRC cells to oxaliplatin by inhibiting ATG10 and thereby inhibiting autophagy Emerging evidence has emphasized that ATG10 displayed higher expression level in tumors of malignancies such as CRC [41] and lung cancer [42]. In addition, increased expression of ATG10 is positively linked with lymphovascular invasion and predicts decreased overall survival times [41]. Further studies showed that ATG10 could promote tumor cells proliferation and malignant transformation [42]. In our study, we demonstrated that miR-27b-3p regulates the expression of ATG10 at the posttranscriptionally level. Moreover, we assessed the effect of miR-27b-3p on inhibiting autophagy, cell proliferation, drug resistance, and even the growth of implanted tumors by suppressing ATG10. Therefore, our data may reveal the therapeutic potential of miR-27b-3p combined with chemotherapy. Discussion Specifically, we found that in oxaliplatin-resistant colorectal cancer cells, the increased chemoresistance and autophagy are due to the enhanced expression of c-Myc, which upregulates the expression of ATG10 by suppressing miR-27b-3p. In fact, previous findings indicated that c-Myc can regulate tumor cell chemoresistance to antitumor platinum drugs [45]. q Autophagy is known to play role in maintaining the survival of tumor cells under a variety of adverse conditions, including nutrient deficiency, chemotherapy and radiation treatment [15]. Accumulating evidence suggests that regulating the autophagic activity could enhance the action of many antitumor agents, including oxaliplatin [10], cisplatin [17], doxorubicin [38] and 5-Fu [39]. Thus, autophagy has been proposed as a potential drug target to reverse drug resistance. Previous reports have shown that a series of miRNAs regulate the drug resistance by modulating of autophagy [10]. Notably, we reported that oxaliplatin-resistant cells showed increased autophagic activity compared to that in the corresponding parental cells. Interestingly, miR-27b-3p has been shown to inhibit PINK expression resulting in autophagy suppression [40]. In the present study, we introduced miR-27b-3p as a novel autophagy regulator in CRC. In oxaliplatin-resistant CRC cells, overexpression of miR-27b-3p inhibited LC3-I to LC3-II conversion, GFP-LC3 accumulation and autophagosome synthesis http://www.thno.org Theranostics 2020, Vol. 10, Issue 5 1995 Furthermore, c-Myc can also trigger autophagy by inducing the expression of multiple ATG genes [46,47]. (2018KJ046, 2017KJ227, 2017KJ204). The funders had no role in the study design, the data collection and analysis, the interpretation of the data, the writing of the report, and the decision to submit this article for publication. In addition to describing the biological importance of miR-27b-3p, the results of our study may be related to the clinical management of CRC patients. For CRC patients, capecitabine (and 5-Fu) has been widely used in combination with platinum-based chemotherapy, which can effectively inhibit tumors and initially improve the survival of patients [5]. However, many patients eventually relapse due to the emergence of chemoresistance [4]. Therefore, it is important to explore the regulatory mechanism of drug resistance and optimize current therapeutic strategies. Given that the expression level of miR-27b-3p is associated with the risk of CRC recurrence, detection of miR-27b-3p may be an effective approach to predict the response of patients to chemotherapy. Furthermore, our work suggested that combining miR-27b-3p with chemotherapeutic agents may elevate the therapeutic effect, providing a potential therapeutic avenue to control CRC, especially in patients who are resistant to chemotherapy. Supplementary Material 16. Levine B, Kroemer G. Autophagy in the Pathogenesis of Disease. Cell. 2008; 132: 27-42. Supplementary figures and tables 1, 3-6. http://www.thno.org/v10p1981s1.pdf Supplementary table 2. 17. Lin KC, Lin MW, Hsu MN, Yu-Chen G, Chao YC, Tuan HY, et al. Graphene oxide sensitizes cancer cells to chemotherapeutics by inducing early autophagy events, promoting nuclear trafficking and necrosis. Theranostics. 2018; 8: 2477-87. http://www.thno.org/v10p1981s2.xlsx http://www.thno.org/v10p1981s2.xlsx 18. Cai Q, Wang S, Jin L, Weng M, Zhou D, Wang J, et al. Long non-coding RNA GBCDRlnc1 induces chemoresistance of gallbladder cancer cells by activating autophagy. Mol Cancer. 2019; 18: 82. Competing Interests The authors have declared that no competing interest exists. References 2011; 144: 646-74. 9. Liu T, Zhang X, Du L, Wang Y, Liu X, Tian H, et al. Exosome-transmitted miR-128-3p increase chemosensitivity of oxaliplatin-resistant colorectal cancer. Mol Cancer. 2019; 18: 43. References 1. Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester R, Barzi A, et al. Colorectal cancer statistics, 2017. CA Cancer J Clin. 2017; 67: 177-93. 1. Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester R, Barzi A, et al. Colorectal cancer statistics, 2017. CA Cancer J Clin. 2017; 67: 177-93. 1. Siegel RL, Miller KD, Fedewa SA, Ahnen DJ, Meester R, Barzi A, et al. Colorectal cancer statistics, 2017. CA Cancer J Clin. 2017; 67: 177-93. 2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68: 394-424. 2. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68: 394-424. 3. Anderson RL, Balasas T, Callaghan J, Coombes RC, Evans J, Hall JA, et al. A framework for the development of effective anti-metastatic agents. Nat Rev Clin Oncol. 2019; 16: 185-204. 3. Anderson RL, Balasas T, Callaghan J, Coombes RC, Evans J, Hall JA, et al. A framework for the development of effective anti-metastatic agents. Nat Rev Clin Oncol. 2019; 16: 185-204. 4. Cremolini C, Schirripa M, Antoniotti C, Moretto R, Salvatore L, Masi G, et al. First-line chemotherapy for mCRC-a review and evidence-based algorithm. Nat Rev Clin Oncol. 2015; 12: 607-19. 4. Cremolini C, Schirripa M, Antoniotti C, Moretto R, Salvatore L, Masi G, et al. First-line chemotherapy for mCRC-a review and evidence-based algorithm. Nat Rev Clin Oncol. 2015; 12: 607-19. 5. Hewish M, Cunningham D. First-line treatment of advanced colorectal cancer. Lancet. 2011; 377: 2060-2. 5. Hewish M, Cunningham D. First-line treatment of advanced colorectal cancer. Lancet. 2011; 377: 2060-2. 6. Ciombor KK, Wu C, Goldberg RM. Recent therapeutic advances in the treatment of colorectal cancer. Annu Rev Med. 2015; 66: 83-95. 6. Ciombor KK, Wu C, Goldberg RM. Recent therapeutic advances in the treatment of colorectal cancer. Annu Rev Med. 2015; 66: 83-95. 7. Gebert L, MacRae IJ. Regulation of microRNA function in animals. Nat Rev Mol Cell Biol. 2019; 20: 21-37. 7. Gebert L, MacRae IJ. Regulation of microRNA function in animals. Nat Rev Mol Cell Biol. 2019; 20: 21-37. 8. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell. Conclusions 10. Yu T, Guo F, Yu Y, Sun T, Ma D, Han J, et al. Fusobacterium nucleatum Promotes Chemoresistance to Colorectal Cancer by Modulating Autophagy. Cell. 2017; 170: 548-63. In summary, we found that c-Myc repressed the transcription of miR-27b-3p, thus indirectly regulated ATG10 expression. Furthermore, we observed that the c-Myc/miR-27b-3p/ATG10 regulatory axis could upregulate autophagy, leading to chemoresistance in colorectal cancer. Our findings providing a novel marker for sensory evaluation of chemotherapy and a potential therapeutic target to reverse chemoresistance in colorectal cancer. 11. Eastlack SC, Dong S, Ivan C, Alahari SK. Suppression of PDHX by microRNA-27b deregulates cell metabolism and promotes growth in breast cancer. Mol Cancer. 2018; 17: 100. 12. Lee JJ, Drakaki A, Iliopoulos D, Struhl K. MiR-27b targets PPARgamma to inhibit growth, tumor progression and the inflammatory response in neuroblastoma cells. Oncogene. 2012; 31: 3818-25. 13. Matsuyama R, Okuzaki D, Okada M, Oneyama C. MicroRNA‐27b suppresses tumor progression by regulating ARFGEF1 and focal adhesion signaling. Cancer Sci. 2016; 107: 28-35. 14. He L, Luo L, Zhu H, Yang H, Zhang Y, Wu H, et al. FEN1 promotes tumor progression and confers cisplatin resistance in non-small-cell lung cancer. Mol Oncol. 2017; 11: 640-54. 15. Levy JMM, Towers CG, Thorburn A. Targeting autophagy in cancer. Nat Rev Cancer. 2017; 17: 528-42. Theranostics 2020, Vol. 10, Issue 5 25. Hu JL, Wang W, Lan XL, Zeng ZC, Liang YS, Yan YR, et al. CAFs secreted exosomes promote metastasis and chemotherapy resistance by enhancing cell stemness and epithelial-mesenchymal transition in colorectal cancer. Mol Cancer. 2019; 18: 91. 25. Hu JL, Wang W, Lan XL, Zeng ZC, Liang YS, Yan YR, et al. CAFs secreted exosomes promote metastasis and chemotherapy resistance by enhancing cell stemness and epithelial-mesenchymal transition in colorectal cancer. Mol Cancer. 2019; 18: 91. 26. Agarwal V, Bell GW, Nam JW, Bartel DP. Predicting effective microRNA target sites in mammalian mRNAs. Elife. 2015; 4. 27. Miranda KC, Huynh T, Tay Y, Ang YS, Tam WL, Thomson AM, et al. A pattern-based method for the identification of MicroRNA binding sites and their corresponding heteroduplexes. Cell. 2006; 126: 1203-17. 28. Backes C, Kehl T, Stockel D, Fehlmann T, Schneider L, Meese E, et al. miRPathDB: a new dictionary on microRNAs and target pathways. Nucleic Acids Res. 2017; 45: D90-6. 29. Kaiser SE, Qiu Y, Coats JE, Mao K, Klionsky DJ, Schulman BA. Structures of Atg7-Atg3 and Atg7-Atg10 reveal noncanonical mechanisms of E2 recruitment by the autophagy E1. Autophagy. 2013; 9: 778-80. y p gy p gy 30. Chang T, Yu D, Lee Y, Wentzel EA, Arking DE, West KM, et al. Widespread microRNA repression by Myc contributes to tumorigenesis. Nat Genet. 2008; 40: 43-50. 31. O'Donnell KA, Wentzel EA, Zeller KI, Dang CV, Mendell JT. c-Myc-regulated microRNAs modulate E2F1 expression. Nature. 2005; 435: 839-43. 32. Matsuyama R, Okuzaki D, Okada M, Oneyama C. MicroRNA-27b suppresses tumor progression by regulating ARFGEF1 and focal adhesion signaling. Cancer Sci. 2016; 107: 28-35. 33. Feng Q, Wu X, Li F, Ning B, Lu X, Zhang Y, et al. miR-27b inhibits gastric cancer metastasis by targeting NR2F2. Protein Cell. 2017; 8: 114-22. 34. Chen D, Si W, Shen J, Du C, Lou W, Bao C, et al. miR-27b-3p inhibits proliferation and potentially reverses multi-chemoresistance by targeting CBLB/GRB2 in breast cancer cells. Cell Death Dis. 2018; 9: 188. 35. Xu Y, Han Y, Ye B, Zhang Y, Dong J, Zhu S, et al. miR-27b-3p is Involved in Doxorubicin Resistance of Human Anaplastic Thyroid Cancer Cells via Targeting Peroxisome Proliferator-Activated Receptor Gamma. Basic Clin Pharmacol. 2018; 123: 670-7. 36. Fang Q, Chen X, Zhi X. Long Non-Coding RNA (LncRNA) Urothelial Carcinoma Associated 1 (UCA1) Increases Multi-Drug Resistance of Gastric Cancer via Downregulating miR-27b. Med Sci Monit. Acknowledgements p gy 19. Stramucci L, Pranteda A, Stravato A, Amoreo CA, Pennetti A, Diodoro MG, et al. MKK3 sustains cell proliferation and survival through p38DELTA MAPK activation in colorectal cancer. Cell Death Dis. 2019; 10: 842. This work was supported by grants from the National Natural Science Foundation of China (Nos. 8170243, 81772629, 81602158, 81602156, 81702275, 81802363, 81702431, 81702437, 81772843) and the Demonstrative Research Platform of Clinical Evaluation Technology for New Anticancer Drugs (No. 2018ZX09201015). This work was also supported by the Tianjin Science Foundation (Nos. 18JCQNJC81900, 18JCYBJC92000, 18JCYBJC25400, 16PTSYJC00170, 18JCYBJC92900) and the Science & Technology Development Fund of the Tianjin Education Commission for Higher Education 20. Huang T, Wan X, Alvarez AA, James CD, Song X, Yang Y, et al. MIR93 (microRNA -93) regulates tumorigenicity and therapy response of glioblastoma by targeting autophagy. Autophagy. 2019; 15: 1100-11. 21. Chen Q, Zhang F, Wang Y, Liu Z, Sun A, Zen K, et al. The Transcription Factor C-Myc Suppresses MiR-23b and MiR-27b Transcription during Fetal Distress and Increases the Sensitivity of Neurons to Hypoxia-Induced Apoptosis. Plos One. 2015; 10: e120217. 22. Sun W, Wang X, Li J, You C, Lu P, Feng H, et al. MicroRNA-181a promotes angiogenesis in colorectal cancer by targeting SRCIN1 to promote the SRC/VEGF signaling pathway. Cell Death Dis. 2018; 9: 438. 23. Howells LM, Sale S, Sriramareddy SN, Irving GRB, Jones DJL, Ottley CJ, et al. Curcumin ameliorates oxaliplatin‐induced chemoresistance in HCT116 colorectal cancer cells in vitro and in vivo. Int J Cancer. 2011; 129: 476-86. 24. Yin Y, Zhang B, Wang W, Fei B, Quan C, Zhang J, et al. miR-204-5p Inhibits Proliferation and Invasion and Enhances Chemotherapeutic Sensitivity of Colorectal Cancer Cells by Downregulating RAB22A. Clin Cancer Res. 2014; 20: 6187-99. http://www.thno.org http://www.thno.org 1996 Theranostics 2020, Vol. 10, Issue 5 2016; 22: 3506-13. g g 37. Zhu Y, He D, Bo H, Liu Z, Xiao M, Xiang L, et al. The MRVI1-AS1/ATF3 signaling loop sensitizes nasopharyngeal cancer cells to paclitaxel by regulating the Hippo-TAZ pathway. Oncogene. 2019; 38: 6065-81. 38. Wang Y, Shi K, Zhang L, Hu G, Wan J, Tang J, et al. Significantly enhanced tumor cellular and lysosomal hydroxychloroquine delivery by smart liposomes for optimal autophagy inhibition and improved antitumor efficiency with liposomal doxorubicin. Autophagy. 2016; 12: 949-62. y p p gy 39. Ji J, Yu Y, Li ZL, Chen MY, Deng R, Huang X, et al. XIAP Limits Autophagic Degradation of Sox2 and Is A Therapeutic Target in Nasopharyngeal Carcinoma Stem Cells. Theranostics. 2018; 8: 1494-510. 40. Kim J, Fiesel FC, Belmonte KC, Hudec R, Wang WX, Kim C, et al. miR-27a and miR-27b regulate autophagic clearance of damaged mitochondria by targeting PTEN-induced putative kinase 1 (PINK1). Mol Neurodegener. 2016; 11: 55. g 41. Jo YK, Kim SC, Park IJ, Park SJ, Jin DH, Hong SW, et al. Increased expression of ATG10 in colorectal cancer is associated with lymphovascular invasion and lymph node metastasis. Plos One. 2012; 7: e52705. 42. Xie K, Liang C, Li Q, Yan C, Wang C, Gu Y, et al. Role of ATG10 expression quantitative trait loci in non-small cell lung cancer survival. Int J Cancer. 2016; 139: 1564-73. 43. Castell A, Larsson LG. Targeting MYC Translation in Colorectal Cancer. Cancer Discov. 2015; 5: 701-3. 44. Ozsolak F, Poling LL, Wang Z, Liu H, Liu XS, Roeder RG, et al. Chromatin structure analyses identify miRNA promoters. Gene Dev. 2008; 22: 3172-83. 45. Sun J, Cai X, Yung MM, Zhou W, Li J, Zhang Y, et al. miR-137 mediates the functional link between c-Myc and EZH2 that regulates cisplatin resistance in ovarian cancer. Oncogene. 2019; 38: 564-80. g 46. Capizzi M, Strappazzon F, Cianfanelli V, Papaleo E, Cecconi F. MIR7-3HG, a MYC-dependent modulator of cell proliferation, inhibits autophagy by a regulatory loop involving AMBRA1. Autophagy. 2017; 13: 554-66. 47. Liu Z, Zeng W, Wang S, Zhao X, Guo Y, Yu P, et al. A potential role for the Hippo pathway protein, YAP, in controlling proliferation, cell cycle progression, and autophagy in BCPAP and KI thyroid papillary carcinoma cells. Am J Transl Res. 2017; 9: 3212-23. http://www.thno.org http://www.thno.org
https://openalex.org/W4324283708	https://storage.googleapis.com/jnl-vt-j-jvs-files/journals/1/articles/364/641066e20e3f8.pdf	English	null	Leg	Journal of veterans studies	2,023	cc-by	603	VOICE Leg K. GORDON TIDBALL VOICE Leg K. GORDON TIDBALL Leg Leg K. GORDON TIDBALL CORRESPONDING AUTHOR: CORRESPONDING AUTHOR: K. Gordon Tidball Cornell University, US kgtidball@cornell.edu This poem is written by a servicemember who is a Gulf War era veteran, served as both enlisted and officer in the US Army National Guard, and after a break in service due to an injury, continues service in a State Defense Force. K. GORDON TIDBALL KEYWORDS: poetry; poem; patriotism; survivor’s guilt; military service TO CITE THIS ARTICLE: Tidball, K. G. (2023). Leg. Journal of Veterans Studies, 9(2), pp. 11–13. DOI: https://doi. org/10.21061/jvs.v9i2.364 TO CITE THIS ARTICLE: Tidball, K. G. (2023). Leg. Journal of Veterans Studies, 9(2), pp. 11–13. DOI: https://doi. org/10.21061/jvs.v9i2.364 12 Tidball Journal of Veterans Studies DOI: 10.21061/jvs.v9i2.364 My clay mug is warm hand-fired artisan artifact announcing the contented contents pretense of a middle class daze. My clay mug is warm hand-fired artisan artifact announcing the contented contents pretense of a middle class daze. …but in my ruck is training and a trust and a portrait of a long lost Leg.* * A “Leg” is a slang term for a non-airborne qualified soldier. * A “Leg” is a slang term for a non-airborne qualified soldier. ARTIST STATEMENT I am a Gulf War era veteran. I served as both enlisted and officer in the US Army National Guard, and after a break in service due to an injury, I continue service in a State Defense Force. I am also an academic working with veterans and military families. I have taken great solace in the arts since my teens, and have been writing poetry and music for over 30 years. I am also a dabbler in and great supporter of the visual arts. This poem was inspired by a recent observation of ROTC (Reserve Officers’ Training Corps) cadets doing PT (physical training) and my reflection upon the more than 30 years that have passed since I raised my right hand in service to my country. It reflects deep tensions among a trio of perspectives. The first being a deep-seated patriotism and sense of service frustrated by feelings of survivor’s guilt and failing to meet personal potential. The second a broad sense of disillusionment with the outcome of post-9/11 wars and the attendant feelings of pessimism and discouragement regarding the manipulation and misappropriation by politicians of selfless service, honor, duty, and valor. The third perspective is that of the long view, of reflecting upon motivations for service today, and of how we can encourage our children to serve, given the first two perspectives, or if we can. Guilt is sparse parsed like powders of saccharin of simulacrum substitutes for mourning the minefield of patriotism paternalism of patsies and privates and platoon after platoon of pretenders in a drama not their own. COMPETING INTERESTS The author has no competing interests to declare. The author has no competing interests to declare. TO CITE THIS ARTICLE: Tidball, K. G. (2023). Leg. Journal of Veterans Studies, 9(2), pp. 11–13. DOI: https://doi.org/10.21061/jvs.v9i2.364 Tidball, K. G. (2023). Leg. Journal of Veterans Studies, 9(2), pp. 11–13. DOI: https://doi.org/10.21061/jvs.v9i2.364 Submitted: 16 June 2022 Accepted: 21 August 2022 Published: 14 March 2023 AUTHOR AFFILIATION K. Gordon Tidball orcid.org/0000-0002-9856-8731 Cornell University, US Tidball Journal of Veterans Studies DOI: 10.21061/jvs.v9i2.364 13 COPYRIGHT: © 2023 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. Journal of Veterans Studies is a peer-reviewed open access journal published by VT Publishing.
https://openalex.org/W4380354571	https://journals.plos.org/digitalhealth/article/file?id=10.1371/journal.pdig.0000235&type=printable	English	null	Improving outcomes for children with malaria, diarrhoea and pneumonia in Mozambique: A cluster randomised controlled trial of the inSCALE technology innovation	PLOS digital health	2,023	cc-by	12,984	PLOS DIGITAL HEALTH PLOS DIGITAL HEALTH RESEARCH ARTICLE Editor: Dylan A. Mordaunt, Flinders University, AUSTRALIA Editor: Dylan A. Mordaunt, Flinders University, AUSTRALIA Received: July 12, 2022 Accepted: March 20, 2023 Published: June 12, 2023 Copyright: © 2023 Soremekun 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: July 12, 2022 Accepted: March 20, 2023 Published: June 12, 2023 Background The majority of post-neonatal deaths in children under 5 are due to malaria, diarrhoea and pneumonia (MDP). The WHO recommends integrated community case management (iCCM) of these conditions using community-based health workers (CHW). However iCCM programmes have suffered from poor implementation and mixed outcomes. We designed and evaluated a technology-based (mHealth) intervention package ‘inSCALE’ (Innovations At Scale For Community Access and Lasting Effects) to support iCCM programmes and increase appropriate treatment coverage for children with MDP. Data Availability Statement: In line with the original consent criteria for the trial, the data for the inSCALE project can be available upon reasonable request from the data manager of the public repository LSHTM Data Compass under DOI https://doi.org/10.17037/DATA.00002559. Researchers wishing to access study data must perform the following actions: 1. Review the data codebook 2. Complete and submit a data request form (available at 10.17037/DATA.00002559) that Data Availability Statement: In line with the original consent criteria for the trial, the data for the inSCALE project can be available upon reasonable request from the data manager of the public repository LSHTM Data Compass under DOI https://doi.org/10.17037/DATA.00002559. Abstract Copyright: © 2023 Soremekun 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. Improving outcomes for children with malaria, diarrhoea and pneumonia in Mozambique: A cluster randomised controlled trial of the inSCALE technology innovation Seyi SoremekunID1☯, Karin Ka¨llander2,3,4☯, Raghu Lingam5, Ana-Cristina Castel Branco6, Neha BaturaID7, Daniel Ll Strachan8, Abel Muiambo6, Nelson Salomao6, Juliao Condoane6, Fenias Benhane6, Frida Kasteng3, Anna Vassall9, Zelee Hill7, Guus ten Asbroek10, Sylvia Meek2†, James Tibenderana2‡, Betty Kirkwood11‡ a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Infection Biology, London School of Hygiene & Tropical Medicine, Keppel Street, London, United Kingdom, 2 Malaria Consortium, The Green House, 244–254 Cambridge Heath Road, London, United Kingdom, 3 Department of Global Public Health, Karolinska Institutet, Stockholm, Sweden, 4 UNICEF Programme Division, Health Section, New York, New York State, United States of America, 5 Population Child Health Research Group, School of Women’s and Children’s Health, University of New South Wales, Australia, 6 Malaria Consortium, Rua Joseph Ki-Zerbo 191, PO Box 3655, Coop, Maputo, Mozambique, 7 Institute for Global Health, University College London, 30 Guilford Street, London, United Kingdom, 8 The Nossal Institute for Global Health, Melbourne School of Population and Global Health, The University of Melbourne Victoria, Australia, 9 Department of Global Health and Development, London School of Hygiene & Tropical Medicine, Keppel Street, London, United Kingdom, 10 Department of Global Health, Amsterdam University Medical Centres, Meibergdreef 9 1105 AZ Amsterdam, The Netherlands and Amsterdam Institute for Global Health and Development, Paasheuvelweg 25, 1105 BP Amsterdam, The Netherlands, 11 Department of Population Health, London School of Hygiene & Tropical Medicine, Keppel Street, London, WC1E 7HT United Kingdom OPEN ACCESS Citation: Soremekun S, Ka¨llander K, Lingam R, Branco A-CC, Batura N, Strachan DL, et al. (2023) Improving outcomes for children with malaria, diarrhoea and pneumonia in Mozambique: A cluster randomised controlled trial of the inSCALE technology innovation. PLOS Digit Health 2(6): e0000235. https://doi.org/10.1371/journal. pdig.0000235 11 Department of Population Health, London School of Hygiene & Tropical Medicine, Keppel Street, London, WC1E 7HT, United Kingdom ☯These authors contributed equally to this work. † Deceased. ‡ JT and BK also contributed equally to this work. seyi.soremekun@lshtm.ac.uk ☯These authors contributed equally to this work. † Deceased. ‡ JT and BK also contributed equally to this work. * seyi.soremekun@lshtm.ac.uk ☯These authors contributed equally to this work. Editor: Dylan A. Mordaunt, Flinders University, AUSTRALIA Methods Researchers wishing to access study data must perform the following actions: 1. Review the data codebook 2. Complete and submit a data request form (available at 10.17037/DATA.00002559) that This superiority cluster randomised controlled trial allocated all 12 districts in Inhambane Province in Mozambique to receive iCCM only (control) or iCCM plus the inSCALE PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 1 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique technology intervention. Population cross-sectional surveys were conducted at baseline and after 18 months of intervention implementation in approximately 500 eligible house- holds in randomly selected communities in all districts including at least one child less than 60 months of age where the main caregiver was available to assess the impact of the inter- vention on the primary outcome, the coverage of appropriate treatment for malaria, diar- rhoea and pneumonia in children 2-59months of age. Secondary outcomes included the proportion of sick children who were taken to the CHW for treatment, validated tool-based CHW motivation and performance scores, prevalence of cases of illness, and a range of secondary household and health worker level outcomes. All statistical models accounted for the clustered study design and variables used to constrain the randomisation. A meta-analy- sis of the estimated pooled impact of the technology intervention was conducted including results from a sister trial (inSCALE-Uganda). outlines the purpose of their research, data variables/measurements that they wish to access, and other information necessary to support their request. Queries about the data transfer agreement/access process can be sent to researchdatamanagement@lshtm ac uk outlines the purpose of their research, data variables/measurements that they wish to access, and other information necessary to support their request. Queries about the data transfer agreement/access process can be sent to researchdatamanagement@lshtm ac uk Funding: The inSCALE study was supported by grants to JT and BK from The Bill and Melinda Gates Foundation, grant number OPP1002407. The BMGF played no role in the design, analysis, decision to publish or preparation of this manuscript. Competing interests: The authors have declared that no competing interests exist. Interpretation The inSCALE intervention led to an improvement in appropriate treatment of common child- hood illnesses when delivered at scale in Mozambique. The programme will be rolled out by the ministry of health to the entire national CHW and primary care network in 2022–2023. This study highlights the potential value of a technology intervention aimed at strengthening iCCM systems to address the largest causes of childhood morbidity and mortality in sub- Saharan Africa. Findings The study included 2740 eligible children in control arm districts and 2863 children in inter- vention districts. After 18 months of intervention implementation 68% (69/101) CHWs still had a working inSCALE smartphone and app and 45% (44/101) had uploaded at least one report to their supervising health facility in the last 4 weeks. Coverage of the appropriate treatment of cases of MDP increased by 26% in the intervention arm (adjusted RR 1.26 95% CI 1.12–1.42, p<0.001). The rate of care seeking to the iCCM-trained community health worker increased in the intervention arm (14.4% vs 15.9% in control and intervention arms respectively) but fell short of the significance threshold (adjusted RR 1.63, 95% CI 0.93–2.85, p = 0.085). The prevalence of cases of MDP was 53.5% (1467) and 43.7% (1251) in the control and intervention arms respectively (risk ratio 0.82, 95% CI 0.78–0.87, p<0.001). CHW motivation and knowledge scores did not differ between intervention arms. Across two country trials, the estimated pooled effect of the inSCALE intervention on cover- age of appropriate treatment for MDP was RR 1.15 (95% CI 1.08–1.24, p <0.001). Introduction Globally more than 5 million mostly preventable deaths occur in children under 5 years of age every year [1]. The vast majority of post neonatal deaths in this group are due to pneumonia, diarrhoea, malaria or malnutrition [2]. It is acknowledged that urgent work is required to address the causes and more widely, the systems within which such deaths occur. However progress has been uneven; under 5 mortality rates in sub Saharan Africa today remain similar to or higher than 1990 mortality rates in most high income countries with an average rate of 78 deaths per 1000 live births across the continent in 2018 [2]. Lack of access to timely and appropriate clinical management of sick children is a key factor driving poor health outcomes [3,4] prompting a World Health Organisation/UNICEF joint statement in 2012 advocating for integrated community case management (iCCM) of malaria, diarrhoea and pneumonia deliv- ered by community-based health workers (CHW), who would be trained to diagnose and treat key childhood conditions in areas with poor access to facility-based services [3]. In the decade following this statement, iCCM programmes have been introduced into more than 50 low- and middle-income countries (LMICs) [5–7]. Whilst a recent multi-country modelling study in DRC, Malawi, Niger, and Nigeria [8] estimated that iCCM may have been responsible for averting nearly 5000 deaths per year in these countries combined due to malaria, diarrhoea and pneumonia, this estimate varied considerably between countries, and was in contrast to a Cochrane systematic review which found no clear impacts of iCCM on morbidity and mortal- ity [9]. Both studies reported poor or uneven implementation of iCCM programmes and a need for further studies that addressed structural deficiencies in health systems and processes that hamper success and scale-up. Central to the challenge in implementation of iCCM has been the lack of sufficient support for CHW networks, high rates of CHW attrition, low rates of reported motivation and poor quality of disease management practices [10]. The WHO calls for “innovative and tailored approaches” to strengthening child health services [1], yet there are few programmes or even research studies which have explicitly addressed structural barriers in the context of scaling up iCCM effectively. Author summary The inSCALE cluster-randomised trial in Mozambique was part of a $10million project funded by the Bill and Melinda Gates Foundation to design and test innovative primary care interventions to improve health outcomes for children with malaria, diarrhoea and pneumonia (MDP), which together are the largest killers of children aged <5yrs. The study aimed to strengthen the primary health care system with a focus on community health workers, representing the most accessible level of care for many underserved 2 / 21 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique populations. We designed a technology-based intervention delivered using low-end smartphones. This intervention was based on mHealth principles and included electronic WHO algorithms with basic AI functions for community health workers (CHWs) to guide correct diagnosis and treatment of MDP, provided personalised feedback to health workers, and alerts to supervising health facilities on stock outs, data tracking and CHW competencies. The study was implemented within the entire province of Inhambane, and districts were randomly assigned to the intervention or to continue with standard care (control). Compared to control districts, we observed significant reductions in the preva- lences of MDP in children under 5 years (reductions of 20% for malaria, 34% for pneumo- nia, and 45% for diarrhoea) and an increase in appropriate treatment of any cases of MDP by 26% (of all cases MDP) and 40% (of all children) in the intervention districts. As a result of this trial, the government of Mozambique incorporated the inSCALE interven- tion into its policy for child health services, and is in the process of scaling up the pro- gramme to all 8000+ CHWs across the country (2022–2023). PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 Results In total 9231 households in the 12 districts were assessed for eligibility and of these, 495 were empty or no one was home, and 4483 did not contain children under 5 years of age. Overall, the main caretaker was available in 92% (3920) of houses with children under 5 years of age. At the endline survey, a total of 2718 children had had symptoms of suspected malaria, diar- rhoea or pneumonia (MDP) in the past month (the impact analysis sample), of which 1467 were in the control arm and 1251 in the intervention arm (Fig 1 flow chart). Household char- acteristics of sick children were fairly well balanced between arms with small variations in some parameters–Table 1. The restricted randomisation method resulted in good balance between intervention arms for balance variables as described in the protocol paper [15], which were care seeking rates, CHW motivation, and cost of seeking care (Table 2). Differences for all variables was either less than 5% (percentages), or less than 0.1 (means). Introduction Digital technologies, and specifically mHealth (the use of mobile technologies in medical and public health practice [11]) is an area of growing interest for the delivery of health services in sub-Saharan Africa [12,13] particularly given the rapid expansion of mobile phone networks and usage in this setting [14]. We designed a systems-strengthening package of programmes 3 / 21 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique collectively called”inSCALE” (interventions at scale for community access and lasting effects). inSCALE was a multi-country study of innovative interventions implemented by the Malaria Consortium in partnership with the governments of Mozambique and Uganda and supported by international funders (the Bill and Melinda Gates Foundation, UNICEF, and the Canadian international Development Agency now Global Affairs Canada (GAC)) and co-designed with academic partners (The London School of Hygiene and Tropical Medicine and University College, London). Reported in this paper are the findings of the inSCALE technology interven- tion, a disease management support package developed following extensive formative research and building on the study conceptual framework [15]. The package was primarily delivered on Samsung Galaxy smartphones (“the inSCALE app”) that included electronic clinical decision algorithms, personalised reports, messaging, and feedback to CHWs and health facility staff. The Mozambique site tested the inSCALE technology intervention only; the Uganda site tested both the technology intervention and a community-based intervention described elsewhere [16]. The technology interventions in both countries sought to specifically address primary health system challenges that could lead to improved management of key childhood illnesses through first improving CHW performance, motivation and reducing attrition. A detailed description of the development of the inSCALE technology intervention, rationale and con- ceptual framework is provided elsewhere [15]. The primary aim of the trial was therefore to assess the impact of the inSCALE technology intervention on the coverage of the appropriate treatment of childhood malaria, diarrhoea and pneumonia. This paper reports on the impacts of the technology intervention in Mozambique to accompany the separate impact analysis for the Uganda inSCALE trial [16] and conducts a meta-analysis of pooled impact of the technol- ogy intervention across its sites in Uganda and Mozambique. Impact of a technology intervention on child health outcomes in Mozambique Impact of a technology intervention on child health outcomes in Mozambique Fig 1. Flow diagram of participant progression through trial phases. Final analysis sample: children aged 2 months to 5 years with suspected malaria, diarrhoea or suspected pneumonia (MDP). * Maxixe and Inhambane, the economic and provincial capitals were excluded from the study (do not implement iCCM). https://doi org/10 1371/journal pdig 0000235 g001 Fig 1. Flow diagram of participant progression through trial phases. Final analysis sample: children aged 2 months to 5 years with suspected malaria, diarrhoea or suspected pneumonia (MDP). * Maxixe and Inhambane, the economic and provincial capitals were excluded from the study (do not implement iCCM). https://doi.org/10.1371/journal.pdig.0000235.g001 https://doi.org/10.1371/journal.pdig.0000235.g001 differences in care seeking rates to other providers or in the prevalence of children for whom no care was sought (Table 5). Appropriate treatment Baseline levels of appropriate treatment were similar for the combined MDP endpoint but var- ied by provider and for individual conditions between intervention arms (Table 6). At endline there was an 26% increase in appropriate treatment of cases of MDP (the primary outcome (Table 7) in the inSCALE arm compared to the control arm (95% confidence interval: 12%- 42%, p<0.001). This rose to 30% when only episodes treated with recommended first line treatments were defined as appropriately treated (Table 7 95% CI: 12%-52%, p = 0.001). A breakdown of appropriate treatment by provider suggests this impact was driven by significant improvements in appropriate treatment coverage in public health facilities (Table 7) which were the first port of call for caregivers for more than 60% of sick children in both arms (Table 5), and in the appropriate treatment of diarrhoea, where a 76% increase (95% CI 20%- 256% p = 0.003) in the use of oral rehydration salts was observed in the intervention arm (Table 8). Note it was not possible to estimate a confidence interval for use of ORS plus zinc due to small cell sizes. Additional improvements came in the use of the recommended first line antibiotic for pneumonia (50% increase in prescribing of amoxicillin in the intervention arm 95% CI: 3%-217%). A whole-child analysis (Table C in S1 Text) estimated an improve- ment of 40% in the proportion of children appropriately treated for all MDP condition(s) reported in the last 4 weeks in the intervention arm (95% CI: 9% - 79% p = 0.009). Intervention coverage, sickness and care seeking By the end of the 18-month implementation period, 68% of CHWs reported having a working inSCALE phone and app, and 45% had sent a report via the app in the last 4 weeks (Table 3). Whilst overall prevalence of MDP was similar between arms at baseline, there were significant reductions in the prevalence of suspected malaria, diarrhoea and pneumonia (reductions rang- ing from 18% to 45%), and a 16% increase in the prevalence of diagnostic testing for malaria in the intervention arm at endline (Table 4). Care seeking to the CHW as the first port of call was 14.4% in the control arm and 15.9% in the intervention arm. After adjusting for the restricted randomisation and clustered design this represented a 63% increase in care-seeking to the CHW, a result that just fell short of significance (Table 5, p = 0.085). There were no significant PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 4 / 21 PLOS DIGITAL HEALTH CHW motivation, identification, clinical knowledge, attrition, and stockouts The mean motivation and the mean social identification scores were relatively high and did not differ between arms (difference in motivation factors score: -0.03 95% CI -0.57, 0.51; 5 / 21 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique Table 1. Characteristics of the inSCALE Mozambique study sample. Characteristic Control % (n) Technology % (n) Eligible children N = 2740 N = 2863 % children with MDP 53.5% (1467) 43.7% (1251) % children with suspected malaria* 48.5% (1300) 39.8% (1118) % children with confirmed malaria* 24.6% (661) 21.8% (612) % children with diarrhoea 7.2% (198) 4.2% (121) % children with suspected pneumonia 16.2% (443) 10.6% (304) Children with MDP N = 1467 N = 1251 % Respondent is mother of sick child 81% (1181) 83% (1036) % children are boys 50% (733) 50% (629) Age of mother 12–19 10% (144) 8% (104) 20–29 38% (552) 37% (467) 30–39 28% (413) 30% (373) 40–49 12% (181) 14% (175) 50+ 8% (113) 6% (76) Not known 4% (64) 4% (56) Highest education level completed (respondent) None 31% (453) 32% (400) Incomplete primary 48% (706) 45% (557) Primary or higher 21% (308) 24% (294) Mother tongue Bitonga 17% (246) 6% (81) Chitsua 54% (793) 72% (900) Chichopi 17% (254) 19% (242) Other 12% (174) 2% (28) Marital status Married 2% (34) 3% (41) Living with partner (not married) 78% (1151) 74% (929) Single/divorced/widowed 19% (282) 22% (281) % Main occupation is farming/fishing 75.9% (1113) 64.0% (801) Denominator is the 5490 children aged 4months– 5 years Denominator is the 5603 children aged 2m – 5 year Table 1. Characteristics of the inSCALE Mozambique study sample. difference in identification factor score: 0.01 95% CI -0.42, 0.43). CHW attrition rates were approximately 18%-19% in both intervention arms with no detected difference (Table 9). Overall scores for CHW clinical knowledge of the signs and management of MDP, also did not differ significantly between arms (Table 9). A breakdown of CHW knowledge scores by condition suggests a trend of improved knowledge in technology arm CHWs particularly in diagnosing pneumonia and diarrhoea—though none reached the threshold for statistical sig- nificance and may be chance findings (Table F in S1 Text). CHW motivation, identification, clinical knowledge, attrition, and stockouts There were no between-arm differ- ences in the rate of stockouts for key MDP treatments in the previous 3 months, though overall rates of stockouts were fairly high at >50% (Table 9). PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique PLOS DIGITAL HEALTH Table 2. Key indicators used to balance arms at baseline. Variables included in the restricted randomisation Control arm 662 children with MDP 128 CHWs Technology 658 children with MDP 128 CHWs Difference Mean (log10) cost of care seeking for children with MDP (SD) 1.11 (0.09) 1.14 (0.14) 0.03 Cluster mean % care seeking to an CHW for children with MDP (SD) 22.5% (23.03) 22.8% (16.92) 0.26 Cluster mean % care seeking to a public facility for children with MDP (SD) 50.96% (21.63) 48.79% (14.80) 2.17 Cluster mean CHW motivation score (SD) 2.29 (0.46) 2.38 (0.50) 0.09 Outcomes are presented as mean percentages or mean costs across all clusters in each arm Table 2. Key indicators used to balance arms at baseline. Variables included in the restricted randomisation Control arm 662 children with MDP 128 CHWs Technology 658 children with MDP 128 CHWs Difference Mean (log10) cost of care seeking for children with MDP (SD) 1.11 (0.09) 1.14 (0.14) 0.03 Cluster mean % care seeking to an CHW for children with MDP (SD) 22.5% (23.03) 22.8% (16.92) 0.26 Cluster mean % care seeking to a public facility for children with MDP (SD) 50.96% (21.63) 48.79% (14.80) 2.17 Cluster mean CHW motivation score (SD) 2.29 (0.46) 2.38 (0.50) 0.09 Outcomes are presented as mean percentages or mean costs across all clusters in each arm https://doi.org/10.1371/journal.pdig.0000235.t002 Table 3. Exposure to intervention components recorded during the endline survey. Intervention component Technology CHWs N = 104 % CHWs had a working phone and inSCALE app at endline (n) 68% (69) % CHWs submitted at least one inSCALE report through their phone in the last 4 weeks 45.2% (44)* data missing for 3 CHWs https://doi.org/10.1371/journal.pdig.0000235.t003 Table 2. Key indicators used to balance arms at baseline. Variables included in the restricted randomisation Table 3. Exposure to intervention components recorded during the endline survey. Intervention component Technology CHWs N = 104 % CHWs had a working phone and inSCALE app at endline (n) 68% (69)* % CHWs submitted at least one inSCALE report through their phone in the last 4 weeks 45.2% (44)* data missing for 3 CHWs https://doi.org/10.1371/journal.pdig.0000235.t003 Table 4. Prevalence of suspected malaria, diarrhoea and pneumonia (MDP) and blood tests for malaria in children under 5 years at baseline and endline. PLOS DIGITAL HEALTH Prevalence of Baseline (reported prevalence in last 2 weeks) Endline (reported prevalence in last 4 weeks) Impact of inSCALE at endline Risk ratio inSCALE/control RR: 95% CI; pᵜ Control arm Technology arm Control arm Technology arm Any of MDP 29.3% (662/2257) 30.4% (658/2165) 53.5% (1467/2740) 43.7% (1251/2863) 0.82 (0.78–0.87) p<0.001 Suspected malaria 25.9% (562/2171) 26.7% (556/2084) 48.5% (1300/2682) 39.8% (1118/2808) 0.80 (0.72–0.89) p<0.001 Blood test for malaria 31.7% (200/631) 29.5% (181/613) 59.7% (937/1570) 62.3% (831/1335) 1.16 (1.06–1.28) p = 0.002 Confirmed malaria* 65.5% (131/200) 68.5% (124/181) 70.5% (661/937) 73.7% (612/831) 1.04 (0.92–1.18) p = 0.514 Diarrhoea 5.1% (116/2257) 5.3% (114/2165) 7.2% (198/2740) 4.2% (121/2863) 0.55 (0.42–0.71) p<0.001 Pneumonia 9.9% (223) 11.8% (256/2165) 16.2% (443/2740) 10.6% (304/2863) 0.66 (0.44–0.99) p = 0.045 Prevalence (n/N) calculated as total eligible children 2 months-59 months of age (N) with condition in question (n), with the exception of fever and malaria where age range is 4 months-59 months in line with treatment guidelines. Prevalence (n/N) calculated as total eligible children 2 months-59 months of age (N) with condition in question (n), with the exception of fever and malaria where age range is 4 months-59 months in line with treatment guidelines. Fevers, excludes fevers with a negative blood test for malaria. denominator includes all children reporting fever in the last two (baseline) or 4 (endline) weeks: the exposure period was extended in the endline survey following recommendations for increasing accuracy of caregiver-reported outcomes published in the interim [17] percentage of all blood tested fevers that were positive. ᵜAdjusted for i) parameters used to balance arms at baseline and ii) the baseline prevalence of prevalence g denominator includes all children reporting fever in the last two (baseline) or 4 (endline) weeks: the exposure period was extended in the endline survey following recommendations for increasing accuracy of caregiver-reported outcomes published in the interim [17] *percentage of all blood tested fevers that were positive. ᵜAdjusted for i) parameters used to balance arms at baseline and ii) the baseline prevalence of prevalence parameter Table 5. Care seeking choices for children with MDP at endline. Sensitivity analyses and robustness tests Sensitivity tests were conducted with two alternative definitions of appropriate treatment: i) only including children with a positive malaria diagnostic test, and ii) including quinine as an PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 6 / 21 mbination therapy for malaria, ORS for diarrhoea, and amoxicillin for pneumonia adjusted for baseline levels of appropriate treatment for the condition(s) in question, and for variables used to balance arms at baseline (rates of facility and CHW care seeking, cost of care seeking, and CHW motivation score) bed disease; n = total episodes where the correct drugs were received PLOS DIGITAL HEALTH Outcome % of children (n) Control (N = 1467) Technology (N = 1251) Risk ratio tech/ control RR: 95% CI p % seeking care at a CHW (first point of call) 14.4% (211) 15.9% (199) 1.63 (0.93–2.85) 0.085 % seeking care at a CHW (at any point) 14.7% (215) 16.2% (203) 1.61 (0.91–2.85) 0.104 % Not seeking care outside the home 18.3% (269) 20.6% (251) 0.98 (0.74–1.30) 0.898 % seeking care at a public facility (at any point) 67.4% (988) 63.7% (797) 1.11 (0.93–1.33) 0.253 % seeking care in the private sector (at any point—private facility, pharmacy, shop, herbalist etc) 3.6% (53) 2.4% (30) 0.94 (0.51–1.71) 0.839 https://doi.org/10.1371/journal.pdig.0000235.t005 Table 5. Care seeking choices for children with MDP at endline. Outcome % of children (n) Table 5. Care seeking choices for children with MDP at endline. 7 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique Table 6. Primary Outcome at baseline: Percentage of episodes of MDP that were appropriately treated. Outcome Control Technology % (n/N) % (n/N) Total children in baseline sample 2257 2165 % children with MDP** 29.3% (662/ 2257) 30.4% (658/ 2165) Appropriate treatment Any episode of MDP 33.1% (298/ 901) 28.2% (261/ 926) Suspected malaria 31.3% (176/ 562) 28.4% (158/ 556) Confirmed malaria 90.1% (118/ 131) 88.7% (110/ 124) Diarrhoea (ORS) 56.0% (65/ 116) 44.7% (51/114) Diarrhoea (ORS and zinc) 2.6% (3/116) 2.6% (3/114) Pneumonia (any antibiotic) 25.6% (57/ 223) 20.3% (52/256) Pneumonia (amoxicillin) 10.3% (23/ 223) 10.6% (27/256) Any episode of MDP at the CHW (first care seeking location) 35.7% (71/ 199) 32.0% (70/219) Any episode of MDP at the Public Facility (first care seeking location) 46.8% (214/ 457) 38.1% (171/ 449) Any episode of MDP at the Private sector (first care seeking location, includes private facility, pharmacy, shop, herbalist) 18.8% (3/16) 19.2% (5/26) Any episode of MDP—no care seeking outside of the home 10.1% (22/ 209) 6.2% (14/226) N = total episodes of the described disease; n = total episodes where the correct drugs were received. MDP: denominator includes all children with suspected or confirmed malaria, diarrhoea or pneumonia https://doi.org/10.1371/journal.pdig.0000235.t006 N = total episodes of the described disease; n = total episodes where the correct drugs were received. PLOS DIGITAL HEALTH This approach estimated similar significant improve- ments in appropriate treatment as the random effects model with the exception of the increase in care seeking to CHWs which did not reach significance. PLOS DIGITAL HEALTH Outcome Control arm Technology arm Freq (%) Freq (%) risk ratio versus control (95% CI) p Appropriate treatment by condition Suspected malaria 54.4% (707/1300) 56.6% (633/1118) 1.08 (0.97–1.20) 0.150 Confirmed malaria 96.4% (637/661) 96.2% (589/612) 1.02 (0.99–1.04) 0.221 Diarrhoea (ORS) 29.3% (58/198) 43.8% (53/121) 1.76 (1.20–2.56) 0.003 Diarrhoea (ORS + zinc) 0.0% (0/198) 1.7% (2/121) - - Suspected pneumonia (any antibiotic) 36.8% (163/443) 31.6% (96/304) 0.80 (0.60–1.08) 0.142 Suspected pneumonia (amoxycillin) 12.2% (54/443) 19.1% (58/304) 1.50 (1.03–2.17) 0.033 https://doi.org/10.1371/journal.pdig.0000235.t008 Table 8. Impact of the inSCALE intervention on appropriate treatment by condition. Table 9. Community health worker (CHW) outcomes: Stockouts, attrition, motivation and knowledge in control and intervention arm clusters. Outcome Control arm (N = 103) Technology arm (N = 104) Effect size (95% CI) p Mean (sd) Mean (sd) % CHWs experienced a stockout of any MDP drug in last 3 months* 52.4% (54) 56.7% (59) RR: 1.17 (0.92–1.49 0.207 Mean CHW motivation score 64.6 (5.9) 66.4 (5.5) RD: +0.44 (-4.96, 5.85) 0.872 Mean CHW social identification score 17.4 (1.7) 17.8 (1.8) RD: -0.03 (-1.52, 1.48) 0.972 Mean CHW knowledge score 19.7 (4.3) 19.8 (4.4) RD: +0.97 (-2.01, 3.94) 0.524 Proportion CHW attrition (%) 18.4% (13.6) 18.5% (10.0) RD: +1.31 (-18.66, 21.30) 0.878 * Stockouts of any of artemisinin combination therapy, amoxycillin, or ORS. RR- risk ratio; RD- risk difference. Minimum and maximum: social identity scores 4, 20; motivation scores 25, 77; knowledge scores 0, 56. tockouts, attrition, motivation and knowledge in control and intervention arm clusters. ealth worker (CHW) outcomes: Stockouts, attrition, motivation and knowledge in control and intervention arm clusters. Table 9. Community health worker (CHW) outcomes: Stockouts, attrition, motivation and knowledge in control and in * Stockouts of any of artemisinin combination therapy, amoxycillin, or ORS. RR- risk ratio; RD- risk difference. Minimum and maximum: social identity scores 4, 20; motivation scores 25, 77; knowledge scores 0, 56. nation therapy, amoxycillin, or ORS. RR- risk ratio; RD- risk difference. Minimum and maximum: social identity scores 4, 20 es 0, 56. and ii)). We performed robustness tests of the key primary and secondary impact outcomes appropriate treatment, care seeking, CHW motivation, social identity, clinical knowledge, stockouts, and attrition by repeating the analyses using a comparison of cluster-level mean outcomes (Tables D1-E2 in S1 Text). PLOS DIGITAL HEALTH *MDP: denominator includes all children with suspected or confirmed malaria, diarrhoea or pneumonia https://doi.org/10.1371/journal.pdig.0000235.t006 N = total episodes of the described disease; n = total episodes where the correct drugs were received. *MDP: denominator includes all children with suspected or confirmed malaria, diarrhoea or pneumonia N = total episodes of the described disease; n = total episodes where the correct drugs were received. appropriate treatment for malaria to account for its use as a second line treatment or during in-patient care (Table B in S1 Text). Both showed a trend of improved coverage of overall appropriate treatment (20% an 14% improvement, p values 0.006 and 0.073 respectively for i) Table 7. Appropriate treatment of MDP at endline: Appropriate treatment of episodes suspected malaria, diarrhoea or suspected pneumonia (MDP). Appropriate treatment Control Technology Technology v Control** % n/N* % n/N* RR (95% CI) p Any episode of MDP 47.8% 928/1941 50.7% 782/1543 1.26 (1.12–1.42) <0.001 Any episode of MDP (first line treatments only)*** 42.2% 819/1941 48.2% 744/1543 1.30 (1.12–1.52) 0.001 Appropriate treatment by first care seeking location (any episode of MDP) CHW 49.5% 146/ 295 61.9% 148/239 1.04 (0.86–1.25) 0.675 Public Facility 58.0% 736/1269 62.2% 594/955 1.26 (1.07–1.48) 0.004 Private sector (private facility, pharmacy, shop, herbalist) 29.4% 10/34 21.7% 5/23 1.66 (0.52–5.34) 0.392 No care seeking outside of the home 10.5% 36/343 10.7% 35/326 1.23 (0.75–2.00) 0.408 N = total episodes of the described disease; n = total episodes where the correct drugs were received adjusted for baseline levels of appropriate treatment for the condition(s) in question, and for variables used to balance arms at baseline (rates of facility and CHW care seeking, cost of care seeking, and CHW motivation score) ** First line treatments: artemisinin combination therapy for malaria, ORS for diarrhoea, and amoxicillin for pneumonia https://doi.org/10.1371/journal.pdig.0000235.t007 t of MDP at endline: Appropriate treatment of episodes suspected malaria, diarrhoea or suspected pneumonia (MDP). riate treatment of episodes suspected malaria, diarrhoea or suspected pneumonia (MDP). Appropriate treatment of MDP at endline: Appropriate treatment of episodes suspected malaria, diarrhoea or suspecte PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 8 / 21 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 8 / 2 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique Table 8. Impact of the inSCALE intervention on appropriate treatment by condition. Meta-analysis of the impact of the inSCALE technology intervention on appropriate treatment for MDP: Uganda and Mozambique Fig 2 shows the results of a fixed effects meta-analysis of the risk ratios (RRs) comparing cover- age of appropriate treatment in control and intervention arms in Uganda and Mozambique. The analysis suggests implementation of the inSCALE technology model could improve appropriate treatment by 15% (8% - 24%) overall. Discussion We have demonstrated that a scalable, innovative technology-based intervention implemented in Mozambique can meaningfully improve the coverage of appropriate treatment for common childhood illnesses covered by the iCCM programme by 26% (any guideline recommended treatment) and 30% (first-line treatments). These changes were mostly driven by significant improvements in the correct treatment of diarrhoea and pneumonia, and in increased appro- priate treatment in primary care facilities. We did not observe between arm differences in CHW outcomes (motivation, social identity, knowledge, and attrition). We finally showed in a PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 9 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique Fig 2. Forest plot displaying an inverse-variance weighted fixed-effect meta-analysis of the effect of the inSCALE technology interventions on appropriate treatment of episodes of suspected malaria, diarrhoea or suspected pneumonia (Uganda and Mozambique). Effect size (ES) is the risk ratio for appropriate treatment. Fixed effects meta analysis (country = fixed effect) Appr. Treat = appropriate treatment of illness episodes (suspected malaria, diarrhoea or suspecter pneumonia). I = intervention arm, C = control arm. ES = baseline-adjusted risk ratio (effect size) of % episodes MDP appropriately treated in intervention versus control arm. https://doi.org/10.1371/journal.pdig.0000235.g002 aying an inverse-variance weighted fixed-effect meta-analysis of the effect of the inSCALE Fig 2. Forest plot displaying an inverse-variance weighted fixed-effect meta-analysis of the effect of the inSCALE technology interventions on appropriate treatment of episodes of suspected malaria, diarrhoea or suspected pneumonia (Uganda and Mozambique). Effect size (ES) is the risk ratio for appropriate treatment. Fixed effects meta analysis (country = fixed effect) Appr. Treat = appropriate treatment of illness episodes (suspected malaria, diarrhoea or suspecter pneumonia). I = intervention arm, C = control arm. ES = baseline-adjusted risk ratio (effect size) of % episodes MDP appropriately treated in intervention versus control arm. https://doi.org/10.1371/journal.pdig.0000235.g002 https://doi.org/10.1371/journal.pdig.0000235.g002 meta-analysis of two countries (Mozambique and Uganda) implementing similar technology- based interventions that an 8–24% improvement in appropriate treatment is likely achievable elsewhere. Our results suggest that iCCM programmes can contribute to the quality of care provided by primary health services by the use of relatively inexpensive technology that priori- tises support, efficiency and performance. Discussion As a result of this work, the government of Mozam- bique has expanded the inSCALE technology intervention and to date seven out of eleven provinces with over 2,500 CHWs and 299 supervisors are using an expanded version of the app. The platform is being rolled out to all 8,800 CHWs and 1,300 supervisors nationally by mid-2023 and a full data integration plan with DHIS2 is ongoing to optimize strategic decision making[18, 19]. The expanded programme name, upSCALE, which includes not only the IMCI sick child module, but also growth monitoring, family planning, adult illness, HIV/TB treatment follow-up and COVID-19, reflects the successful and rapid uptake of our research into national policy and the creation of a national CHW mHealth system[19]. p y y Our conceptual framework for the design of the inSCALE innovation expected that differ- ences in CHW motivation, attrition and social identification would be one of the drivers of improvement in appropriate treatment[15, 20]. As noted in our formative research and in other settings, ownership of local programmes can be fragmented, CHW interventions are often only tenuously linked with the wider health system and therefore poorly supervised, and their contribution under-recognised[21]. The CHWs we surveyed however were in general highly motivated and reported a strong degree of connectedness to the wider health system in both arms at baseline and at the end of follow-up. Control arm CHWs will have received some support offered as standard practice—supervision, review of consultation records and refresher training as required. Local facility records estimated CHW attrition was moderate at 18% over the implementation period and did not differ by intervention arm. Our findings sug- gest that in contrast to our conceptual framework, alternative routes to impact may be more important in this setting. Though coverage of appropriate treatment in children taken to the CHW was similar across arms, we observed the largest overall increase in coverage of appro- priate treatment occurred at public health facilities and in children with diarrhoea and in first line treatments for pneumonia, very similar to the pattern of impact observed in the sister inSCALE trial in Uganda [16]. That these patterns were largely consistent across two countries with separate intervention designs and implementation teams is a persuasive indicator of the alternative impact pathways and mechanisms of impact through which inSCALE operated— and warrants further exploration in future studies. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 Discussion This may highlight the integrated nature of PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 10 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique inSCALE intervention where training and usage of the app by both CHWs and supervising staff at primary care facilities will have increased facility-based health worker exposure to guideline-led recommendations for disease management, alerts for low CHW stock for key commodities, motivational messaging, and reporting on prevalence of key childhood condi- tions. Diarrhoea in particular is often perceived simply as a symptom of a more highly priori- tised condition on which treatment is focused [22] and may therefore be overlooked by health workers even if ORS is available [22–24]. These findings point to the gains that may be made by adopting an approach that not only strengthens community based iCCM programmes, but can also positively impact more broadly on the primary health care system. Coverage of the inSCALE innovation after 18 months of follow-up was moderate to good (68% CHWs had a working phone and inSCALE app and 45% had sent one or more reports in the last month). It is difficult to assess this intervention coverage rate in context of similar interventions due to the variability in intervention components, and in endpoint definitions. A SMS (short message service) message-based randomised controlled trial in Tanzania deliv- ered 7 messages to drug shop vendors with recommendations for correct treatment of malaria and reported 70% of dispensers received at least 75% of the intended SMS messages. Whilst this appears comparable to our reported rates, the difference in the health provider type and in the content of the intervention precludes a direct comparison [25]. Nonetheless a review of a selection of trials of child health-based mHealth interventions in low and middle income country settings (LMICs) report widely varying intervention coverage rates between 24% and 85% [26, 27]. Lack of medicinal stock for common childhood conditions has been a barrier to prompt and appropriate treatment in other settings [28], and affected our study sites in Mozambique during and after the period of implementation [8, 21]. Key issues were the national stock outs of key malaria and antibiotic medications and diagnostic tests within the last 6 months of the study, which may have supressed the full potential impact of the technology intervention for these outcomes. Discussion Research by Save the Children in this setting suggests that households who were aware of stockouts at the iCCM CHW may go elsewhere for treatment [21]. More than 50% of CHWs across both arms reporting at least one stockout of supplies for the management of MDP in the previous 3 months; such commodity supply barriers were outside the control of our intervention nonetheless it is encouraging that the intervention remained impactful in the context of these persistent issues and moderate intervention coverage. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 Wider implications for case prevalence, fatality, productivity, user costs and transmission The highest case fatality rates for pneumonia, malaria and diarrhoea occur in sub-Saharan Africa [29, 30]; and important proximal outcomes of community-based interventions like inSCALE may include a potential to reduce community transmission of causative pathogens and/or the risk of recurrent MDP infections [31] and lowering cost of care seeking and increasing equitable access to care [24] through the strengthening and raised profile of iCCM- trained CHWs. There was a decrease in prevalence of MDP over time across the study site in Mozambique, and the additional significantly faster fall in prevalence in intervention arm dis- tricts. The primary aim of inSCALE was to deliver more appropriate treatment for MDP through a strengthening of the health system. A secondary aim of the intervention was to increase community awareness of MDP through the community-based actions of the health workers. Decreased prevalence of illness may arise through the timely use of appropriate medi- cations, and increased awareness of preventative measures; however we cannot rule out the presence of unmeasured confounding including the influence of secular trends. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 11 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique Our study had several limitations. Our estimates of prevalence of MDP and appropriate treatment were based on caregiver reports which may be subject to misclassification. Studies of caregiver recall for pneumonia and malaria symptoms have sensitivities and specificities in the range of 31%-91% depending on the setting [17, 32]. Recommended strategies to improve the accuracy of disease and treatment estimates include setting the length of the recall period to four weeks (versus the 2 week recall period used in the inSCALE baseline survey), the use of pictures of available treatments to respondents and the development of survey questions which prioritise local terms and are based on well-validated survey instruments [17, 32, 33] including the United Nations- and USAID-supported Multiple Indicator Cluster Survey and Demographic Health Surveys [34, 35]. We incorporated all these strategies in our surveys and used tailored definitions of disease and treatments in line with national guidelines, including the presumptive use of ACT to treat fever, a practice compounded by nationwide stockouts of iCCM drugs and tests at the time. Wider implications for case prevalence, fatality, productivity, user costs and transmission Our sensitivity analyses of the main outcome with alterna- tive definitions of appropriate treatment [see Table B in S1 Text] nonetheless produced similar results–of note, restricting cases of fever to those with a confirmed malaria test estimated a 20% improvement in appropriate treatment which remained highly significant. The inSCALE study evaluated the impact of strategies to improve scale-up and coverage of iCCM pro- grammes using a randomised controlled design as we believed this method would provide the most convincing effectiveness estimates for stakeholders. The number of district clusters avail- able for randomisation was however determined by iCCM programme readiness (iCCM- trained CHWs in place) and lack of concurrent interventions in the area at baseline. This resulted in twelve districts available for randomisation. We tested the robustness of our ran- dom-effects models with cluster-averaged t-tests of our primary outcomes as recommended by Hayes and Moulton [36], and whilst less powerful, we observed similar estimates of impact on appropriate treatment (See Table D1 in S1 Text). We are missing information for 495 households (250 and 245 households where no one was at home in the control and interven- tion arms respectively), which if we assume a similar proportion contain eligible children to the proportion observed in visited household, about half of the 495 may have contained one or more eligible children under 60 months. Any impacts on generalisability are difficult to quan- tify however these are likely to be minimal given the missing represents 6% of all households/ eligible households. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 Setting The study took place in the Inhambane Province of southern Mozambique between November 2013 and July 2016. The province covers a geographical area of 68,775km2 with a population of approximately 1.4 million of which 17% are under 5 years of age [41]. The Inhambane mor- tality rate in this age group at the start of the study was 76.4 deaths per 1000 livebirths [42], with more than 60% of all deaths in the province caused by a combination of malaria, diar- rhoeal diseases and pneumonia [43, 44]. Since 2011, iCCM had been included in the Inham- bane programme of work for Agentes Polivalentes Elementares (APE), the name for the local cadre of community health worker and referred to as CHWs in this paper. Government and donor funding for the programme supported the training, supervision and provision of iCCM commodities for all CHWs in the province throughout the study period. A detailed study pro- tocol for the inSCALE trial in Mozambique has been published elsewhere [15]. Details of the setting and study design for the inSCALE study in Uganda, used for the metanalysis presented in this paper, is published elsewhere [16]. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 Conclusions The body of research evidence for digital and mHealth programmes is increasing, however there remains a significant gap for evidence from good quality studies of the potential for avail- able and emerging technologies to transform health outcome in LMICs for children, particu- larly when delivered via community-based health workers [37]. Mobile phone penetration is predicted to increase in sub-Saharan Africa [38] and is estimated to be a key medium by which health care access and service delivery can be improved for the underserved. The WHO’s recently published set of global recommendations for digital interventions for health services delivery highlight the potential of mHealth interventions for a range of uses and includes the key components of the inSCALE technology intervention–clinical decision support, stock tracking and provider-to-provider telemedicine [39]. Significantly, the 2019 WHO/UNICEF iCCM strategy meeting in Addis Ababa recommended “Interventions to improve quality, including supportive supervision and mentoring of CHWs in designated health facilities, are essential to ensure high-quality iCCM and should be budgeted for and included in district plans”[40]. This renewed encouragement for implementation of iCCM this decade may encourage relevant stakeholders looking to efficiently scale up programmes to explore digital PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 12 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique solutions for their context. Our study and meta-analysis together with the expansion of the inSCALE programme in Mozambique [18, 19] highlight the potential scalability of an mHealth intervention aimed at strengthening iCCM systems to address the largest causes of childhood morbidity and mortality in sub-Saharan Africa. Study procedures Sample size calculations. The baseline survey was conducted in all households in 2–8 randomly-selected communities (known as enumeration areas or EAs) in each district in order to cover approximately 500 households and 350 children per district (mean of 91 house- holds per village). In the absence of site-specific data, these estimates were based on equivalent baseline data on prevalence of illness and between-cluster correlation from the sister inSCALE trial in Uganda. Following the baseline survey in Mozambique, sample size calculations for the impact analysis were updated using local data on MDP prevalence and the proportion of households with eligible children; indicating that by sampling 530 households with a rate of 48% containing eligible children we would have 90% power to detect an impact of the inter- vention of a 20% or higher increase in appropriate treatment given an estimated coefficient of variation in the outcome between clusters of 0.16 and a rate of 40% of children with MDP receiving appropriate treatment in the control group. Parameters and calculations are described in more detail in the inSCALE protocol paper [15]. Approximately 21 CHWs were also surveyed per district at baseline (1–2 per village). Community sampling frame data were based on complete enumeration information provided by the District Offices and EA location, size and households were verified by field workers. Data collection. The baseline survey was administered to caregivers of children under 5 years of age, and collected information on symptoms of malaria, diarrhoea, and pneumonia (MDP) in these children in the last 2 weeks, care seeking for these illnesses, costs of care, socio- economic data at the household level. Household questionnaires were based on standard health survey instruments used in the country (Mozambique DHS [34] and Malaria Indicator Cluster Surveys [35]). The CHW survey included data on CHWs’ clinical knowledge, time use, motivation and ‘social identity’–or sense and degree of connectedness to the local and national CHW and health services systems. The CHW clinical knowledge instrument was developed based on the format and example clinical event scenarios in the WHO Health Facility Survey Tool template [47]. The likert scale-based CHW motivation and identity tool used a pictorial scale indicating degree of agreement with statements about enthusiasm for the job, motivation and identification with the local and national iCCM programme, and was developed based on extensive formative research by the study team on health worker motivation and social identity [48, 49]. Study design and participants We conducted a superiority open label two-arm cluster-randomised controlled trial in 12 dis- tricts in Inhambane Province in southern Mozambique. Six districts (Funhalouro, Govuro, Jangamo, Panda, Vilankulo, and Zavala) were randomised to the inSCALE intervention and six (Homoine, Inharrime, Inhassouro, Mabote, Massinga, and Morrumbene) to the control arm. The provincial and economic capitals of Inhambane: Inhambane City and Maxixe respec- tively, was excluded from the randomisation and Maxixe was instead used for piloting of the intervention in its rural areas during the design stage. Each district was defined as a cluster and randomly allocated to either intervention or control; the rationale being the district was the lowest unit of administration and training for the CHW iCCM programme and therefore for implementation of the inSCALE intervention. Eligible households within the district were defined as those with at least one resident child under five years of age at any point during the study in a community served by at least one iCCM-trained CHW. Local data collected by Malaria Consortium in the area in 2011 suggested that 45% of households within each com- munity fit the eligibility criteria. Ethical approval for the study was obtained from the Mozambique National Bioethics Committee for Health (reference 345-CNBS-10) and the London School of Hygiene & Tropical Medicine Ethics Committee in the UK (reference 5762). Approval for the random allocation of districts to intervention or control groups and implementation of the interven- tions was obtained from the Inhambane Provincial Office responsible for health administra- tion for the province. Written informed consent was obtained from the main caregiver of each child under 5 years of age for baseline and follow up data collection. Consent forms included an explanation of the purpose of the data collection and confirmed participants were free to withdraw at any time. The inSCALE trial is registered on clinicaltrials.gov with identifier NCT01972321. 13 / 21 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique Randomisation and blinding In order to reduce the likelihood of chance imbalances between intervention arms at baseline given the number of clusters available for randomisation [36, 45]., we performed a randomisa- tion that was restricted with respect to the percentages of households with sick children seek- ing care at a public health facility, the percentage seeking care to a CHW, average CHW motivation (composite) score and the log cost of care seeking for sick children. Data for these parameters was obtained from a baseline survey conducted between November- December 2012. We used the statistical software Stata (version 13) to generate the 924 possible randomi- sation schemes for the 12 districts and picked one at random from the subsection of 84 schemes that fitted our balance criteria [46] [see online S3 Text]. Due to the nature of the tech- nology intervention, it was not possible to blind participants or fieldworkers to the interven- tion allocation, however allocation was masked in the database and all analyses were initially performed on data with intervention allocation removed and repeated on unblinded data after agreement on the final analysis methods with study partners. Intervention The inSCALE technology intervention was developed in collaboration with Dimagi a social enterprise corporation focused on the development of digital solutions for health workers in low and middle income settings [50]. Steering and input was received from the Mozambique ministry of health, community and facility-based stakeholders, funding bodies and the inSCALE trial steering committee [15, 51]. The aim of the innovation was to leverage techno- logical strategies to strengthen links between fragmented CHW programmes and the primary care health services including links between CHW peers and health facility staff, and by doing so to improve CHW outcomes and child health outcomes for children. The inSCALE technol- ogy intervention development process and conceptual framework linking intervention com- ponents, outputs and outcomes is described in detail in the protocol paper [15]. The technology intervention was implemented in July 2013, and after a 6 month roll-out and embedding period, follow-up to impact analysis continued to July 2015 (18 months). iCCM-trained CHWs and their health facility supervisors in the intervention arm were pro- vided with a Samsung Galaxy smartphone installed with the inSCALE mHealth application (with CHW and facility supervisor versions), a solar charger with a number of pin adapters, and a solar lamp for night-time consultations. Intervention arm CHWs and health facility supervising staff were trained in the use of the mHealth tools. Training was conducted using a cascade approach where 7 master trainers trained 18 facility- and community-based trainers who in turn trained 47 health facility supervisors and 132 CHWs in the technology arm over 4 days with follow-up support provided by the project team during the implementation period. The inSCALE mHealth mobile app consisted of a sick child decision support algorithm in a user-friendly interface including multimedia, audio and images to improve adherence to iCCM treatment protocols. CHWs were additionally able to use the phones to track consulta- tions with sick children, make diagnoses and management choices, report key statistics to a central server, request stock from their supervising facilities, and maintain a register of patients seen. These reports were forwarded on a weekly basis to health facility supervising staff who received tailored follow-up supervision prompts. The inSCALE app also worked offline, there- fore where there were network coverage issues, CHWs and health facility supervising staff had the option to delay sending of reports until they were in a location with better coverage. Study procedures Both tools were adapted to the Mozambique setting prior to usage with the input of facility staff and CHWs in an iterative process that developed wording, accompanying graphics and reference to local health system structures and clinical guidelines. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 14 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique Household and CHW surveys were also extensively field piloted with field staff including workshops role playing and refining of translations, and all tools were delivered in Portuguese. A quality control check of 10% of the weekly survey households was undertaken by field worker supervisors. Pictures of locally available medications were used in the household sur- veys to improve the accuracy of reports of treatment for MDP [17]. At the end of the follow-up period an endline impact survey was conducted in May-June 2015 in the same villages visited at baseline in households with children meeting inclusion cri- teria at endline, and again covering all active CHWs serving the selected villages. The survey collected data across the same themes to estimate study impact and included additional ques- tions to CHWs and caregivers on coverage of the inSCALE innovation components in the intervention arm. The recall period for recent illness in children (caregiver reported) was extended to 4 weeks in the endline survey following the publication of a series of seminal papers recommending increasing the exposure period to 28 days to improve accuracy of reports of suspected pneumonia [17] household and CHW data collection tools are available in S2 Text. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 Intervention CHWs and supervisors were allocated to local closed user groups which enabled complimen- tary calls with the inSCALE smartphones to be made without charge to fellow members in the CHWs and supervisors were allocated to local closed user groups which enabled complimen- tary calls with the inSCALE smartphones to be made without charge to fellow members in the PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 15 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique group. Finally, CHWs and their health facility supervisors received weekly (CHWs only) and monthly (CHWs and facility supervisors) automated motivational messages tailored to their report results with the tone and emphasis based on formative work. In brief this included pre- testing of messages with subgroups of CHWs and supervisors where versions of the app were evaluated on acceptability, comprehension, and usefulness, and phone functionalities were adapted accordingly. Email report formats were pretested with supervisors and iteratively updated. A series of action-oriented tips were developed based on a range of common perfor- mance-related scenarios, and all materials were adapted to Mozambique’s context and android phone software, and translated into Portuguese. After an initial 6-month roll-out and embedding phase, the technology intervention contin- ued to be implemented in intervention arm districts for an additional 18 months. Standard care during this period was available in both intervention and control arm districts, where CHWs and health facilities provided guideline-led management for childhood illnesses via the underlying iCCM programme and integrated management of neonatal and childhood illnesses (IMNCI) services [9, 52] respectively. Data on CHW recruitment, training, resignations, and replacements were tracked by super- vising health facilities at district level and this data was regularly extracted into the inSCALE database during the follow-up period by the study team in order to estimate attrition rates. Standard practice in control arm Control arm CHWs did not receive the inSCALE package and accessories. CHWs in both arms received training in iCCM, health promotion and education, supplies of commodities including rapid diagnostic tests and drugs for common childhood illnesses, monthly supervi- sion, and a $40 monthly subsidy. CHWs in both arms maintained paper based registers. Addi- tional details of the components of the intervention arms is provide in Table I in S1 Text Outcomes All study outcomes were based on data collected on questionnaires during the cross sectional survey at the end of the intervention follow-up period as described above (Data Collection). The primary outcome for the trial was the percentage of reported recent cases of MDP in the last 4 weeks in children 2 months to 5 years of age that were treated using appropriate medica- tion [15]. Secondary outcomes were the prevalence of MDP in children at endline, the percent- age of cases of MDP appropriately treated with recommended first line medications, the percentage of children who sought care for MDP by provider type, the percentage of CHWs who had had a stock-out of iCCM medications in the past 3 months, CHW attrition rate calcu- lated as the proportion of CHWs who left their post during the follow-up period out of the total CHWs trained at baseline, CHW clinical knowledge scores (overall and by condition sub- group), and composite scores describing CHW enthusiasm and drive for the job (motivation) and sense of connectedness to the local and national iCCM programme (social identification). CHW knowledge scores were calculated as the sum of correct clinical decisions mentioned by the CHW in response to a series of scenarios involving children with symptoms of mild and severe disease. Points for correct items were weighted according to an algorithm defined in Tables G1-G6 in S1 Text. CHW motivation and social identification scores were calculated by a factor analysis and empirical item reduction process applied to original field tool responses. The methods have been detailed previously [53], and resulted in a three-factor CHW motiva- tion scale of 17 items measuring general motivation, feelings of reward for effort, and retention in role. An additional single factor CHW scale of 4 items was identified which explained the majority of the variation in social identification between CHWs. Retained items from both the PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 16 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique motivation and social identification tools showed good internal consistency (alpha = 0.67 for CHW motivation and alpha = 0.69 for CHW social identification). The final motivation scores were calculated as the product of the rotated factor analysis regression coefficients and the Likert value for each original item. Statistical analysis All analyses were based on intention-to-treat principles and accounted for the cluster-rando- mised design. Multivariate mixed-effects models were specified for all impact outcomes, which account for the clustering of outcomes by inclusion of a random intercept at the district level. Intervention arm, the cluster mean percentage of children who were appropriately treated at baseline, and the parameters used to balance intervention arms during randomisation were included as fixed effects. Unadjusted analyses of impact were also performed, and the results included in Table A in S1 Text. For the primary outcome and other binary outcomes, odds ratios were converted to risk ratios using the marginal standardisation technique and 95% CIs estimated using the delta method [54]. Additional sensitivity analyses of the primary outcome were conducted to account for alternative treatments available for MDP [see Table H in S1 Text for definitions]. Robustness checks of the primary outcomes were conducted by estimat- ing cluster averaged results using t-tests following the procedures of Hayes and Moulton [36], recommended when the number of clusters per arm is less than 10[36] [see Tables D1-E2 in S1 Text]. CHW attrition was estimated using cluster-averaged mean rates as human resources information was provided at this level only from district health offices. In order to estimate a pooled effect of the inSCALE technology intervention on appropriate treatment across both Uganda and Mozambique, we performed a meta-analysis of effect sizes by combining the results of the Mozambique (this study) and Uganda [16] trials of the inSCALE intervention. As there was no significant heterogeneity (<50% estimated from exploratory meta-analysis models) between studies, we present the pooled result from a fixed effects meta-analysis. Outcomes The final social identification scores were calculated as the sum of the original 4 items (after reverse coding as appropriate). Additional impact measures included the percentage of children who were appropriately treated overall whether they had symptoms of one or more of MDP in the last 4 weeks (i.e. a per-child analysis), and two sensitivity analyses of the main impact outcome using alternative definitions of appropriate treatment. All definitions of appropriate treatment and medications used in primary secondary and additional endpoints are provided in Table H in S1 Text. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 Acknowledgments We wish to thank the local research team who collected and managed the data, diMaggi for support in designing the app software, the CHWs and supervisors who delivered the interven- tion, and the participants of the study for their time and insight. SS, KK, RL, NB, FK and BK had full access to all the data and SS had final responsibility for the decision to submit for publication. Supporting information S1 Text. Table A. Unadjusted analyses of primary outcomes. Table B. Sensitivity analyses for primary outcome at endline (any episode of MDP): Alternative definitions of appropriate malaria treatment. Table C. Whole Child Analysis–Appropriate treatment. Table D1. Robust- ness tests (cluster-averaged outcomes)–Primary Outcomes. Table D2. Robustness tests (clus- ter-averaged outcomes)–Secondary Outcomes: appropriate treatment stratified by condition or provider Table E1. Robustness tests (cluster-averaged outcomes)–Secondary outcomes: prevalence of suspected and confirmed malaria, malaria blood testing, diarrhoea, and pneu- monia. Table E2. Robustness tests (cluster-averaged outcomes)–Secondary outcomes: preva- lence of care-seeking for suspected malaria, diarrhoea and pneumonia. Table F. CHW clinical knowledge by subgroup (diagnosis of MDP, treatment of MDP). Table G1. Item weighting– Case study: Francis/Alberte (pneumonia and diarrhoea). Table G2. Item weighting–Case study: Hope/Tina (malaria). Table G3. Item weighting–Case study: Beatrice/Janete (severe pneumonia). Table G4. Item weighting–Case study: Muteesa/Kizito (severe disease/malaria). Table G5. Item weighting–Case study: James (severe disease/malaria). Table G6. CHW PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 17 / 21 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique Knowledge scoring for subcategories: diagnosis and treatment of suspected malaria, diarrhoea and pneumonia. Table H. Definitions of appropriate treatment and other key child health out- comes. Table I. Intervention and control arm components—inSCALE Mozambique. (DOCX) Knowledge scoring for subcategories: diagnosis and treatment of suspected malaria, diarrhoea and pneumonia. Table H. Definitions of appropriate treatment and other key child health out- comes. Table I. Intervention and control arm components—inSCALE Mozambique. (DOCX) References 1. WHO. Children: improving survival and well-being. Sep 2020 [cited 29 Sep 2021]. Available: https:// www.who.int/news-room/fact-sheets/detail/children-reducing-mortality 1. WHO. Children: improving survival and well-being. Sep 2020 [cited 29 Sep 2021]. Available: https:// www.who.int/news-room/fact-sheets/detail/children-reducing-mortality 2. UNICEF. Levels and trends in child mortality 2019: Estimates developed by the UN Inter-agency group for child mortality estimation. Sep 2019. Available: https://www.unicef.org/reports/levels-and-trends- child-mortality-report-2019 2. UNICEF. Levels and trends in child mortality 2019: Estimates developed by the UN Inter-agency group for child mortality estimation. Sep 2019. Available: https://www.unicef.org/reports/levels-and-trends- child-mortality-report-2019 3. WHO, UNICEF. WHO/UNICEF JOINT STATEMENT: Integrated Community Case Management (iCCM). 2012. Available: https://www.who.int/maternal_child_adolescent/documents/statement_child_ services_access_whounicef.pdf 4. Kallander K, Burgess DH, Qazi SA. Early identification and treatment of pneumonia: a call to action. Lancet Glob Health. 2016; 4: e12–e13. https://doi.org/10.1016/S2214-109X(15)00272-7 PMID: 26577842 5. Diaz T, Aboubaker S, Young M. Current scientific evidence for integrated community case management (iCCM) in Africa: Findings from the iCCM Evidence Symposium. J Glob Health. 2014; 4: 020101. https://doi.org/10.7189/jogh.04.020101 PMID: 25520783 6. Boschi-Pinto C, Labadie G, Dilip TR, Oliphant N, Dalglish SL, Aboubaker S, et al. Global implementa- tion survey of Integrated Management of Childhood Illness (IMCI): 20 years on. BMJ Open. 2018; 8: e019079. https://doi.org/10.1136/bmjopen-2017-019079 PMID: 30061428 7. Allen KC, Whitfield K, Rabinovich R, Sadruddin S. The role of governance in implementing sustainable global health interventions: review of health system integration for integrated community case manage- ment (iCCM) of childhood illnesses. BMJ Glob Health. 2021; 6: e003257. https://doi.org/10.1136/ bmjgh-2020-003257 PMID: 33789866 8. Prosnitz D, Herrera S, Coelho H, Moonzwe Davis L, Zalisk K, Yourkavitch J. Evidence of Impact: iCCM as a strategy to save lives of children under five. J Glob Health. 2019; 9: 010801. https://doi.org/10. 7189/jogh.09.010801 PMID: 31263547 9. Oliphant NP, Manda S, Daniels K, Odendaal WA, Besada D, Kinney M, et al. Integrated community case management of childhood illness in low- and middle-income countries. Cochrane Database Syst Rev. 2021 [cited 22 Sep 2021]. https://doi.org/10.1002/14651858.CD012882.pub2 PMID: 33565123 10. The Global Fund for HIV TB and Malaria, WHO, UNICEF. Integrated Community Case Management (iCCM) in Sub-Saharan Africa: Successes & Challenges with Access, Speed & Quality. Nairobi, Kenya; 2018 Aug p. 145. Available: https://www.theglobalfund.org/media/9754/core_ integratedcommunitycasemanagementsub-saharanafrica_review_en.pdf 11. WHO Global Observatory for eHealth. mHealth: new horizons for health through mobile technologies: second global survey on eHealth. 2011 [cited 5 Feb 2022]. Available: https://apps.who.int/iris/handle/ 10665/44607 12. Braun R, Catalani C, Wimbush J, Israelski D. Community Health Workers and Mobile Technology: A Systematic Review of the Literature. Author Contributions Conceptualization: Seyi Soremekun, Karin Ka¨llander, Raghu Lingam, Daniel Ll Strachan, Frida Kasteng, Anna Vassall, Zelee Hill, Guus ten Asbroek, Sylvia Meek, James Tibenderana, Betty Kirkwood. Data curation: Seyi Soremekun, Neha Batura, Abel Muiambo, Nelson Salomao, Juliao Condoane, Fenias Benhane, Frida Kasteng. Formal analysis: Seyi Soremekun, Raghu Lingam, Neha Batura, Daniel Ll Strachan, Frida Kasteng. Funding acquisition: James Tibenderana, Betty Kirkwood. Funding acquisition: James Tibenderana, Betty Kirkwood. Investigation: Seyi Soremekun, Karin Ka¨llander, Raghu Lingam, Ana-Cristina Castel Branco, Neha Batura, Daniel Ll Strachan, Abel Muiambo, Nelson Salomao, Juliao Condoane, Fenias Benhane, Frida Kasteng, Anna Vassall, Guus ten Asbroek, Sylvia Meek. Methodology: Seyi Soremekun, Karin Ka¨llander, Raghu Lingam, Ana-Cristina Castel Branco, Neha Batura, Nelson Salomao, Frida Kasteng. Project administration: Ana-Cristina Castel Branco. Resources: Karin Ka¨llander, Raghu Lingam, Ana-Cristina Castel Branco. Supervision: Seyi Soremekun, Raghu Lingam, Zelee Hill, Guus ten Asbroek, Sylvia Meek, Betty Kirkwood. Validation: Seyi Soremekun. Validation: Seyi Soremekun. Writing – original draft: Seyi Soremekun. Writing – original draft: Seyi Soremekun. Writing – review & editing: Seyi Soremekun, Karin Ka¨llander, Daniel Ll Strachan, Abel Muiambo, Nelson Salomao, Juliao Condoane, Fenias Benhane, Anna Vassall, Zelee Hill, Guus ten Asbroek, James Tibenderana, Betty Kirkwood. 18 / 21 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique References Treating diarrhoeal disease in chil- dren under five: the global picture. Arch Dis Child. 2014; 99: 273–278. https://doi.org/10.1136/ archdischild-2013-304765 PMID: 24197873 23. Nsabagasani X, Ogwal-Okeng J, Mbonye A, Ssengooba F, Muhumuza S, Hansen EH. Availability and utilization of the WHO recommended priority lifesaving medicines for under five-year old children in pub- lic health facilities in Uganda: a cross-sectional survey. J Pharm Policy Pract. 2015; 8: 18. https://doi. org/10.1186/s40545-015-0038-2 PMID: 25995847 24. Soremekun S, Kasteng F, Lingam R, Vassall A, Kertho E, Settumba S, et al. Variation in the quality and out-of-pocket cost of treatment for childhood malaria, diarrhoea, and pneumonia: Community and facil- ity based care in rural Uganda. PLOS ONE. 2018; 13: e0200543. https://doi.org/10.1371/journal.pone. 0200543 PMID: 30475808 25. Bruxvoort K, Festo C, Kalolella A, Cairns M, Lyaruu P, Kenani M, et al. Cluster Randomized Trial of Text Message Reminders to Retail Staff in Tanzanian Drug Shops Dispensing Artemether-Lumefan- trine: Effect on Dispenser Knowledge and Patient Adherence. Am J Trop Med Hyg. 2014; 91: 844–853. https://doi.org/10.4269/ajtmh.14-0126 PMID: 25002300 26. Zurovac D, Sudoi RK, Akhwale WS, Ndiritu M, Hamer DH, Rowe AK, et al. The effect of mobile phone text-message reminders on Kenyan health workers’ adherence to malaria treatment guidelines: a clus- ter randomised trial. Lancet Lond Engl. 2011; 378: 795–803. https://doi.org/10.1016/S0140-6736(11) 60783-6 PMID: 21820166 27. Friedman W, Woodman B, Chatterji M. Can mobile phone messages to drug sellers improve treatment of childhood diarrhoea?—A randomized controlled trial in Ghana. Health Policy Plan. 2015; 30 Suppl 1: i82–92. https://doi.org/10.1093/heapol/czu122 PMID: 25759456 28. Mubiru D, Byabasheija R, Bwanika JB, Meier JE, Magumba G, Kaggwa FM, et al. Evaluation of Inte- grated Community Case Management in Eight Districts of Central Uganda. PLOS ONE. 2015; 10: e0134767. https://doi.org/10.1371/journal.pone.0134767 PMID: 26267141 29. Marangu D, Zar HJ. Childhood pneumonia in low-and-middle-income countries: An update. Paediatr Respir Rev. 2019; 32: 3–9. https://doi.org/10.1016/j.prrv.2019.06.001 PMID: 31422032 30. WHO. Child mortality and causes of death. Geneva, Switzerland; 2022. Available: https://www.who.int/ data/gho/data/themes/topics/topic-details/GHO/child-mortality-and-causes-of-death 31. Information NC for B, Pike USNL of M 8600 R, MD B, Usa 20894. Malaria transmission and antimalarial medicines. Guidelines for the Treatment of Malaria. 3rd edition. World Health Organization; 2015. Avail- able: https://www.ncbi.nlm.nih.gov/books/NBK294437/ 32. Eisele TP, Silumbe K, Yukich J, Hamainza B, Keating J, Bennett A, et al. Measuring coverage in MNCH: accuracy of measuring diagnosis and treatment of childhood malaria from household surveys in Zambia. PLoS Med. 2013; 10: e1001417. https://doi.org/10.1371/journal.pmed.1001417 PMID: 23667337 33. References Bullen C, editor. PLoS ONE. 2013; 8: e65772. https://doi.org/10. 1371/journal.pone.0065772 PMID: 23776544 13. Ka¨llander K, Tibenderana JK, Akpogheneta OJ, Strachan DL, Hill Z, ten Asbroek AHA, et al. Mobile Health (mHealth) Approaches and Lessons for Increased Performance and Retention of Community Health Workers in Low- and Middle-Income Countries: A Review. J Med Internet Res. 2013; 15: e17. https://doi.org/10.2196/jmir.2130 PMID: 23353680 14. World Bank. Mobile cellular subscriptions (per 100 people)—Sub-Saharan Africa. 2020 [cited 5 Feb 2022]. Available: https://data.worldbank.org/indicator/IT.CEL.SETS.P2?locations=ZG 15. Ka¨llander K, Strachan D, Soremekun S, Hill Z, Lingam R, Tibenderana J, et al. Evaluating the effect of innovative motivation and supervision approaches on community health worker performance and reten- tion in Uganda and Mozambique: study protocol for a randomised controlled trial. 2015; 16: 157. https:// doi.org/10.1186/s13063-015-0657-6Trials. PMID: 25873093 16. Ka¨llander K, Soremekun S, Strachan D, Hill Z, Kasteng F, Kertho E, et al. Improving community health worker treatment for malaria, diarrhoea and pneumonia in Uganda through inSCALE community and mHealth innovations: A cluster randomised controlled trial. PLOS Digit Health. 2023. https://doi.org/10. 1371/journal.pdig.0000217 17. Campbell H, el Arifeen S, Hazir T, O’Kelly J, Bryce J, Rudan I, et al. Measuring Coverage in MNCH: Challenges in Monitoring the Proportion of Young Children with Pneumonia Who Receive Antibiotic Treatment. PLOS Med. 2013; 10: e1001421. https://doi.org/10.1371/journal.pmed.1001421 PMID: 23667338 18. Feldman M, Krylova VL, Farrow P, Donovan L, Zandamela E, Rebelo J, et al. Community health worker knowledge, attitudes and practices towards COVID-19: Learnings from an online cross-sectional survey 19 / 21 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique using a digital health platform, UpSCALE, in Mozambique. PLOS ONE. 2021; 16: e0244924. https://doi. org/10.1371/journal.pone.0244924 PMID: 33566850 using a digital health platform, UpSCALE, in Mozambique. PLOS ONE. 2021; 16: e0244924. https://doi. org/10.1371/journal.pone.0244924 PMID: 33566850 19. Malaria Consortium. upSCALE mHealth system strengthening for case management and disease sur- veillance. 2018. Available: https://www.malariaconsortium.org/upscale/. Accessed 17 Sep 2021. 20. Global Fund to Fight AIDS, Tuberculosis and Malaria. Global Fund Thematic Review on Community Health. 2020 Jun p. 46. Available: https://www.theglobalfund.org/media/10128/core_ endasantecommunityhealthsynthesis_review_en.pdf 21. Save the Children. Systems Effects of Integrated Community Case Management Projects: Mozam- bique Country Report. In: Resource Centre [Internet]. 8 Apr 2019 [cited 11 Jun 2021]. Available: https:// resourcecentre.savethechildren.net/library/systems-effects-integrated-community-case-management- projects-mozambique-country-report 22. Unger CC, Salam SS, Sarker MSA, Black R, Cravioto A, Arifeen SE. References Walker CLF, Fontaine O, Black RE. Measuring Coverage in MNCH: Current Indicators for Measuring Coverage of Diarrhea Treatment Interventions and Opportunities for Improvement. PLOS Med. 2013; 10: e1001385. https://doi.org/10.1371/journal.pmed.1001385 PMID: 23667330 34. Demographic Health Surveys. The DHS Program—Questionnaires and Manuals. In: USAID [Internet]. [cited 11 Jun 2021]. Available: https://dhsprogram.com/Methodology/Questionnaires.cfm 35. UNICEF. Tools—UNICEF MICS. [cited 11 Jun 2021]. Available: https://mics.unicef.org/tools 36. Hayes R, Moulton L. Cluster Randomised Trials. 2nd ed. Chapman and Hall/CRC; Available: https:// www.routledge.com/Cluster-Randomised-Trials/Hayes-Moulton/p/book/9781498728225 37. Feroz A, Jabeen R, Saleem S. Using mobile phones to improve community health workers performance in low-and-middle-income countries. BMC Public Health. 2020; 20: 49. https://doi.org/10.1186/s12889- 020-8173-3 PMID: 31931773 20 / 21 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 PLOS DIGITAL HEALTH Impact of a technology intervention on child health outcomes in Mozambique 38. Crul S, Diop J. The mHealth opportunity in Sub-Sahara Africa: The path towards practical application. Deloitte; 2014. Available: https://www2.deloitte.com/content/dam/Deloitte/nl/Documents/technology- media-telecommunications/deloitte-nl-mhealth.pdf 39. WHO. WHO Guideline: recommendations on digital interventions for health system strengthening. World Health Organization; 2019. Available: http://www.who.int/reproductivehealth/publications/digital- interventions-health-system-strengthening/en/ 40. WHO, UNICEF. Institutionalising integrated community case management (iCCM) to end preventable child deaths: Technical consultation and country action planning 22–26 July 2019, Elilly International Hotel, Addis Ababa, Ethiopia. 41. National Institute of Statistics Mozambique. Inhambane—Instituto Nacional de Estatistica. 2013 [cited 11 Jun 2021]. Available: http://www.ine.gov.mz/estatisticas/estatisticas-territorias-distritais/inhambane 42. UN Inter-agency Group for Child Mortality Estimation. Under five Mortality Rate—Mozambique. [cited 11 Jun 2021]. Available: https://childmortality.org/data/Mozambique 43. Fernandes QF, Wagenaar BH, Anselmi L, Pfeiffer J, Gloyd S, Sherr K. Effects of health-system strengthening on under-5, infant, and neonatal mortality: 11-year provincial-level time-series analyses in Mozambique. Lancet Glob Health. 2014; 2: e468–e477. https://doi.org/10.1016/S2214-109X(14) 70276-1 PMID: 25103520 44. Ka¨llander K, Counihan H, Cerveau T, Mbofana F. Barriers on the pathway to survival for children dying from treatable illnesses in Inhambane province, Mozambique. J Glob Health. 9. https://doi.org/10. 7189/jogh.09.010809 PMID: 31275569 45. Ivers NM, Halperin IJ, Barnsley J, Grimshaw JM, Shah BR, Tu K, et al. Allocation techniques for balance at baseline in cluster randomized trials: a methodological review. Trials. 2012; 13: 120. https://doi.org/ 10.1186/1745-6215-13-120 PMID: 22853820 46. Moulton LH. Covariate-based constrained randomization of group-randomized trials. Clin Trials Lond Engl. 2004; 1: 297–305. https://doi.org/10.1191/1740774504cn024oa PMID: 16279255 47. WHO. Health facility survey: tool to evaluate the quality of care delivered to sick children attending out- patient facilities. World Health Organization; 2003. Available: https://apps.who.int/iris/handle/10665/ 42643 48. Chandler CIR, Chonya S, Mtei F, Reyburn H, Whitty CJM. PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 References Motivation, money and respect: a mixed- method study of Tanzanian non-physician clinicians. Soc Sci Med 1982. 2009; 68: 2078–2088. https:// doi.org/10.1016/j.socscimed.2009.03.007 PMID: 19328607 49. Franco LM, Bennett S, Kanfer R, Stubblebine P. Determinants and consequences of health worker motivation in hospitals in Jordan and Georgia. Soc Sci Med 1982. 2004; 58: 343–355. https://doi.org/10. 1016/s0277-9536(03)00203-x PMID: 14604620 50. DIMAGI. Dimagi \| Technology for Social Impact. In: Dimagi [Internet]. [cited 9 May 2022]. Available: https://www.dimagi.com/ 51. Strachan DL, Ka¨llander K, Nakirunda M, Ndima S, Muiambo A, Hill Z, et al. Using theory and formative research to design interventions to improve community health worker motivation, retention and perfor- mance in Mozambique and Uganda. Hum Resour Health. 2015; 13: 25. https://doi.org/10.1186/s12960- 015-0020-8 PMID: 25925007 52. WHO UNICEF, editors. Handbook IMCI: integrated management of childhood illness. Geneva: World Health Organization; 2005. 53. Strachan DL. Ugandan community health worker motivation: using the Social Identity Approach to explore an accepted constraint to scaled up health strategies. Doctoral thesis, UCL (University College London). Doctoral, UCL (University College London). 2019. pp. 1–410. Available: https://discovery.ucl. ac.uk/id/eprint/10073256/ 54. Localio AR, Margolis DJ, Berlin JA. Relative risks and confidence intervals were easily computed indi- rectly from multivariable logistic regression. J Clin Epidemiol. 2007; 60: 874–882. https://doi.org/10. 1016/j.jclinepi.2006.12.001 PMID: 17689803 PLOS Digital Health \| https://doi.org/10.1371/journal.pdig.0000235 June 12, 2023 21 / 21
https://openalex.org/W3005463761	https://europepmc.org/articles/pmc7033319?pdf=render	English	null	A dataset for the effect of earthworm abundance and functional group diversity on plant litter decay and soil organic carbon level	Data in brief	2,020	cc-by	7,114	Wei Huang a, b, Grizelle Gonzalez c, Xiaoming Zou a, b, * Wei Huang a, b, Grizelle Gonzalez c, Xiaoming Zou a, b, * Wei Huang a College of Biology and the Environment, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, Jiangsu, China b Department of Environmental Sciences, College of Natural Sciences, University of Puerto Rico, P.O. Box 70377, San Juan, PR 00936-8377, USA c International Institute of Tropical Forestry, USDA Forest Service, Jardín Botanico Sur, 1201 Calle Ceiba, Río Piedras, PR 00926-1119, USA Contents lists available at ScienceDirect Contents lists available at ScienceDirect DOI of original article: https://doi.org/10.1016/j.apsoil.2019.103473. * Corresponding author. College of Biology and the Environment, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, Jiangsu, China. E-mail address: Xiaoming.zou@upr.edu (X. Zou). https://doi.org/10.1016/j.dib.2020.105263 2352-3409/© 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/). Data in brief 29 (2020) 105263 Data in brief 29 (2020) 105263 DOI of original article: https://doi.org/10.1016/j.apsoil.2019.103473. * Corresponding author. College of Biology and the Environment, Nanjing Forestry University, 159 Longpan Road, Nanjing 210037, Jiangsu, China. E-mail address: Xiaoming.zou@upr.edu (X. Zou). Value of the Data To date, no dataset has provided a comprehensive synthesis of existing experimental data about the effect of ear on litter decomposition and soil organic carbon (SOC) levels at global scale. To date, no dataset has provided a comprehensive synthesis of existing experimental data on litter decomposition and soil organic carbon (SOC) levels at global scale. p g ( ) g Data can be used to quantify the effect of earthworms on litter decomposition and SOC levels at global scale. Data can be used to quantify the effect of earthworms on litter decomposition and SOC levels at global scale. Data can be used to identify effects of earthworm functional group diversity, vegetation types, litter quality, litterbag mesh size, soil C/N, soil aggregate size, experiment types and length of experimental time on earthworm induced plant litter and SOC decay. q y p g Data can be used to identify effects of earthworm functional group diversity, vegetation types, litter quality, litterbag mesh size, soil C/N, soil aggregate size, experiment types and length of experimental time on earthworm induced plant litter and SOC decay. a r t i c l e i n f o 18 countries over ﬁve continents With the article Wei Huang, Grizelle Gonzalez, Xiaoming Zou, Earthworm Abundance and Functional Group Diversity Regulate Plant Litter Decay and Soil Organic Carbon Level: A Global Meta-analysis, Applied Soil Ecology, in press, https://doi.org/10.1016/j.apsoil.2019. 103473. [1] Wei Huang, Grizelle Gonzalez, Xiaoming Zou, Earthworm Abundance and Functional Group Diversity Regulate Plant Litter Decay and Soil Organic Carbon Level: A Global Meta-analysis, Applied Soil Ecology, in press, https://doi.org/10.1016/j.apsoil.2019. 103473. [1] 1. Data description Data were extracted from peer-reviewed journal papers published between 1985 and 2018. Totally 340 observations from 69 studies were included. Detailed data are listed in Tables 1e5, giving the following information: location, ecosystem, earthworm density, annual litter decomposition rate, earthworm function group, the response ratio (R), mean annual temperature, mean annual precipi- tation, experimental type, experimental duration, litter quality, forest ﬂoormass thickness and carbon stock, soil carbon concentration, soil C/N, soil aggregate size, and literature reference. a r t i c l e i n f o Article history: Received 5 January 2020 Received in revised form 1 February 2020 Accepted 3 February 2020 Available online 8 February 2020 Keywords: Anecic worms Endogeic worms Epigeic worms Forest ﬂoor mass Litter decomposition Soil carbon Article history: Received 5 January 2020 Received in revised form 1 February 2020 Accepted 3 February 2020 Available online 8 February 2020 This paper describes data of earthworm abundance and functional group diversity regulate plant litter decay and soil organic carbon (SOC) level in global terrestrial ecosystems. The data also describes the potential effect of vegetation types, litter quality, litterbag mesh size, soil C/N, soil aggregate size, experimental types and length of experimental time on earthworm induced plant litter and SOC decay. The data were collected from 69 studies published between 1985 and 2018, covering 340 observations. This data article is related to the paper “Earthworm Abundance and Func- tional Group Diversity Regulate Plant Litter Decay and Soil Organic Carbon Level: A Global Meta-analysis” [1]. Keywords: Anecic worms Endogeic worms Epigeic worms Forest ﬂoor mass Litter decomposition Soil carbon © 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4.0/). W. Huang et al. / Data in brief 29 (2020) 105263 2 Subject Ecology, Soil Science Speciﬁc subject area Earthworm ecology, litter decomposition, soil carbon Type of data Table How data were acquired Systematic review of the literature Data format Raw Parameters for data collection We used three different combinations of keywords: earthworm and litter decomposition; earthworm and forest ﬂoor; earthworm and soil carbon. Description of data collection Data were collected from the ISI-Web of Science and Google Scholar. Data source location 18 countries over ﬁve continents Data accessibility With the article Related research article Wei Huang, Grizelle Gonzalez, Xiaoming Zou, Earthworm Abundance and Functional Group Diversity Regulate Plant Litter Decay and Soil Organic Carbon Level: A Global Meta-analysis, Applied Soil Ecology, in press, https://doi.org/10.1016/j.apsoil.2019. 103473. [1] Ecology, Soil Science Earthworm ecology, litter decomposition, soil carbon Table Systematic review of the literature Raw We used three different combinations of keywords: earthworm and litter decomposition; earthworm and forest ﬂoor; earthworm and soil carbon. Data were collected from the ISI-Web of Science and Google Scholar. 2. Experimental design, materials, and methods A data set was compiled using literature search of peer-reviewed publications about the effects of earthworms on litter decomposition or SOC from the ISI-Web of Science and Google Scholar research database. We used three different combinations of keywords: earthworm and litter decomposition; earthworm and forest ﬂoor; earthworm and soil carbon. A total of 69 studies published between 1985 and 2018 were found (Tables 1e5). An Engauge Digitizer (Free Software Foundation, Inc., Boston, MA, United States of America) was used to extract numerical values from ﬁgures in selected articles in which data were graphically presented. For Table 1, we included studies that reported earthworm density and litter decomposition/decay rate; 40 observations from 13 studies were found. For Table 3, we included studies that reported earthworm density and forest ﬂoor thickness or carbon stock; 32 observations from 12 studies were found. For Table 4, we included studies that reported earthworm density and soil carbon content (%, g C/kg soil or mg C/g soil); 70 observations from 12 studies were found. For Tables 1, 3 and 4, we included studies that reﬂected earthworm density under ﬁeld conditions (i.e. earthworms were not reduced or added), and plant litter from the vegetation currently under the experimental sites so that these ob- servations can reﬂect the balance between earthworm density and turnover of plant litter, SOC under ﬁeld conditions. W. Huang et al. / Data in brief 29 (2020) 105263 3 3 Table 1 Location, earthworm density, plant litter decomposition rate, and earthworm functional group in crop ﬁelds, tree plantations and forests worldwide for curve estimation. 2. Experimental design, materials, and methods Location Ecosystem Earthworm density (no./m2) Annual litter decomposition rate (y1) Earthworm function group Reference Georgia, USA Crop Soy bean 176 1.67 Mixture [3] Rye 176 1.45 Mixture Queensland, Australia Sugarcane 199 1.88 Endogeic [4] Plantation Dublin, Ireland Salix 189 1.69 Mixture [5] Carlshead, UK Short Rotation Forestry 152 0.91 Mixture [6] Natural forest Puerto Rico, USA Tabonuco (Upland) 45 1.47 Mixture [7] Tabonuco (Riparian) 16 0.94 Mixture Anduze, France Chestnut 86 1.50 Mixture [8,9] 86 0.55 Mixture 86 1.10 Mixture 86 0.64 Mixture 4 0.71 Anecic 4 0.56 Anecic 4 0.50 Anecic 4 0.37 Anecic 28 0.52 Mixture 28 0.52 Mixture 28 0.48 Mixture 28 0.25 Mixture Skane, Sweden Beech 2.5 0.33 Epigeic [10] 39.8 0.60 Mixture 219.7 2.15 Mixture Hawaii, USA Metrosiderus 21 0.37 Mixture [11,12] Puerto Rico, USA Tabonuco (Control) 168.8 1.12 Mixture [13] Tabonuco (Fertilization) 29.33 0.84 Endogeic Subtropical lower montane rain forest (Control) 12 0.7 mixture Subtropical lower montane rain forest (Fertilization) 19 1.49 Mixture Ontario, Canada Sugar maple and American beech 67.675 0.39 Mixture [14] Colorado, USA Aspen Forest 44.44 0.36 Mixture [15] 44.44 0.31 Mixture Pine Forest 0.77 0.29 Epigeic 0.77 0.25 Epigeic New York State, USA Sugar maple 79.6 1.05 Mixture [16] 26.5 0.51 Mixture 99.4 1.27 Mixture 26.1 0.6 Mixture Oak 81.6 0.96 Mixture 26.4 0.53 Mixture 92.6 1.16 Mixture 21.5 0.63 Mixture Table 1 i rthworm density, plant litter decomposition rate, and earthworm functional group in crop ﬁelds, tree plantations an dwide for curve estimation. recipitation (MAP), experimental type, experimental duration, earthworm functional group, earthworm numbers, litter omposition in the meta-analysis. MAP (mm) Experimental type Experimental period (days) Earthworm functional group Litter type Litter C/N Litter bag mesh size (mm) Effect size References 3500 Field 365 Endogeic Leaf 26 1 2.62 [17] 3500 Field 365 Endogeic Root 101 1 1.10 3456 Field 365 Mixture Leaf 32 1 1.22 3456 Field 365 Mixture Root 101 1 1.12 Field 240 Mixture Leaf 10 2.29 [18] Field 240 Mixture Leaf 1 1.12 1212 Field 760 Mixture Leaf 5 2.33 [8] [9] Field 760 Mixture Leaf 5 1.75 Field 760 Mixture Leaf 5 2.42 Field 760 Mixture Leaf 5 1.492 Field 365 Leaf 4 33.76 [19] Field 365 Leaf 4 2.32 Field 365 Leaf 4 1.95 Field 365 Leaf 4 1.64 Field 365 Leaf 4 9.81 Field 365 Leaf 4 3.73 Field 365 Leaf 4 2.33 Field 365 Leaf 4 2.56 Field 365 Leaf 4 2.79 Field 365 Leaf 4 0.77 Field 365 Leaf 4 1.73 Field 365 Leaf 4 0.94 Lab 56 Epigeic Leaf 10.1 2.53 [20] Field 56 Epigeic Leaf 10.1 1.98 1000 Field 190 Mixture Leaf 10 0.98 [21] Field 190 Mixture Leaf 10 1.077 Field 190 Mixture Leaf 10 1.027 Field 190 Mixture Leaf 10 1.11 Field 340 Mixture Leaf 10 1.35 Field 340 Mixture Leaf 10 1.51 Field 340 Mixture Leaf 10 2.58 Field 340 Mixture Leaf 10 1.53 Field 540 Mixture Leaf 10 1.68 Field 540 Mixture Leaf 10 2.41 Field 540 Mixture Leaf 10 1.56 Field 540 Mixture Leaf 10 2.59 W. Huang et al. / Data in brief 29 (2020) 105263 4 r W. Huang et al. / Data in brief 29 (2020) 105263 W. Huang et al. / Data in brief 29 (2020) 105263 g [ ] Lab 97 Endogeic Leaf 32 1.03 Lab 97 Endogeic Leaf 34 1.07 Lab 97 Endogeic Leaf 42 1.04 Lab 97 Endogeic Leaf 27 0.78 Lab 97 Endogeic Leaf 32 0.89 Lab 97 Endogeic Leaf 34 1.00 Lab 97 Endogeic Leaf 42 0.98 Lab 84 Endogeic Leaf 34.7 0.96 [25] Lab 84 Epigeic Leaf 34.7 1.00 Lab 84 Epigeic Leaf 34.7 1.43 Lab 84 Mixture Leaf 34.7 1.02 Lab 84 Mixture Leaf 34.7 1.09 Lab 84 Epigeic Leaf 34.7 1.12 Lab 84 Epigeic Leaf 34.7 1.32 Lab 84 Endogeic Leaf 34.7 1.11 Lab 84 Endogeic Leaf 27.2 0.95 Lab 84 Epigeic Leaf 27.2 1.04 Lab 84 Epigeic Leaf 27.2 1.97 Lab 84 Mixture Leaf 27.2 1.02 Lab 84 Mixture Leaf 27.2 1.31 Lab 84 Epigeic Leaf 27.2 1.25 Lab 84 Epigeic Leaf 27.2 2.05 Lab 84 Endogeic Leaf 27.2 1.56 Field 123 Anecic Leaf 4.62 [26] Lab 42 Anecic Leaf 1.50 [27] Lab 42 Epigeic Leaf 2.35 Lab 42 Mixture Leaf 2.80 Field 82 Anecic Leaf 1.06 Field 82 Epigeic Leaf 1.47 Field 82 Mixture Leaf 1.37 Lab 28 Epigeic Leaf 1.07 [28] Lab 28 Epigeic Leaf 1.11 Lab 28 Epigeic Leaf 1.17 Lab 28 Epigeic Leaf 1.21 (continued on next page) W. Huang et al. / Data in brief 29 (2020) 105263 5 [23] [24] Lab 126 Lab 126 Lab 63 Lab 63 Lab 63 Lab 97 Lab 97 Lab 97 Lab 97 Lab 97 Lab 97 Lab 97 Lab 97 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 126 Lab 126 Lab 63 Lab 63 Lab 63 Lab 97 Lab 97 Lab 97 Lab 97 Lab 97 Lab 97 Lab 97 Lab 97 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 Lab 84 MAP (mm) Experimental type Experimental period (days) Earthworm functional group Litter type Litter C/N Litter bag mesh size (mm) Effect size References Lab 56 Anecic Leaf 2.12 [29] Lab 56 Anecic Leaf 2.68 Lab 56 Anecic Leaf 3.15 Lab 56 Anecic Leaf 3.26 Lab 56 Anecic Leaf 2.67 Lab 56 Anecic Leaf 4.00 Lab 56 Anecic Leaf 13.28 Lab 56 Anecic Leaf 6.28 Lab 56 Anecic Leaf 1.34 Lab 56 Anecic Leaf 1.06 Lab 56 Anecic Leaf 35.85 Lab 56 Anecic Leaf 2.15 Lab 56 Anecic Leaf 5.95 Lab 56 Anecic Leaf 1.33 Lab 56 Anecic Leaf 2.18 Lab 56 Anecic Leaf 4.72 Lab 56 Anecic Leaf 9.63 Lab 56 Anecic Leaf 1.16 Lab 56 Anecic Leaf 1.20 Lab 56 Anecic Leaf 1.56 Lab 56 Anecic Leaf 1.80 Lab 56 Anecic Leaf 3.34 Lab 56 Anecic Leaf 11.36 Lab 56 Anecic Leaf 6.97 Lab 56 Anecic Leaf 12.36 Lab 22 Mixture Leaf 2.10 [30] 630 Field 365 Mixture Leaf 32.5 2.26 [31] Field 365 Mixture Leaf 39.5 1.51 1000 Field 365 Mixture Leaf 39.5 5 5.28 [6] Field 365 Mixture Leaf 52 5 8.15 Field 365 Mixture Leaf 33 5 12.44 Field 365 Mixture Leaf 32.5 5 10.41 Field 261 Mixture Leaf 18.2 5 17.56 Lab 120 Epigeic Leaf 1.35 [32] Lab 120 Epigeic Leaf 1.07 Lab 120 Epigeic Leaf 2.50 Lab 90 Epigeic Leaf 1.24 [33] W. Huang et al. / Data in brief 29 (2020) 105263 6 W. Huang et al. / Data in brief 29 (2020) 105263 7 Table 3 Location, earthworm density, and forest ﬂoormass thickness and carbon stock in forests worldwide for curve estimation. Location Earthworm density (no./m2) Forest ﬂoormass References Thickness (cm) Carbon stock (g/m2) Minnesota, USA 592.00 0.60 [34] Minnesota, USA 821.47 1.14 [35] Ontario, Canada 99.50 2.70 [36] Alberta, Canada 622.72 4.19 [37] 181.59 3.66 108.14 3.57 136.42 3.49 162.75 2.64 214.18 1.01 196.08 0.97 623.02 0.20 458.67 0.12 661.73 0.04 Maryland, USA 212.00 1.00 116.00 [38] Maryland, USA 38.00 6.25 [39] Michigan, USA 9.10 895.60 [40] 247.80 316.20 New York State, USA 106.30 211.20 [41] 76.83 70.40 New York State, USA 150.00 196.34 [42] 89.20 295.39 Puerto Rico, USA 32.67 785.10 [43] 56.00 406.40 8.76 563.90 Jilin, China 780 1.0 [44] 336 2.5 153 2.0 52 1.5 Yunan, China 28.5 1.5 [45] 12.35 0.5 7.5 1 ensity, and forest ﬂoormass thickness and carbon stock in forests worldwide for curve estimation. To be included in the meta-analysis, the paper had to report the means, standard deviation (SDs) and replicate numbers of litter percent mass loss or SOC for the control treatment (C, with no earthworms or reduced earthworm number) and the experimental treatment (E, with earthworms or earthworm number do not reduce). For studies that did not report SD or standard error (SE), we conservatively estimated SD values as 150% of the average variance across the dataset [2]. To evaluate the signiﬁcance of the earthworm-induced effect on litter decomposition, 113 observations from 20 studies were found (Table 2). For the magnitude of the earthworm-induced effect on SOC content, 120 observations from 22 studies were found (Table 5). Because most of the studies do not report soil bulk density, we therefore converted SOC stocks with known bulk density (20 obser- vations) to SOC concentrations. Besides earthworm functional groups, other details of experimental conditions were also speciﬁed in our analyses. We included studies that reported climate, vege- tation types (naturally-grown forest, plantation, pastureland and crop), litter quality (litter C/N ratio and leaf versus root litter), litterbag mesh size, time length of experiment, soil depth, soil aggregate size, soil C/N ratio and experimental types (ﬁeld versus laboratory). These parameters were the controlling factors that we considered for the earthworm effect on litter decay and SOC. The magnitude of the earthworm-induced effect on litter decay and SOC were calculated as the response ratio (R), R ¼ E/C, where E and C are the means of experimental and control treatments, respectively. ation in 12 sites of crop ﬁelds, pasture, and forests worldwide used for curve estimation. Earthworm density (no./m2) Soil depth (cm) Soil organic C concentration (%) Earthworm functional group References 17.9 0e10 16.1 Mixture [46] 10e20 12.4 20e30 12.3 30e40 8.8 30 0e20 8.04 Anecic [47] 9.09 y 9.33 2.26 [48] 14.76 2.16 9.33 2.16 13.33 2.10 26.67 2.53 0e15 [49] 46.05 1.2 Mixture 52.85 1.4 Mixture 40.5 1.0 Mixture 45.8 1.1 Mixture 75.5 1.1 Mixture 27.35 1.3 Mixture 36.75 1.1 Mixture 12 0e25 7.55 Endogeic [50] 151 8.52 Endogeic 154 8.80 Endogeic 398 9.86 Endogeic 119.3 0e10 1.56 Mixture [51] 10e20 1.52 20e30 0.87 113.3 0e10 1.79 Mixture 10e20 1.22 20e30 0.75 160 0e10 1.94 Mixture 10e20 1.23 20e30 0.74 132.7 0e10 1.71 Mixture 10e20 1.14 20e30 0.68 157.3 0e10 1.75 Mixture 10e20 1.15 20e30 0.67 W. Huang et al. / Data in brief 29 (2020) 105263 ntration in 12 sites of crop ﬁelds, pasture, and forests worldwide used for curve estimation. Earthworm density (no./m2) Soil depth (cm) Soil organic C concentration (%) Earthworm functional group References 17.9 0e10 16.1 Mixture [46] 10e20 12.4 20e30 12.3 30e40 8.8 30 0e20 8.04 Anecic [47] 9.09 rley 9.33 2.26 [48] 14.76 2.16 9.33 2.16 13.33 2.10 26.67 2.53 0e15 [49] 46.05 1.2 Mixture 52.85 1.4 Mixture 40.5 1.0 Mixture 45.8 1.1 Mixture 75.5 1.1 Mixture 27.35 1.3 Mixture 36.75 1.1 Mixture 12 0e25 7.55 Endogeic [50] 151 8.52 Endogeic 154 8.80 Endogeic 398 9.86 Endogeic ey, 119.3 0e10 1.56 Mixture [51] 10e20 1.52 20e30 0.87 113.3 0e10 1.79 Mixture 10e20 1.22 20e30 0.75 160 0e10 1.94 Mixture 10e20 1.23 20e30 0.74 132.7 0e10 1.71 Mixture 10e20 1.14 20e30 0.68 157.3 0e10 1.75 Mixture 10e20 1.15 20e30 0.67 W. Huang et al. / Data in brief 29 (2020) 105263 ef 29 (2020) 105263 0e30 4.94 Mixture [52] 0e10 3.74 Mixture [53] 3.12 Mixture 2.56 Epigeic 3.21 Mixture 2.68 Mixture 3.06 Epigeic 0e7.5 0.93 [54] 0.94 0.96 0e5 3.98 Mixture [55] 5e10 4.10 10e18 3.30 18e26 3.20 0e10 7.58 Mixture [53] 5.79 8.07 Mixture 0e5 5.75 Mixture [39,40] 5e10 2.63 10e15 1.65 15e20 1.43 0e5 6.97 Mixture 5e10 4.12 10e15 1.93 15e20 1.71 0e15 3.59 Endogeic [56] 0e30 5.24 Mixture [52] 0e10 3.53 Endogeic [53] 4.45 Mixture 5.62 Endogeic 5.51 Mixture 0e25 8.90 Mixture [50] 9.43 Mixture W. Huang et al. / Data in brief 29 (2020) 105263 9 0e10 3.74 Mixture [53] 3.12 Mixture 2.56 Epigeic 3.21 Mixture 2.68 Mixture 3.06 Epigeic 0e7.5 0.93 [54] 0.94 0.96 0e5 3.98 Mixture [55] 5e10 4.10 10e18 3.30 18e26 3.20 0e10 7.58 Mixture [53] 5.79 8.07 Mixture 0e5 5.75 Mixture [39,40] 5e10 2.63 10e15 1.65 15e20 1.43 0e5 6.97 Mixture 5e10 4.12 10e15 1.93 15e20 1.71 0e15 3.59 Endogeic [56] 0e30 5.24 Mixture [52] 0e10 3.53 Endogeic [53] 4.45 Mixture 5.62 Endogeic 5.51 Mixture 0e25 8.90 Mixture [50] 9.43 Mixture W. Huang et al. / Data in brief 29 (2020) 105263 9 m functional group, earthworm number, soil depth, soil C/N and soil aggregate size for observations about the effects of Experimental type Earthworm functional group Soil depth (cm) Experimental period Soil C/N Soil aggregate size Effect size of soil organic carbon References Field Mixture 0 - 5 730 13.3 0.62 [41] Mixture 5 - 10 730 11.6 0.81 Mixture 10 - 15 730 10.1 0.62 Mixture 15 - 20 730 10.0 0.65 Mixture 0 - 5 730 0.75 Mixture 5 - 10 730 1.27 Mixture 10 - 15 730 0.72 Mixture 15 - 20 730 0.78 Field Mixture 0 - 5 730 0.86 [57] Mixture 5 - 10 730 1.10 Mixture 10 - 15 730 0.62 Mixture 15 - 20 730 0.72 Field Anecic 0 - 5 10950 0.82 [55] 5 - 10 10950 0.75 10 - 18 10950 0.58 18 - 26 10950 0.82 0 - 5 7300 0.98 5 - 10 7300 1.06 10 - 18 7300 1.05 18 - 26 7300 1.24 Field 0 - 3 18.73 1.34 [42] 3 - 6 17.53 1.14 6 - 9 16.80 1.08 9 - 12 15.84 0.96 0 - 3 13.59 1.17 3 - 6 11.83 0.99 6 - 9 11.59 1.05 9 - 12 11.18 0.95 Lab 0 - 8 110 1.06 [58] Lab Endogeic 26 >250 2.05 [59] Endogeic 26 53e250 0.78 Endogeic 26 <53 1.30 Epigeic 26 >250 3.60 Epigeic 26 53e250 0.96 Epigeic 26 <53 1.13 Field Mixture 0 - 10 1075 1.11 [46] Mixture 10 - 20 1075 1.19 Mixture 20 - 30 1075 1.01 Mixture 30 - 40 1075 1.02 W. Huang et al. / Data in brief 29 (2020) 105263 10 W. Huang et al. / Data in brief 29 (2020) 105263 W Huang et al / Data in brief 29 (2020) 105263 eld Anecic 0 - 20 2555 8.30 1.02 [47] 2555 1.02 eld 0e10 14.00 1.56 [60] 10e20 13.30 1.50 eld Endogeic 0e5 600 11.80 0.94 [61] 5e15 600 11.80 1.05 0e5 600 11.80 0.72 5e15 600 11.80 1.45 Endogeic 0e10 0.67 [62] 10e20 1.31 20e30 1.00 b Endogeic 20 >2000 3.42 [63] 20 250e2000 0.52 b Endogeic 20 >2000 3.12 [64] 20 250e2000 0.78 20 53e250 0.71 20 <53 0.61 b Epigeic 23 0.92 [65] 23 0.89 23 >2000 10.25 23 >2000 5.32 23 250e2000 0.59 23 250e2000 0.80 23 53e250 0.08 23 53e250 0.66 b Mixture 42 14.88 1.01 [66] 42 14.31 1.06 42 15.25 0.99 42 15.25 1.03 b Endogeic 0e3.5 37 1.03 [67] Epigeic 3.5e7 37 1.09 Endogeic 0e3.5 37 0.98 Epigeic 3.5e7 37 1.08 b Epigeic 1e4 28 1.03 [68] 1e4 56 0.89 1e4 84 0.96 1e4 28 0.73 1e4 56 0.89 1e4 84 0.70 4e7 28 0.94 4e7 56 0.90 4e7 84 1.00 4e7 28 0.79 4e7 56 1.00 (continued on next page) W. Huang et al. / Data in brief 29 (2020) 105263 11 mental Earthworm functional group Soil depth (cm) Experimental period Soil C/N Soil aggregate size Effect size of soil organic carbon References 4e7 84 0.68 >7 28 1.16 >7 56 1.29 >7 84 1.04 >7 28 1.60 >7 56 1.23 >7 84 1.94 0e2.5 30 0.95 [69] 0e2.5 30 1.12 0e2.5 30 0.94 0e2.5 30 1.18 2.5e5 30 1.03 2.5e5 30 0.77 2.5e5 30 0.95 2.5e5 30 1.14 Anecic 40 0.96 [70] 40 0.77 40 <250 1.10 40 250e1000 0.79 40 1000e2000 1.21 40 >2000 1.19 compost 18 13.04 1.04 [71] 18 13.04 1.15 18 13.04 1.04 35 14.09 1.12 35 14.09 1.10 35 14.09 1.08 Anecic 22 0.98 [30] Endogeic 22 1.01 Endogeic 22 0.94 Mixture 22 0.99 Mixture 22 0.97 Mixture 22 0.97 Mixture 22 0.97 Endogeic 365 1.02 [72] Endogeic 365 0.82 Endogeic 365 0.81 W. Huang et al. / Data in brief 29 (2020) 105263 12 ganic 8 6 9 W. Huang et al. / Data in brief 29 (2020) 105263 13 Acknowledgments This work was ﬁnancially supported by a cooperative agreement between the USDA-Forest Service International Institute of Tropical Forestry and the University of Puerto Rico [14-JV-11120101-018, 2015]. Grizelle Gonzalez was supported by the Luquillo Critical Zone Observatory [EAR-1331841] and the Luquillo Long-Term Ecological Research Site [DEB-1239764]. Conﬂict of Interest The authors declare that they have no known competing ﬁnancial interests or personal relation- ships that could have appeared to inﬂuence the work reported in this paper. References [14] B.W. Jennings, S.A. Watmough, The impact of invasive earthworms on soil respiration and soil carbon with hardwood forests, Ecosystems 19 (2016) 942e954. , y ( ) [15] G. Gonzalez, T.R. Seastedt, Z. Donato, Earthworms, arthropods and plant litter decomposition in aspen (Populus trem- uloides) and lodgepole pine (Pinus contorta) forests in Colorado, USA, Pedobiologia 47 (2003) 863e869. [15] G. Gonzalez, T.R. Seastedt, Z. Donato, Earthworms, arthropods and plant litter decomposition in aspen (P uloides) and lodgepole pine (Pinus contorta) forests in Colorado, USA, Pedobiologia 47 (2003) 863e869. , , , , p p p p ( p oides) and lodgepole pine (Pinus contorta) forests in Colorado, USA, Pedobiologia 47 (2003) 863e869. [16] E.R. Suarez, T.J. Fahey, J.B. Yavitt, P.M. Groffman, P.J. Bohlen, Patterns of litter disappearance in a northern hardwood forest invaded by exotic earthworms, Ecol. Appl. 16 (2006) 154e165. [17] Z. Liu, X. Zou, Exotic earthworms accelerate plant litter decomposition in a Puerto Rican pasture and a we Appl. 12 (2002) 1406e1417. [18] K. Szlavecz, M. McCormick, L. Xia, J. Saunders, T. Morcol, et al., Ecosystem effects of non-native earthworms in Mid-Atlantic deciduous forests, Biol. Invasions 13 (2011) 1165e1182. [19] L. Heneghan, J. Steffen, K. Fagen, Interactions of an introduced shrub and introduced earthworms in an Illinois urban woodland: impact on leaf litter decomposition, Pedobiologia 50 (2007) 543e551. [20] C.O. Adejuyigbe, G. Tian, G.O. Adeoye, Microcosmic study of soil microarthropod and earthworm int decomposition and nutrient turnover, Nutrient Cycl. Agroecosyst. 75 (2006) 47e55. p y g y ( ) [21] P.J. Bohlen, P.M. Groffman, T.J. Fahey, M.C. Fisk, E. Suarez, et al., Ecosystem consequences of exotic earthworm invasion of north temperate forests, Ecosystems 7 (2004) 1e12. [ ] h l f b l h l f l b p y ( ) [22] J. Wu, H. Li, W. Zhang, F. Li, J. Huang, et al., Contrasting impacts of two subtropical earthworm species on leaf litter carbon sequestration into soil aggregates, J. Soils Sediments 17 (2017) 1672e1681. [23] N. Eisenhauer, S. Marhan, S. Scheu, Assessment of anecic behavior in selected earthworm species: effects o burial, seedling establishment, wheat growth and litter incorporation, Appl. Soil Ecol. 38 (2008) 79e82. [24] Y. Araujo, F.J. Luiz~ao, E. Barros, Effect of earthworm addition on soil nitrogen availability, microbial biom decomposition in mesocosms, Biol. Fertil. Soils 39 (2004) 146e152. p ( ) [25] J. Seeber, S. Scheu, E. References Rajapaksha, K.R. Butt, E. Vanguelova, A. Moffat, Effects of short rotation forestry on earthworm community develop- ment in the UK, For. Ecol. Manag. 309 (2013) 96e104. [6] N. Rajapaksha, K.R. Butt, E. Vanguelova, A. Moffat, Effects of short rotation forestry on earthworm community develop- ment in the UK, For. Ecol. Manag. 309 (2013) 96e104. [7] J. Dechaine, H. Ruan, L.Y. Sanchez-de, X. Zou, Correlation between earthworms and plant litter decomposition in a tropical wet forest of Puerto Rico, Pedobiologia 49 (2005) 601e607. wet forest of Puerto Rico, Pedobiologia 49 (2005) 601e6 g ( ) [8] J. Cortez, M. Bouche, Field decomposition of leaf litters: earthwormemicroorganism interactionsdthe plough Soil Biol. Biochem. 30 (1998) 795e804. ( ) [9] J. Cortez, Field decomposition of leaf litters: relationships between decomposition rates and soil moisture, soil temper- ature and earthworm activity, Soil Biol. Biochem. 30 (1998) 783e793. [10] H. Staaf, Foliage litter turnover and earthworm populations in three beech forests of contrasting soil and vegetation types, Oecologia 72 (1987) 58e64. g ( ) et, Alteration of earthworm community biomass by the alien Myrica faya in Hawai'i, Oecologia 82 (1990) 414e41 g ( ) [11] G. Aplet, Alteration of earthworm community biomass by the alien Myrica faya in Hawai'i, Oecologia 82 (1990) 414e416. [12] P.M. Vitousek, D.R. Turner, W.J. Parton, R.L. Sanford, Litter decomposition on the Mauna Loa environmental matrix, Hawai'i: patterns, mechanisms, and models, Ecology 75 (1994) 418e429. g ( ) [11] G. Aplet, Alteration of earthworm community biomass by the alien Myrica faya in Hawai'i, Oecologia 82 (19 [12] P.M. Vitousek, D.R. Turner, W.J. Parton, R.L. Sanford, Litter decomposition on the Mauna Loa environmental matrix, Hawai'i: patterns, mechanisms, and models, Ecology 75 (1994) 418e429. [13] X. Yang, M. Warren, X. Zou, Fertilization responses of soil litter fauna and litter quantity, quality, and turnov high elevation forests of Puerto Rico, Appl. Soil Ecol. 37 (2007) 63e71. [13] X. Yang, M. Warren, X. Zou, Fertilization responses of soil litter fauna and litter quantity, quality, and turnover in low and high elevation forests of Puerto Rico, Appl. Soil Ecol. 37 (2007) 63e71. [14] B.W. Jennings, S.A. Watmough, The impact of invasive earthworms on soil respiration and soil carbon within temperate high elevation forests of Puerto Rico, Appl. Soil Ecol. 37 (2007) 63e71. [14] B.W. Jennings, S.A. Watmough, The impact of invasive earthworms on soil respiration and soil carbon within temperate hardwood forests, Ecosystems 19 (2016) 942e954. References [1] W. Huang, G. Gonzalez, X. Zou, Earthworm abundance and functional group diversity regulate plant litter decay and soil organic carbon level: a global meta-analysis, Appl. Soil Ecol. (2019), https://doi.org/10.1016/j.apsoil.2019.103473. [1] W. Huang, G. Gonzalez, X. Zou, Earthworm abundance and functional group diversity regulate plant litter decay and soil organic carbon level: a global meta-analysis, Appl. Soil Ecol. (2019), https://doi.org/10.1016/j.apsoil.2019.103473. [2] I.M. Lubbers, K.J. van Groenigen, S.J. Fonte, J. Six, L. Brussaard, et al., Greenhouse-gas emissions from soils earthworms, Nat. Clim. Change 3 (2013) 187e194. [2] I.M. Lubbers, K.J. van Groenigen, S.J. Fonte, J. Six, L. Brussaard, et al., Greenhouse-gas emissions from soils increased by earthworms, Nat. Clim. Change 3 (2013) 187e194. [3] R. Parmelee, M. Beare, W. Cheng, P. Hendrix, S. Rider, et al., Earthworms and enchytraeids in conventional and no-tillage agroecosystems: a biocide approach to assess their role in organic matter breakdown, Biol. Fertil. Soils 10 (1990) 1e10. [4] A. Spain, M. Hodgen, Changes in the composition of sugarcane harvest residues during decomposition as a surface mulch, Biol. Fert. soils 17 (1994) 225e231. agroecosystems: a biocide approach to assess their role in organic matter breakdown, Biol. Fertil. Soils 10 (1990) 1e10. [4] A. Spain, M. Hodgen, Changes in the composition of sugarcane harvest residues during decomposition as a surface mulch, Biol. Fert. soils 17 (1994) 225e231. agroecosystems: a biocide approach to assess their role in organic matter breakdown, Biol. Fertil. Soils 10 (1990) 1e10. [4] A. Spain, M. Hodgen, Changes in the composition of sugarcane harvest residues during decomposition as a surface mulch, Biol. Fert. soils 17 (1994) 225e231. ( ) [5] J. Curry, M. Kelly, T. Bolger, Role of invertebrates in the decomposition of Salix litter in reclaimed cutover peat, in: A.H. Fitter (Ed.), Ecological Interactions in the Soil Special Publication Number 4 of the British Ecological Society, Blackwell Scientiﬁca Publications, London, 1985, pp. 393e397. [5] J. Curry, M. Kelly, T. Bolger, Role of invertebrates in the decomposition of Salix litter in reclaimed cutover peat, in: A.H. Fitter (Ed.), Ecological Interactions in the Soil Special Publication Number 4 of the British Ecological Society, Blackwell Scientiﬁca Publications, London, 1985, pp. 393e397. [5] J. Curry, M. Kelly, T. Bolger, Role of invertebrates in the decomposition of Salix litter in reclaimed cutover peat, in: A.H. Fitter (Ed.), Ecological Interactions in the Soil Special Publication Number 4 of the British Ecological Society, Blackwell Scientiﬁca Publications, London, 1985, pp. 393e397. [6] N. References [43] M W W X Z S il f d li i i h i l l i di b d i i P [43] M.W. Warren, X. Zou, Soil macrofauna and litter nutrients in three tropical tree plantations on a disturbed site in Puerto Rico, For. Ecol. Manag. 170 (2002) 161e171. g ( ) [44] Y. Xiu, Q. Li, Y. Ling, S. Bo, The relation and difference of nutritional elements in forest litter-macrofaun Chinese Geo. Res. 25 (2006) 320e326. ( ) [45] S. Wang, H. Wang, W. Li, Effect of different land use types on spatial-temporal distribution of earthworm denstiy and biomass, Chinese J. Ecol. 36 (2017) 118e123. [46] W.D. Shuster, S. Subler, E. McCoy, Deep-burrowing earthworm additions changed the distribution of soil organic carbon in a chisel-tilled soil, Soil Biol. Biochem. 33 (2001) 983e996. [47] J. Zhang, F. Hu, H. Li, Q. Gao, X. Song, et al., Effects of earthworm activity on humus composition and humic acid char- acteristics of soil in a maize residue amended riceewheat rotation agroecosystem, Appl. Soil Ecol. 51 (2011) 1e8. [48] M. Iordache, I. Borza, Relation between chemical indices of soil and earthworm abundance under chemical fertilization, [47] J. Zhang, F. Hu, H. Li, Q. Gao, X. Song, et al., Effects of earthworm activity on humus composition and humic acid char- acteristics of soil in a maize residue amended riceewheat rotation agroecosystem, Appl. Soil Ecol. 51 (2011) 1e8. [48] M. Iordache, I. Borza, Relation between chemical indices of soil and earthworm abundance under chemical fertilization, Plant Soil Environ. 56 (2010) 401e407. [48] M. Iordache, I. Borza, Relation between chemical indices of soil and earthworm abundance under chemic Plant Soil Environ. 56 (2010) 401e407. [49] A.J. Ashworth, F.L. Allen, D.D. Tyler, D.H. Pote, M.J. Shipitalo, Earthworm populations are affected from long-term crop sequences and bio-covers under no-tillage, Pedobiologia 60 (2017) 27e33. [50] X. Zou, M. Bashkin, Soil carbon accretion and earthworm recovery following revegetation in abandoned sugarcane ﬁelds, Soil Biol. Biochem. 30 (1998) 825e830. ( ) [51] G. Ernst, C. Emmerling, Impact of ﬁve different tillage systems on soil organic carbon content and the densit community composition of earthworms after a ten year period, Eur. J. Soil Biol. 45 (2009) 247e251. [52] T.S.H. Kumar, M. Siddaraju, C.H.K. Bhat, K.S. References ] l d d h ff h l d [37] N. Eisenhauer, S. Partsch, D. Parkinson, S. Scheu, Invasion of a deciduous forest by earthworms: Changes in soil chemistry, microﬂora, microarthropods and vegetation, Soil Biol. Biochem. 39 (2007) 1099e1110. [38] K. Szlavecz, C. Csuzdi, Land use change affects earthworm communities in Eastern Maryland, USA, Eur. J. Soil Biol. 43 8] K. Szlavecz, C. Csuzdi, Land use change affects earthworm communities in Eastern Maryland, USA, Eur. J. S (2007) S79eS85. ( ) [39] Y. Ma, T.R. Filley, C.T. Johnston, S.E. Crow, K. Szlavecz, et al., The combined controls of land use legacy and earthworm activity on soil organic matter chemistry and particle association during afforestation, Org. Geochem. 58 (2013) 56e68. a, T.R. Filley, C.T. Johnston, S.E. Crow, K. Szlavecz, et al., The combined controls of land use legacy and earthw vity on soil organic matter chemistry and particle association during afforestation, Org. Geochem. 58 (2013) 56e l l f l d [40] K.J. McFarlane, M.S. Torn, P.J. Hanson, R.C. Porras, C.W. Swanston, et al., Comparison of soil organic matter dynamics at ﬁve temperate deciduous forests with physical fractionation and radiocarbon measurements, Biogeochemistry 112 (2013) 457e476. [40] K.J. McFarlane, M.S. Torn, P.J. Hanson, R.C. Porras, C.W. Swanston, et al., Comparison of soil organic matter dynamics at ﬁve temperate deciduous forests with physical fractionation and radiocarbon measurements, Biogeochemistry 112 (2013) 457e476. [41] T J Fahey J B Yavitt R E Sherman J C Maerz PM Groffman et al Earthworm effects on the incorporation of litter C and N [ ] J , , J , , , , p g y temperate deciduous forests with physical fractionation and radiocarbon measurements, Biogeochemistry 112 (2013) 457e476. [41] T.J. Fahey, J.B. Yavitt, R.E. Sherman, J.C. Maerz, P.M. Groffman, et al., Earthworm effects on the incorporation of litter C and N i il i i l f E l A l 23 (2013) 1185 1201 [41] T.J. Fahey, J.B. Yavitt, R.E. Sherman, J.C. Maerz, P.M. Groffman, et al., Earthworm effects on the incorporation o into soil organic matter in a sugar maple forest, Ecol. Appl. 23 (2013) 1185e1201. g g p pp ( ) [42] P.J. Bohlen, D.M. Pelletier, P.M. Groffman, T.J. Fahey, M.C. Fisk, Inﬂuence of earthworm invasion on redistribution and retention of soil carbon and nitrogen in northern temperate forests, Ecosystems 7 (2004) 13e27. References Meyer, Effects of macro-decomposers on litter decomposition and soil properties in alpine pastureland: a mesocosm experiment, Appl. Soil Ecol. 34 (2006) 168e175. [26] J. Qiu, M.G. Turner, Effects of non-native Asian earthworm invasion on temperate forest and prairie soils in the Mid- western US, Biol. Invasions 19 (2016) 73e88. 14 W. Huang et al. / Data in brief 29 (2020) 105263 7] H.G. Greiner, D.R. Kashian, S.D. Tiegs, Impacts of invasive Asian (Amynthas hilgendorﬁ) and European (Lumbric earthworms in a North American temperate deciduous forest, Biol. Invasions 14 (2012) 2017e2027. p ( ) [28] F. Kitz, M. Steinwandter, M. Traugott, J. Seeber, Increased decomposer diversity accelerates and potentially stabilises litter decomposition, Soil Biol. Biochem. 83 (2015) 138e141. [29] G. Patoine, M.P. Thakur, J. Friese, C. Nock, L. Honig, et al., Plant litter functional diversity effects on litter mass loss depend on the macro-detritivore community, Pedobiologia 65 (2017) 29e42. [30] C. Huang, G. Gonzalez, P.F. Hendrix, Resource utilization by native and invasive earthworms and their effects on soil carbon and nitrogen dynamics in Puerto Rican soils, Forests 7 (2016) 277. 1] N.S.S. Rajapaksha, K.R. Butt, E.I. Vanguelova, A.J. Moffat, Short rotation forestry e earthworm interactions: a mesocosm experiment, Appl. Soil Ecol. 76 (2014) 52e59. [32] J. Seeber, G. Seeber, R. Langel, S. Scheu, E. Meyer, The effect of macro-invertebrates and plant litter of different quality on the release of N from litter to plant on alpine pastureland, Biol. Fertil. Soils 44 (2008) 783e790. [33] D. Grubert, O. Butenschoen, M. Maraun, S. Scheu, Understanding earthworm e Collembola interactions and their importance for ecosystem processes needs consideration of species identity, Eur. J. Soil Biol. 77 (2016) 60e67. 4] D.H. Alban, E.C. Berry, Effects of earthworm invasion on morphology, carbon, and nitrogen of a forest soil, Appl (1994) 243e249. ( ) [35] C.M. Hale, L.E. Frelich, P.B. Reich, J. Pastor, Exotic earthworm effects on hardwood forest ﬂoor, nutrient availability and native plants: a mesocosm study, Oecologia 155 (2008) 509e518. p y g ( ) [36] T.E. Sackett, S.M. Smith, N. Basiliko, Indirect and direct effects of exotic earthworms on soil nutrient and carbon pools in North American temperate forests, Soil Biol. Biochem. 57 (2013) 459e467. 7] N. Eisenhauer, S. Partsch, D. Parkinson, S. Scheu, Invasion of a deciduous forest by earthworms: Changes in soi microﬂora, microarthropods and vegetation, Soil Biol. Biochem. 39 (2007) 1099e1110. References Sreepada, Seasonal distribution and abundance of earthworms (Annelida: Oligochaeta) in relation to the edaphic factors around Udupi Power Corporation Limited (UPCL), Udupi District, south- western coast of India, J. Threat. Taxa 10 (2018) 11432. J ( ) [53] R.J. Haynes, C.S. Dominy, M.H. Graham, Effect of agricultural land use on soil organic matter status and the composition of earthworm communities in KwaZulu-Natal, South Africa, Agric. Ecosyst. Environ. 95 (2003) 453e464. [ ] i ff f i ill f i il i bi l bi i d h [54] P. Haines, N. Uren, Effects of conservation tillage farming on soil microbial biomass, organic matter and earthworm populations, in north-eastern Victoria, Aust. J. Exp. Agric. 30 (1990) 365e371. [55] N.L. Schon, A.D. Mackay, R.A. Gray, M.B. Dodd, The action of an anecic earthworm (Aporrectodea longa) on vertical soil carbon distribution in New Zealand pastures several decades after their introduction, Eur. J. Soil Biol. 62 (2014) 101e104. [56] S.J. Fonte, E. Barrios, J. Six, Earthworms, soil fertility and aggregate-associated soil organic matter dynamics in the Quesungual agroforestry system, Geoderma 155 (2010) 320e328. g g y y ( ) [57] T.J. Fahey, J.B. Yavitt, R.E. Sherman, J.C. Maerz, P.M. Groffman, et al., Earthworms, litter and soil carbon in a northern hardwood forest, Biogeochemistry 114 (2012) 269e280. [58] L. Cole, R.D. Bardgett, P. Ineson, Enchytraeid worms (Oligochaeta) enhance mineralization of carbon in organic upland soils, Eur. J. Soil Sci. 51 (2000) 185e192. [59] L.Y. Sanchez-de, J. Lugo-Perez, D.H. Wise, J.D. Jastrow, M.A. Gonzalez-Meler, Aggregate formation and carbon sequestration by earthworms in soil from a temperate forest exposed to elevated atmospheric CO2: a microcosm experiment, Soil Biol. Biochem. 68 (2014) 223e230. ( ) nen, T.R. Moore, Exotic earthworm invasion increases soil carbon and nitrogen in an old-growth forest in southern Can. J. For. Res. 36 (2006) 845e854. ( ) [60] M. Wironen, T.R. Moore, Exotic earthworm invasion increases soil carbon and nitrogen in an old-growth for Quebec, Can. J. For. Res. 36 (2006) 845e854. W. Huang et al. / Data in brief 29 (2020) 105263 15 X. Zou, D.A. Schaefer, Alteration of soil labile organic carbon by invasive earthworms (Pontoscolex corethrurus) l rubber plantations, Eur. J. Soil Biol. 46 (2010) 74e79. [61] M. Zhang, X. Zou, D.A. Schaefer, Alteration of soil labile organic carbon by invasive earthworms (Pontosc in tropical rubber plantations, Eur. J. Soil Biol. 46 (2010) 74e79. [62] P. Lavelle, A. References Martin, Small-scale and large-scale effects of endogeic earthworms on soil organic matter dynamics in soils of the humid tropics, Soil Biol. Biochem. 24 (1992) 1491e1498. p ( ) [63] H. Bossuyt, J. Six, P. Hendrix, Rapid incorporation of carbon from fresh residues into newly formed stable microaggregates within earthworm casts, Eur. J. Soil Sci. 55 (2004) 393e399. [64] H. Bossuyt, J. Six, P.F. Hendrix, Protection of soil carbon by microaggregates within earthworm casts, Soil Biol. Biochem. 37 (2005) 251e258. ) ang, P.F. Hendrix, L.E. Dame, R.A. Burke, J. Wu, et al., Earthworms facilitate carbon sequestration through unequ ﬁcation of carbon stabilization compared with mineralization, Nat. Commun. 4 (2013) 2576. [66] G. Ernst, I. Henseler, D. Felten, C. Emmerling, Decomposition and mineralization of energy crop residues governed by earthworms, Soil Biol. Biochem. 41 (2009) 1548e1554. [67] Q. Zhang, P.F. Hendrix, Earthworm (Lumbricus rubellus and Aporrectodea caliginosa) effects on carbon ﬂux in soil, Soil Sci. Soc. Am. J. 59 (1995) 816e823. J ( ) [68] S. Scheu, D. Parkinson, Effects of earthworms on nutrient dynamics, carbon turnover and microorganisms cool temperate forests of the Canadian Rocky Mountains d laboratory studies, Appl. Soil Ecol. 1 (1994) 113 J ( ) eu, D. Parkinson, Effects of earthworms on nutrient dynamics, carbon turnover and microorganisms in soils fro emperate forests of the Canadian Rocky Mountains d laboratory studies, Appl. Soil Ecol. 1 (1994) 113e125. eu, D. Parkinson, Effects of earthworms on nutrient dynamics, carbon turnover and microorganisms in soils fro emperate forests of the Canadian Rocky Mountains d laboratory studies, Appl. Soil Ecol. 1 (1994) 113e125. p y y pp ( ) [69] Y. Guo, A. Liang, Y. Zhang, S. Zhang, X. Chen, et al., Evaluating the contributions of earthworms to soil organic carbon decomposition under different tillage practices combined with straw additions, Ecol. Indicat. 109 (2018) 516e524. [ ] h b h f h h f f b l p g p ( ) [70] Y. Wu, M. Shaaban, Q.A. Peng, A. Zhou, R. Hu, Impacts of earthworm activity on the fate of straw carbon in soil: a microcosm experiment, Environ. Sci. Pollut. Res. Int. 25 (2018) 11054e11062. [71] X. Zhu, L. Chang, J. Li, J. Liu, L. Feng, et al., Interactions between earthworms and mesofauna affect CO emissions from soils under long-term conservation tillage, Geoderma 332 (2017) 153e160. References [ ] , g, J , J , g, , emissions from soils under long-term conservation tillage, Geoderma 332 (2017) 153e160. [72] PT Ngo C Rumpel T T Doan P Jouquet The effect of earthworms on carbon storage and soil organic matter composition [72] P.T. Ngo, C. Rumpel, T.T. Doan, P. Jouquet, The effect of earthworms on carbon storage and soil organic matte in tropical soil amended with compost and vermicompost, Soil Biol. Biochem. 50 (2012) 214e220.
https://openalex.org/W4281760314	https://link.springer.com/content/pdf/10.1007/s00018-022-04325-y.pdf	English	null	Mitochondrial matrix-localized Src kinase regulates mitochondrial morphology	Cellular and molecular life sciences	2,022	cc-by	10,842	Abstract The architecture of mitochondria adapts to physiological contexts: while mitochondrial fragmentation is usually associated to quality control and cell death, mitochondrial elongation often enhances cell survival during stress. Understanding how these events are regulated is important to elucidate how mitochondrial dynamics control cell fate. Here, we show that the tyrosine kinase Src regulates mitochondrial morphology. Deletion of Src increased mitochondrial size and reduced cellular respiration independently of mitochondrial mass, mitochondrial membrane potential or ATP levels. Re-expression of Src targeted to the mitochondrial matrix, but not of Src targeted to the plasma membrane, rescued mitochondrial morphology in a kinase activity-dependent manner. These findings highlight a novel function for Src in the control of mitochondrial dynamics. Keywords Mitochondrial dynamics · Cellular respiration · Mitochondria-shaping protein · Oxidative Cellular and Molecular Life Sciences (2022) 79:327 https://doi.org/10.1007/s00018-022-04325-y Cellular and Molecular Life Sciences (2022) 79:327 https://doi.org/10.1007/s00018-022-04325-y Cellular and Molecular Life Sciences ORIGINAL ARTICLE Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology Olivier Lurette1,2 · Hala Guedouari1,2 · Jordan L. Morris3 · Rebeca Martín‑Jiménez1,2 · Julie‑Pier Robichaud1,2 · Geneviève Hamel‑Côté1,2 · Mehtab Khan1,2 · Nicholas Dauphinee1,2 · Nicolas Pichaud4 · Julien Prudent3 · Etienne Hebert‑Chatelain1,2 Received: 15 November 2021 / Revised: 19 April 2022 / Accepted: 22 April 2022 / Published online: 30 May 2022 © The Author(s) 2022 * Etienne Hebert‑Chatelain etienne.hebert.chatelain@umoncton.ca Introduction the metabolic state of the cell, to respond to cellular cues and maintain cellular homeostasis [1, 2]. For instance, mito- chondria elongate during mild stress [3, 4], whereas inhibi- tion of OXPHOS or mitochondrial depolarization induces mitochondrial fragmentation [5, 6]. These morphological transition states are mainly regulated by dynamin GTPase proteins with Dynamin-related protein 1 (Drp1), the main actor of mitochondrial division, Mitofusins 1 and 2 (Mfn1/2) and Optic Atrophy 1 (OPA1) controlling outer mitochon- drial membrane (OMM) and inner mitochondrial membrane (IMM) fusion, respectively [1, 7].i Mitochondria are considered as the cellular powerhouse due to their role in oxidative phosphorylation (OXPHOS) and ATP production, but they are also involved in other key physiological processes including Ca2+ homeostasis, apop- tosis and steroidogenesis. Mitochondria form networks into the cytosol which are constantly modified by fission and fusion events, a process known as mitochondrial dynamics [1]. Morphology of individual organelle can shift from tubu- lar to more fragmented or elongated structures according to The tyrosine kinase Src was among the first oncogenes to be described [8]. It is involved in various physiologi- cal and oncogenic processes, ranging from metabolism, proliferation and differentiation to survival, motility and angiogenesis [8, 9]. Src has been found at multiple sub- cellular compartments including endosomes and plasma membrane [10], the Golgi apparatus [11, 12] as well as mitochondria [13, 14]. Src targets various mitochondrial proteins involved in different steps of metabolism includ- ing OXPHOS, fatty acid oxidation, pyruvate metabolism, ketone body production and production of reactive oxygen species [13, 15–17]. We recently observed that metastatic triple-negative breast cancer cells show higher activity of intramitochondrial Src [16]. Interestingly, metastatic Olivier Lurette and Hala Guedouari share the first authorship. * Etienne Hebert‑Chatelain etienne.hebert.chatelain@umoncton.ca 1 Canada Research Chair in Mitochondrial Signaling and Physiopathology, Moncton, NB, Canada 2 Department of Biology, University of Moncton, Moncton, NB, Canada 3 Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, UK 4 Department of Chemistry and Biochemistry, University of Moncton, Moncton, NB, Canada Olivier Lurette and Hala Guedouari share the first authorship. Olivier Lurette and Hala Guedouari share the first authorship. * Etienne Hebert‑Chatelain etienne.hebert.chatelain@umoncton.ca 1 Canada Research Chair in Mitochondrial Signaling and Physiopathology, Moncton, NB, Canada 2 Department of Biology, University of Moncton, Moncton, NB, Canada 3 Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, UK 4 Department of Chemistry and Biochemistry, University of Moncton, Moncton, NB, Canada 4 Department of Chemistry and Biochemistry, University of Moncton, Moncton, NB, Canada Src decreases mitochondrial size Although indirect evidence suggests that Src might impact mitochondrial shape, its exact role in the regulation of mitochondrial dynamics remains unknown. To address this, the morphology of the mitochondrial network was first examined using confocal microscopy in (1) Src++ mouse embryonic fibroblasts (MEFs) expressing Src but knocked-out for Yes and Fyn (two other members of the Src kinases family) and (2) SYF MEFs knocked-out for Src, Yes and Fyn [19], named hereafter Src+/+ and Src−/− MEFs, respectively. We observed that the dele- tion of Src (Fig. S1a) decreased the percentage of cells with tubular mitochondria and increased the number of cells with elongated mitochondria (Fig. 1a, b). Quantita- tive analyses of regions of interest (ROIs) showed that the deletion of Src also decreased the number of mito- chondria and increased the area of individual organelles in ROIs (Fig. 1a, c), supporting the mitochondrial elon- gated phenotype observed in Src−/− MEFs. To examine whether the downregulation of Src increases mitochon- drial size independently of Yes and Fyn, Src was silenced using shRNA in naive MEFs (Fig. S1b). Similar to what was observed in Src−/− MEFs, shSrc increased the per- centage of MEFs with elongated mitochondria (Fig. S1c, d), decreased the number of individual mitochondria and increased the area of mitochondria in ROIs (Fig. S1c, e). Src was also silenced using siRNA in the human cell line HeLa (Fig. S1f). Again, we observed that lower expres- sion of Src increased the percentage of HeLa cells with elongated mitochondria (Fig. 1d, e), decreased the num- ber of individual mitochondria and increased the area of individual organelles in ROIs (Fig. 1d, f). The impact of Src deletion on mitochondrial morphology was confirmed using transmission electron microscopy since individual organelles of Src−/− MEFs had higher area and perimeter as compared to Src+/+ mitochondria (Fig. 1g, h). Strik- ingly, upregulation of Src had opposite effects as compared to its deletion. Indeed, over-expression of Src-HA in Hela cells increased the percentage of cells with fragmented mitochondria (Fig. 1i, j) and increased the number of mito- chondria with smaller area in ROIs (Fig. 1i, k). Overall, these findings reveal for the first time that Src reduces mitochondrial size. To understand how the downregulation of Src leads i properties of these cells depend on mitochondrial dynam- ics. Results Fig. 1 Src reduces mitochondrial size. a Representative micrographs (n = 3) of the mitochondrial protein TOM20 labeling in Src+/+ and Src−/− mouse embryonic fibroblasts (MEFs). Scale bars: 20 μm. b, c Quantitative analysis of mitochondrial morphology showing b the distribution of cells among the different mitochondrial phenotypes, c the number of mitochondria and the area of individual mitochon- dria in regions of interest (ROI) in Src+/+ and Src−/− MEFs as shown in a. d Representative micrographs (n = 3) of the mitochondrial pro- tein TOM20 labeling in HeLa cells transfected with scramble siRNA (siCTL) or siRNA targeting Src (siSrc). Scale bars: 20 μm. e, f Quan- titative analysis of mitochondrial morphology showing e the distri- bution of cells among the different mitochondrial phenotypes, f the number of mitochondria and the area of individual mitochondria in ROI in HeLa cells as shown in d. g, h Representative g transmission electron microscopy images of mitochondria and h quantification of mitochondrial area and perimeter in Src+/+ and Src−/− MEFs. Scale bar: 500 nm. i Representative micrographs (n = 3) of the mitochon- drial protein TOM20 and HA labeling in HeLa cells over-expressing control vector or Src-HA. j, k Quantitative analysis of mitochondrial morphology showing j the distribution of cells among the different mitochondrial phenotypes, k the number of mitochondria and the area of individual mitochondria in ROI in HeLa cells as shown in i. Data are presented as mean ± S.E.M. For the analyses of mitochon- drial phenotype, data with different letters are statistically different (p < 0.05), according to two-way ANOVA followed by Tukey’s mul- tiple comparison test. For all other analyses, p < 0.05, p < 0.01, *p < 0.001 according to Student’s t test ◂ Introduction Etienne Hebert‑Chatelain etienne.hebert.chatelain@umoncton.ca 1 Canada Research Chair in Mitochondrial Signaling and Physiopathology, Moncton, NB, Canada 2 Department of Biology, University of Moncton, Moncton, NB, Canada 3 Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, UK 4 Department of Chemistry and Biochemistry, University of Moncton, Moncton, NB, Canada Olivier Lurette and Hala Guedouari share the first authorship. * Etienne Hebert‑Chatelain etienne.hebert.chatelain@umoncton.ca 1 Canada Research Chair in Mitochondrial Signaling and Physiopathology, Moncton, NB, Canada 1 Canada Research Chair in Mitochondrial Signaling and Physiopathology, Moncton, NB, Canada 4 Department of Chemistry and Biochemistry, University of Moncton, Moncton, NB, Canada (0121 3456789) 3 327 Page 2 of 17 O. Lurette et al. 1 3 3 Page 3 of 17 327 Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology Src decreases mitochondrial size These findings indicate that the mitochondrial elongation induced by deletion of Src is associated to lower levels of specific pro-fission proteins (Drp1 and MiD51) and higher levels of the pro-fusion protein OPA1. observed in FIS1 or MiD49 levels (Fig. 2a, b). Levels of Drp1 were also lower in cytosolic and mitochondrial frac- tions of Src−/− MEFs, but no differences were observed when normalized to the total level of Drp1 in TCL (Fig. S1h), suggesting no defects in Drp1 recruitment to mito- chondrial membranes in Src−/− MEFs. Activity and trans- location of Drp1 to mitochondria are partly controlled by phosphorylation at its S616 and S637 residues [20–22]. When normalized to Drp1 levels, phosphorylation of both S616- and S637-Drp1 were not different between Src+/+ and Src−/− MEFs (Fig. 2a, b), further corroborating that Src does not affect activity or subcellular distribution of Drp1. Among pro-fusion proteins, levels of the IMM fusion regulator OPA1 were increased in Src−/− MEFs without changing the ratio between long and short OPA1 isoforms (Fig. 2a, c) or OPA1 oligomerization (Fig. S1i). The levels of the OMM fusion protein MFN2 did not change upon deletion of Src (Fig. 2a, c). Surprisingly, levels of the OMM fusion protein MFN1 were decreased in Src−/− MEFs (Fig. 2a, c), suggesting it is not involved in Src-mediated regulation of mitochondrial morphology. These findings indicate that the mitochondrial elongation induced by deletion of Src is associated to lower levels of specific pro-fission proteins (Drp1 and MiD51) and higher levels of the pro-fusion protein OPA1. Next, we observed that the changes of mitochondrial mor- phology induced by downregulation of Src were not associ- ated with alterations of mitochondrial mass, as shown by immunoblotting several mitochondrial proteins (Fig. 3a, b), assessing citrate synthase activity (Fig. 3c), and by staining Src+/+ and Src−/− MEFs with Mitotracker™ green which labels mitochondria independently of the mitochondrial membrane potential (Fig. 3d).i However, deletion of Src significantly decreased cellular respiration in MEFs (Fig. 3e). Similarly, silencing of Src in HeLa cells also decreased oxygen consumption (Fig. S1j), suggesting that the changes of mitochondrial size induced by Src downregulation could be due to defects in OXPHOS. However, staining Src+/+ and Src−/− MEFs with tetrameth- ylrhodamine (TMRM) showed that deletion of Src had no impact on mitochondrial membrane potential (Fig. 3f). Finally, cytosolic ATP levels, as measured by the ATP probe GoAteam2 [26], were not different between Src+/+ and Src−/− MEFs (Fig. Src decreases mitochondrial size 3g). ATP levels were also comparable after treatment with the ATP synthase inhibitor oligomycin, used as a proxy to measure cytosolic ATP generated by the mitochondrial ATP synthase (Fig. 3g, h). Thus, these find- ings suggest that the Src-dependent decrease in oxygen con- sumption does not lead to altered mitochondrial membrane potential nor ATP production. Overall, we propose that the Src-dependent changes in mitochondrial size are not due to a global mitochondrial dysfunction. Src decreases mitochondrial size Indeed, metastatic breast cancer cells display frag- mented mitochondria, and inhibition of mitochondrial division via silencing of Drp1 or promotion of mitochon- drial fusion by over-expression of Mfn1 blocks metastasis in these cells [18]. These findings suggest that Src could affect mitochondrial dynamics to control oncogenic prop- erties, at least in specific cancer cells. However, the con- tribution of Src to adjustments of mitochondrial shape is currently not well understood. The aim of this study was to examine and describe the potential role of Src in the regulation of mitochondrial dynamics. We observed that deletion or silencing of Src increased mitochondrial size, whereas its over-expression had the opposite effect. Deletion of Src also reduced cel- lular respiration and affected the levels of the mitochon- dria-shaping proteins Drp1, MiD51 and OPA1, whereas no effect was observed on mitochondrial mass, mitochondrial membrane potential and ATP levels. Re-expression of Src rescued alterations of mitochondrial morphology but not cel- lular respiration or levels of mitochondria-shaping proteins. Mechanistically, we propose that mitochondrial matrix- localized Src modulates mitochondrial shape independently of OXPHOS. Indeed, re-expression of Src targeted to the mitochondrial matrix rescued mitochondrial morphology, in a kinase activity-dependent manner, whereas re-expression of Src targeted to plasma membrane had no impact on mito- chondrial shape. Overall, these findings highlight a direct contribution of intramitochondrial Src and its kinase activity to the control of mitochondrial dynamics. To understand how the downregulation of Src leads to mitochondrial elongation, we first examined the levels of a panel of mitochondria-shaping proteins by immu- noblotting (Fig. 2a–c). Results showed that levels of the pro-fission Drp1 and its mitochondrial receptor MiD51 were decreased in Src−/− MEFs, whereas no change was 1 3 3 O. Lurette et a 1 327 Page 4 of 17 327 Page 4 of 17 O. Lurette et al. 1 3 Page 5 of 17 327 Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology 327 the mitochondrial matrix (mtGFP) by live cell confocal microscopy, as previously described [23]. Our findings suggest no difference in fusion and fission rates upon deletion of Src (Fig. 2d; Movies 1 and 2). Similar results were obtained when these events were quantified in HeLa cells treated with siSrc (Fig. S1g; Movies 3 and 4). Mito- chondrial fusion was further evaluated using Src+/+ and Src−/− MEFs expressing mitochondria-targeted photo- activable GFP (mt-PAGFP) [24]. Src decreases mitochondrial size Quantification of GFP fluorescence in stimulated ROIs of cells co-expressing mt-PAGFP and the mitochondrial marker mtDsRed indi- cated similar mitochondrial fusion rates between Src+/+ and Src−/− MEFs after 10 min (Fig. 2e, f). We also treated Src+/+ and Src−/− MEFs expressing mtGFP with the uncou- pler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to induce Drp1-dependent mitochondrial fission [25]. We observed that Src+/+ and Src−/− MEFs express- ing mtGFP have similar fragmentation levels upon treat- ment with the uncoupler FCCP, suggesting no defects in the fission machinery per se, at least under this specific stress (Fig. 2g, h). These findings indicate that while we observed a dysregulation of some of the main regulators of mitochondrial morphology, including Drp1 and OPA1, the downregulation of Src increases mitochondrial size without affecting the overall mitochondrial fusion or fis- sion rates. Fig. 2 Src impacts on specific mitochondria-shaping proteins but not on mitochondrial fusion and fission rates. a Representative immu- noblotting (n = 3–6) of the pro-fission proteins pS616-Drp1, pS637- Drp1, Drp1, FIS1, MiD49 and MiD51, of the pro-fusion proteins MFN1, MFN2 and OPA1, and of the total protein load (TPL) used as loading control, in Src+/+ and Src−/− MEFs. b, c Quantification of the immunoblotting shown for b pro-fission and c pro-fusion mito- chondria-shaping proteins. Protein levels were normalized by TPL. d Quantification of mitochondrial fusion and fission events in live Src+/+ and Src−/− MEFs expressing mtGFP (n = 3) during 450 s vid- eos. See also Movies 1 and 2. e Representative micrographs (n = 5) of Src+/+ and Src−/− MEFs expressing the mitochondria-targeted photo- activable GFP (mt-PAGFP) and mtDsRed after photoactivation of mt-PAGFP. f Quantification of GFP normalized to DsRed in region of interest (ROI) upon photoactivation as shown in e. g Live cell micrographs of Src+/+ and Src−/− MEFs expressing mtDsRed treated with FCCP (50 μM) as indicated. h Quantification of the number of cells with fragmented mitochondria as shown in g. Data are presented as mean ± S.E.M. A.U.: arbitrary unit. For b, c and d, p < 0.05, p < 0.01, according to Student’s t test. For f and h, data were ana- lyzed with two-way ANOVA. Scale bars: 20 μm ◂ Fig. 2 Src impacts on specific mitochondria-shaping proteins but not on mitochondrial fusion and fission rates. Src decreases mitochondrial size a Representative immu- noblotting (n = 3–6) of the pro-fission proteins pS616-Drp1, pS637- Drp1, Drp1, FIS1, MiD49 and MiD51, of the pro-fusion proteins MFN1, MFN2 and OPA1, and of the total protein load (TPL) used as loading control, in Src+/+ and Src−/− MEFs. b, c Quantification of the immunoblotting shown for b pro-fission and c pro-fusion mito- chondria-shaping proteins. Protein levels were normalized by TPL. d Quantification of mitochondrial fusion and fission events in live Src+/+ and Src−/− MEFs expressing mtGFP (n = 3) during 450 s vid- eos. See also Movies 1 and 2. e Representative micrographs (n = 5) of Src+/+ and Src−/− MEFs expressing the mitochondria-targeted photo- activable GFP (mt-PAGFP) and mtDsRed after photoactivation of mt-PAGFP. f Quantification of GFP normalized to DsRed in region of interest (ROI) upon photoactivation as shown in e. g Live cell micrographs of Src+/+ and Src−/− MEFs expressing mtDsRed treated with FCCP (50 μM) as indicated. h Quantification of the number of cells with fragmented mitochondria as shown in g. Data are presented as mean ± S.E.M. A.U.: arbitrary unit. For b, c and d, p < 0.05, p < 0.01, according to Student’s t test. For f and h, data were ana- lyzed with two-way ANOVA. Scale bars: 20 μm ◂ observed in FIS1 or MiD49 levels (Fig. 2a, b). Levels of Drp1 were also lower in cytosolic and mitochondrial frac- tions of Src−/− MEFs, but no differences were observed when normalized to the total level of Drp1 in TCL (Fig. S1h), suggesting no defects in Drp1 recruitment to mito- chondrial membranes in Src−/− MEFs. Activity and trans- location of Drp1 to mitochondria are partly controlled by phosphorylation at its S616 and S637 residues [20–22]. When normalized to Drp1 levels, phosphorylation of both S616- and S637-Drp1 were not different between Src+/+ and Src−/− MEFs (Fig. 2a, b), further corroborating that Src does not affect activity or subcellular distribution of Drp1. Among pro-fusion proteins, levels of the IMM fusion regulator OPA1 were increased in Src−/− MEFs without changing the ratio between long and short OPA1 isoforms (Fig. 2a, c) or OPA1 oligomerization (Fig. S1i). The levels of the OMM fusion protein MFN2 did not change upon deletion of Src (Fig. 2a, c). Surprisingly, levels of the OMM fusion protein MFN1 were decreased in Src−/− MEFs (Fig. 2a, c), suggesting it is not involved in Src-mediated regulation of mitochondrial morphology. Src does not impact on mitochondrial fusion and fission rates g OFP/GFP ratio from the ATP FRET sensor GoAteam2 expressed in Src+/+ and Src−/− MEFs before (time 0) and during treat- ment with oligomycin (1 μg mL−1) as indicated (n = 5). h Difference in OFP/GFP ratio from the ATP FRET sensor GoAteam2 expressed in Src+/+ and Src−/− MEFs before (time 0) and after 25 min of treat- ment with oligomycin (1 μg mL−1) (n = 5), as shown in e. Data are presented as mean ± S.E.M. A.U.: arbitrary unit. Data were analyzed using Student’s t test (b–d, f, h) or two-way ANOVA followed by Tukey’s multiple comparison test (e, g). p < 0.01, *p < 0.001 Src does not impact on mitochondrial fusion and fission rates To examine whether the mitochondrial elongation induced by Src downregulation was associated with deregula- tion of mitochondrial fusion or fission, we first quanti- fied real-time mitochondrial fusion and fission events in Src+/+ and Src−/− MEFs expressing a GFP targeted to 1 3 327 Page 6 of 17 O. Lurette et al. 327 Page 6 of 17 Fig. 3 Silencing of Src does not affect mitochondrial mass, mem- brane potential and ATP levels. a Representative immunoblotting (n = 6–7) of mitochondrial proteins and of the total protein load (TPL) used as loading control in Src+/+ and Src−/− MEFs. b Quan- tification of immunoblotting shown in a. Protein levels were normal- ized by TPL. c Citrate synthase activity in Src+/+ and Src−/− MEFs. d Mitochondrial mass of Src+/+ and Src−/− MEFs as measured by staining with Mitotracker Green™ (n = 3). e Cellular respiration of Src+/+ and Src−/− MEFs (n = 10). f Mitochondrial membrane potential of Src+/+ and Src−/−MEFs as measured using staining with TMRM (n = 3). g OFP/GFP ratio from the ATP FRET sensor GoAteam2 expressed in Src+/+ and Src−/− MEFs before (time 0) and during treat- ment with oligomycin (1 μg mL−1) as indicated (n = 5). h Difference in OFP/GFP ratio from the ATP FRET sensor GoAteam2 expressed in Src+/+ and Src−/− MEFs before (time 0) and after 25 min of treat- ment with oligomycin (1 μg mL−1) (n = 5), as shown in e. Data are presented as mean ± S.E.M. A.U.: arbitrary unit. Data were analyzed using Student’s t test (b–d, f, h) or two-way ANOVA followed by Tukey’s multiple comparison test (e, g). p < 0.01, *p < 0.001 Fig. 3 Silencing of Src does not affect mitochondrial mass, mem- brane potential and ATP levels. a Representative immunoblotting (n = 6–7) of mitochondrial proteins and of the total protein load (TPL) used as loading control in Src+/+ and Src−/− MEFs. b Quan- tification of immunoblotting shown in a. Protein levels were normal- ized by TPL. c Citrate synthase activity in Src+/+ and Src−/− MEFs. d Mitochondrial mass of Src+/+ and Src−/− MEFs as measured by staining with Mitotracker Green™ (n = 3). e Cellular respiration of Src+/+ and Src−/− MEFs (n = 10). f Mitochondrial membrane potential of Src+/+ and Src−/− MEFs as measured using staining with TMRM (n = 3). The kinase activity of mitochondrial matrix‑localized Src is sufficient to impact on mitochondrial size present in mitochondria-enriched fractions obtained from MEFs, HEK293 and HeLa (Fig. 4a), as previously observed [13, 15, 16, 29]. Trypsin sensitivity assay then confirmed that Src is present inside mitochondria since it was protected from degradation when mitochondrial-enriched fractions were treated with trypsin, in contrast to the OMM protein TOM20 (Fig. 4b). To further determine the localization of Src at different mitochondrial subcompartments, mito- chondria-enriched fractions were treated with increasing Src was previously shown to localize to different cellular compartments including mitochondria in several cellular and animal models, using multiple techniques such as transmis- sion electron microscopy [13, 15, 27, 28]. Subcellular frac- tionation followed by immunoblotting showed that Src is 3 Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology Page 7 of 17 327 327 reversed the mitochondrial elongation induced by siSrc in HeLa cells (Fig. 6c, d), suggesting that mitochondrial matrix-localized Src affects mitochondrial shape in a kinase activity-dependent manner. The same constructs were then over-expressed in HeLa cells: mtSrc(CA)-FLAG reduced mitochondrial size, whereas the inactive mutant mtSrc- (KD)-FLAG had no effect (Fig. S3a, b). This gain-of-func- tion experiment confirmed that the kinase activity of Src inside the mitochondrial matrix is sufficient to induce the Src-dependent alterations of mitochondrial shape. concentrations of digitonin, which dose-dependently release the intermembrane space protein Smac and the IMM matrix- facing protein ATP5a (Fig. 4c). We observed that the endog- enous Src appears to be located in different mitochondrial compartments including the IMM/mitochondrial matrix compartment (Fig. 4b), as previously observed [13, 15]. Immunoblotting of the ER lumen protein ERp57 excluded bias linked to the well-known contamination of mitochon- dria-enriched fractions with ER since ERp57 was released differently than Src (Fig. 4c). f To test whether the impact of Src on mitochondrial mor- phology depends on its subcellular localization, we gener- ated different tagged Src constructs: (i) untargeted Src, (ii) Src targeted to the mitochondrial matrix (mtSrc), and (iii) Src targeted to the plasma membrane (pmSrc). The con- structs Src-V5, mtSrc-V5 and pmSrc-V5 were then used to rescue levels of Src in Src−/− MEFs in specific subcellular compartments (Fig. 4d and Fig. S2a). Expression of Src- V5 or mtSrc-V5 both reversed the changes of mitochondrial shape induced by the deletion of Src, whereas no change was observed upon expression of pmSrc-V5 (Fig. 4d, e). Simi- larly, over-expression of Src-V5 and mtSrc-V5 in HeLa cells decreased mitochondrial size (Fig. S2b–d). Over‑expression of mitochondrial matrix‑localized Src prevents mitochondrial elongation during starvation During starvation, mitochondria elongate to protect them- selves from autophagic degradation and prevent cell death [4, 31]. We thus hypothesized that the Src-dependent modulation of mitochondrial dynamics could be involved in this process. To address this, we first treated Src+/+ and Src−/− MEFs with Hank’s balanced salt solution (HBSS) during 5 h, a treatment known to induce starvation-induced mitochondrial elongation [4]. We observed more Src+/+ MEFs with elongated mitochondria upon starvation, whereas no change was observed in Src−/− MEFs (Fig. 7a, b), con- firming that the mitochondrial network of Src−/− MEFs was already in an elongated state before starvation. The mito- chondrial morphology was also examined in HeLa cells over-expressing different Src constructs and treated with HBSS during 5 h. As expected, the HBSS treatment induced mitochondrial elongation in control HeLa cells (Fig. 7c, d). Mitochondrial elongation induced by starvation was, how- ever, inhibited in cells over-expressing mtSrc-FLAG but not in cells expressing the inactive mtSrc(KD)-FLAG (Fig. 7c, d), suggesting that the activity of intramitochondrial Src is decreased during starvation to allow mitochondrial elonga- tion. However, we observed higher phosphorylation of the activating residue of Src (i.e., pY416-Src), indicating that Src activity increased upon HBSS treatment in both total cell lysates and mitochondria-enriched fractions (Fig. 7e, f). This finding indicates that the regulation of mitochondrial shape by endogenous mitochondrial matrix-localized Src is likely not involved in starvation-induced mitochondrial elongation. fif Since re-expression of Src in Src−/− MEFs rescued mitochondrial morphology, we tested whether it would also reverse the alterations of Drp1, MiD51 and OPA1 levels. Surprisingly, expression of Src-HA and mtSrc-HA in Src−/− MEFs did not reverse the decrease of Drp1 and MiD51 levels or the increase of OPA1 levels induced by the deletion of Src (Fig. 5a, b). Considering that downregulation of Src decreases cellular respiration (Fig. 3e and Fig. S1j), we also examined whether expression of Src-HA and mtSrc- HA would affect oxygen consumption in Src−/− MEFs. Simi- lar to Drp1, MiD51 and OPA1 levels, expression of Src- HA and mtSrc-HA in Src−/− MEFs did not rescue cellular respiration (Fig. 5c). Altogether, these results suggest that Src affects mitochondrial size and cellular respiration via different mechanisms. f Expression of Src-HA and mtSrc-HA both reversed the mitochondrial elongation phenotype induced by siSrc in HeLa cells (Fig. 6a, b). The kinase activity of mitochondrial matrix‑localized Src is sufficient to impact on mitochondrial size Over-expression of Src targeted to the plasma membrane had no effect on the number and the area of individual mitochondria in ROIs although pmSrc-V5 changed the number of cells between the different mitochondrial phenotypes. (Fig. S2b–d). Globally, our results suggest that Src localized in the mitochondrial matrix is sufficient to affect mitochondrial shape. Over‑expression of mitochondrial matrix‑localized Src prevents mitochondrial elongation during starvation To examine if these morphologi- cal changes depend on the kinase activity of Src, we per- formed similar rescue experiments in HeLa cells treated with siSrc using constitutively active (CA) and kinase-dead (KD) mutants of mtSrc [30], named mtSrc(CA)-FLAG and mtSrc(KD)-FLAG, respectively. Interestingly, mtSrc(CA)- FLAG, but not the inactive mutant mtSrc(KD)-FLAG, Discussion d Representative micrographs (n = 3) of the plasma membrane marker wheat germ agglutinin (WGA), V5 and TOM20 labeling in Src+/+ and Src−/− mouse embryonic fibroblasts (MEFs) express- ing pcDNA, Src-V5, mitochondria-targeted mtSrc-V5 and plasma membrane-targeted pmSrc-V5. e Quantitative analysis of mitochon- drial morphology in region of interest (ROI) as shown in c. Scale bars: 20 μm. Data are presented as mean ± S.E.M. Data with differ- ent letters are statistically different (p < 0.05), according to one-way ANOVA followed by Tukey’s post hoc test ◂ Mitochondrial activity and morphology are tightly con- nected and treatment with inhibitors of OXPHOS leads to mitochondrial fragmentation [5, 6]. For instance, the uncoupler FCCP or the mitochondrial ATP synthase inhibi- tor oligomycin induce mitochondrial fragmentation via the cleavage of OPA1 by the protease OMA1, leading to inhibi- tion of mitochondrial fusion and unopposed mitochondrial fragmentation [32]. Previous works showed that pharmaco- logical inhibition or genetic downregulation of Src decreases OXPHOS, mitochondrial membrane potential and ATP pro- duction [13, 15, 33, 34]. Here, the downregulation of Src did not decrease mitochondrial membrane potential or ATP levels, at least in the cytosol, suggesting that the alterations of oxygen consumption induced by downregulation of Src observed here and in previous work [13, 15, 33–35] did not translate into decreased mitochondrial membrane potential or ATP levels in MEFs and HeLa cells. Our results thus suggest that the Src-dependent regulation of mitochondrial morphology is independent of OXPHOS defects. In con- trast, mitochondria can elongate upon various stress stimuli. Starvation triggers cAMP-PKA signaling and inhibition of Drp1-mediated mitochondrial fragmentation leading to unopposed mitochondrial fusion [4, 31]. UV irradiation, actinomycin D and cycloheximide also induce mitochon- drial elongation. In such conditions, mitochondrial fusion increases due to stabilization of the pro-fusion long OPA1 isoforms by SLP-2 [3]. This stress-induced mitochondrial hyperfusion is independent of mitochondrial depolarization or changes in ATP levels [3, 4], similar to our observations about Src-dependent mitochondrial elongation. However, our findings suggest that the regulation of mitochondrial size by endogenous Src is not involved in starvation-induced mitochondrial elongation. Overall, the functional impact of the Src-mediated modulation of mitochondrial shape is still unclear, and future works will be necessary to address this. Src is present in several subcellular compartments, including mitochondria [10, 13, 15]. Our experiments con- firm previous observations showing that a pool of Src is, at least partly, localized inside the organelle [13, 15, 28]. Discussion The present work examined the impact of the tyrosine kinase Src on mitochondrial morphology. We show that lower expression of Src increases mitochondrial size, whereas its over-expression shortens mitochondria. We suggest that the kinase activity of Src inside the mitochondrial matrix adjusts mitochondrial shape. 1 3 1 3 O. Lurette et a 327 Page 8 of 17 O. Lurette et al. 327 Page 8 of 17 327 Page 8 of 17 1 3 Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology Page 9 of 17 327 Page 9 of 17 327 Page 9 of 17 327 Our findings suggest that Src regulates mitochondrial shape downstream of mitochondria-shaping proteins. Indeed, although levels of Drp1, MiD51 and OPA1 were different between Src+/+ and Src−/− MEFs, re-expression of Src in Src−/− MEFs rescued the changes of mitochondrial morphology but not of the levels of these mitochondria- shaping proteins. Thus, the different levels of Drp1, MiD51 and OPA1 between Src+/+ and Src−/− MEFs could have been indirect consequence of the deletion of the kinase itself, to compensate for the chronic deficiency of Src. Similarly, deletion of Src did not affect processing and oligomeriza- tion of OPA1. Thus, Src could control mitochondrial shape downstream of the canonical fission/fusion machineries. Drp1-independent mitochondrial division can occur during hypoxia or deferiprone-induced mitophagy in mammalian cells [36]. Elevated cytosolic Ca2+ can also shorten mito- chondria independently of Drp1 [37, 38]. Moreover, some types of mitochondria-derived vesicles are released from mitochondria in a Drp1-independent manner [39]. To our knowledge, there is, however, no evidence for any mecha- nism leading to mitochondrial elongation independently of the pro-fusion proteins OPA1 and Mfn1/2. Although this was not the focus of this work, it would be critical to identify the mechanisms linking Src and mitochondrial dynamics in future studies. Fig. 4 Mitochondrial matrix-localized Src regulates mitochondrial shape. a Representative immunoblotting (n = 3) of Src, cytosolic tubulin and mitochondrial SDHA in total cell lysate (TCL), cyto- sol- (cyto) and mitochondria- (mito) enriched fractions of Src+/+ and Src−/− MEFs, HEK293 and HeLa. b Representative immunoblotting (n = 3) of Src, the IMM protein SDHA and the OMM protein TOM20 in mitochondria-enriched fractions of HeLa cells treated as indicated. c Representative immunoblotting (n = 4) of Src, Smac-diablo, ATP5a and ERp57 in supernatant (SN) and pellet obtained from mitochon- dria-enriched fractions of HeLa cells treated with digitonin as indi- cated. Discussion Targeting Src to the mitochondrial matrix was sufficient to reduce mitochondrial size. These findings suggest that Src could target proteins inside the organelle to control its shape. We recently identified several potential targets of intramitochondrial Src which could be involved in the morphological changes observed in the present work [17]. For example, it has been shown that Src could interact with SLP-2 [17] which is required for stress-induced mitochon- drial hyperfusion [3]. Similarly, we showed potential inter- action of intramitochondrial Src with prohibitin 2 and the mitochondrial contact site and cristae organizing system Mic60 [17] which are also key players in the regulation of mitochondrial architecture [40, 41]. It is also possible that Src is present in other compartments of the organelle from where it could impact on mitochondrial architecture and functions. Similarly, over-expression of Src targeted to the plasma membrane impacted the number of cells among the different mitochondrial phenotypes, suggesting that the dif- ferent subcellular pools of Src could impact on mitochon- drial morphology via specific pathways. Further investiga- tion could thus allow to identify new pathways important for the regulation of mitochondrial shape and examine whether Src modulates mitochondrial morphology and functions 1 3 327 Page 10 of 17 O. Lurette et al. Fig. 5 Mitochondrial matrix-localized Src modulates mitochondrial size independently of Drp1, MiD51, OPA1 and cellular respiration. a Representative immunoblotting (n = 3) of Src, Drp1, MiD51, OPA1 and total protein load (TPL), used as loading control, in Src+/+ and Src−/− MEFs expressing pcDNA, Src-HA and mtSrc-HA. b Quantifi- cation of immunoblotting shown in e. Protein levels were normalized by TPL. c Cellular respiration of Src+/+ and Src−/− MEFs expressing pcDNA, Src-HA and mtSrc-HA (n = 3). Data with different letters are statistically different (p < 0.05), according to one-way ANOVA (for b) or two-way ANOVA (for c) followed by Tukey’s post hoc test. A.U. arbitrary unit by TPL. c Cellular respiration of Src+/+ and Src−/− MEFs expressing pcDNA, Src-HA and mtSrc-HA (n = 3). Data with different letters are statistically different (p < 0.05), according to one-way ANOVA (for b) or two-way ANOVA (for c) followed by Tukey’s post hoc test. A.U. arbitrary unit by TPL. c Cellular respiration of Src+/+ and Src−/− MEFs expressing pcDNA, Src-HA and mtSrc-HA (n = 3). Constructs and site‑directed mutagenesis The constructs encoding hemagglutinin (HA)- or V5-tagged mouse Src (Src-HA and Src-V5) and HA- or V5-tagged mouse Src targeted to the mitochondria (mtSrc- HA and mtSrc-V5) were generated by amplification of the sequence of Src from pCMV5-Src (#13663, Addgene, MA, USA) and fused to HA or V5 with or without the mitochondrial leading sequence (MLS) of cytochrome c oxidase VIIIa, as described [16]. Constructs encoding FLAG-tagged human wild-type Src, constitutively active (CA) Src and kinase-dead (KD) Src fused to the MLS of the very long chain acyl-CoA dehydrogenase (named here- after mtSrc-FLAG, mtSrc(CA)-FLAG and mtSrc(KD)- Flag, respectively) were obtained from Addgene (#44652, #44654 and # 44653, respectively). Src-V5 was targeted to the plasma membrane (pmSrc-V5) using the first 26 amino acids of Lck (derived from Lck-GFP obtained from Addgene #61099) as previously shown [42, 43]. Discussion Data with different letters are statistically different (p < 0.05), according to one-way ANOVA (for b) or two-way ANOVA (for c) followed by Tukey’s post hoc test. A.U. arbitrary unit Fig. 5 Mitochondrial matrix-localized Src modulates mitochondrial size independently of Drp1, MiD51, OPA1 and cellular respiration. a Representative immunoblotting (n = 3) of Src, Drp1, MiD51, OPA1 and total protein load (TPL), used as loading control, in Src+/+ and Src−/− MEFs expressing pcDNA, Src-HA and mtSrc-HA. b Quantifi- cation of immunoblotting shown in e. Protein levels were normalized FuGENE HD (Promega, WI, USA) or Lipofectamine™ RNAiMAX (Invitrogen, CA, USA), whereas MEFs were electroporated with the Neon™ transfection system (Inv- itrogen, CA, USA) in accordance with the manufactur- er’s instructions. Cells were analyzed 24–48 h following transfection. FuGENE HD (Promega, WI, USA) or Lipofectamine™ RNAiMAX (Invitrogen, CA, USA), whereas MEFs were electroporated with the Neon™ transfection system (Inv- itrogen, CA, USA) in accordance with the manufactur- er’s instructions. Cells were analyzed 24–48 h following transfection. through SLP-2, Mic60 and other classical and non-classical mitochondria-shaping proteins. In conclusion, the present work highlights a novel func- tion for Src as a regulator of mitochondrial morphology. This may provide insights into the contribution of Src sign- aling in several cellular processes and diseases such as the involvement of Src in different pathologies, including cancer. The role of Src in oncogenesis and the metabolic rewiring observed in cancer cells is well described [8]. Considering that metastasis of some cancer cells depends on appropriate regulation of mitochondrial dynamics, the intramitochon- drial Src signaling and its impact on mitochondrial morphol- ogy could represent a novel therapeutic avenue. RNAi Designed individual or SMARTpool siRNAs were used to specifically knockdown Src in HeLa cells. Single siRNA was purchased from Sigma-Aldrich (MO, USA) and were based on the following sequences: (sense: 5′-GGAAACACCUGUAGGCAGAUU-3′; antisense: 5′-UCUGCCUACAGGUGUUUCCUU-3′). SMARTpool siRNAs were obtained from Dharmacon (L-003175-00- 0005 ON-TARGETplus Human SRC siRNA). Scrambled siRNA (siCTL) was obtained from ThermoFisher Scien- tific (#4390843). HeLa cells were transfected with siRNA (20 nM) using Lipofectamine™ RNAiMax (Invitrogen, CA, USA) according to the manufacturer’s protocol. For rescue experiments, cells were transfected with plasmids encoding empty vector or Src constructs 48 h after trans- fection with siSrc. Cells were then analyzed 24 h later. For the evaluation of mitochondrial membrane poten- tial and mitochondrial mass, Src+/+ and Src−/− MEFs were treated with 200 nM tetramethylrhodamine methyl ester (TMRM, Invitrogen, CA, USA) and with 100 nM Mitotracker green™ (Invitrogen, CA, USA), respectively, during 30 min at 37 °C. Cells were then rinsed and directly imaged with the EVOS Fl Auto 2 (Invitrogen, CA, USA) at 37 °C in 5% CO2 and 95% humidity using a 40 × objective (LPLAN 40 ×, 0.65NA, EVOS), and appropriate excitation and emission filters. For each independent experiment, fluorescence intensity was quantified in 30 cells using Fiji software (NIH, MD, USA). For analysis of mitochondrial morphology, cells seeded on 12- or 18-mm glass round coverslips were fixed in 4–5% (w/v) paraformaldehyde for 15 min at RT and then incubated with 50 mM ammonium chloride in PBS for 10 min at RT. Cells were permeabilized with 0.1–0.5% triton X-100/PBS for 10–15 min and blocked in 10% BSA or FBS in PBS for 45 min, as previously described [46]. Cells were labeled between 1 h and overnight with rabbit anti-HA (Cell Signal- ing; #2367), rabbit anti-TOM20 (Proteintech 11802-1-AP), rabbit anti-myc (Cell Signaling: #2278), mouse anti-HA (Biolegend; #901513), mouse anti-myc (Cell Signaling; #2276), mouse anti-FLAG (Sigma-Aldrich; #F1804) and mouse anti-TOM20 (Santa Cruz; sc-17764) antibodies, and then with anti-rabbit IgG or anti-mouse IgG conjugated with Alexa Fluor®488, Alexa Fluor®546, Alexa Fluor®633 or Alexa Fluor®647 (Invitrogen, CA, USA) for 1 h at RT in either PBS-BSA or PBS-FBS. Plasma membrane was labeled with wheat germ agglutinin conjugated with Alexa Fluor®647 (Invitrogen, CA, USA). For Figs. 1a, d, i, 7a, c, Figs. RNAi S1c, S2c images were acquired using a 60 × or 100 ×/1.4 apochromat objective of the Nikon Eclipse Ti-E microscope, coupled to an Andor Dragonfly spinning disk confocal system equipped with an Andor sCMOS camera, exciting with appropriate lasers. Fusion software (Andor) was used to acquire images. For Figs. 2e, g, 4d, 6a, c and Fig. S3a, coverslips were mounted on slides and placed on the stage of the Olympus FV3000 confocal microscope (Olympus, Japan) and imaged using a 60 × oil objective (UPLAN 60 × oil, 1.35NA, Olympus, Japan), and appro- priate excitation/emission parameters. Stacks separated by 0.2 μm along the z axis were acquired. Images were then complied by “max projection” using Fiji software. Control shRNA (shCTL, #SCH016) and shRNA against mouse Src (shSrc, # TRCN0000278660) were purchased from Sigma-Aldrich. Naive MEFs were elec- troporated with mtGFP and either shCTL or shSrc, as described above. MEFs were analyzed 24 h later. Cell culture and transfection Src++ and SYF MEFs [19] (named here Src+/+ and Src−/−, respectively), naive MEFs, HeLa and HEK293 cells were cultured in high glucose (4.5 g L−1) Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 2 mM glu- tamine, 1 mM pyruvate, 10% (v/v) of fetal bovine serum (FBS), 100 units mL−1 penicillin and 100 g mL−1 strepto- mycin. Cells were cultured at 37 °C in 5% CO2 and 95% humidity. HeLa and HEK293 cells were transiently trans- fected with polyethylenimine (PolySciences, PA, USA), 1 3 Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology Page 11 of 17 327 17 327 Subcellular fractionation a Representative micrographs (n = 3) of TOM20 and HA labeling in HeLa cells transfected with scramble siRNA (siCTL) or siRNA targeting Src (siSrc) with pcDNA, Src- HA or mitochondria-targeted mtSrc-HA. b Quantitative analysis of mitochondrial morphology showing the distribution of cells among the different mitochondrial phenotypes, the number of mitochondria and the area of individual mitochondria in region of interest (ROI) in HeLa cells as shown in a. c Representative micrographs (n = 3) of TOM20 and FLAG labeling in HeLa cells transfected with scramble siRNA (siCTL) or siRNA targeting Src (siSrc) and over-expressing pcDNA, mitochondria-targeted constitutively active mtSrc(CA)- FLAG or mitochondria-targeted kinase-dead mtSrc(KD)-FLAG. d Quantitative analysis of mitochondrial morphology showing the distribution of cells among the different mitochondrial pheno- types, the number of mitochondria and the area of individual mito- chondria in ROI in HeLa cells as shown in c. Data are presented as mean ± S.E.M. Data with different letters are statistically different (p < 0.05), according to one-way ANOVA followed by Tukey’s post hoc test. Scale bars: 20 μm ◂ ATP levels were evaluated using the ATP FRET sen- sor GoAteam2, as described [26]. Src+/+ and Src−/− MEFs expressing GoAteam2 were analyzed 24 h post-transfection, with excitation at 488 nm and emission at 500–535 nm for GFP and 545–625 nm for OFP. To determine ATP lev- els depending on the mitochondrial ATP synthase, MEFs were treated with oligomycin (1 µg/ml). The OFP/GFP ratio was measured every 5 min in a randomly selected ROI of 25 μm2 within peripheral regions of cells using Fiji in a minimum of 5 cells in 5 independent experiments (total of 25 cells per condition). least 30 cells were analyzed in each independent experiment (n = 3) using Fiji software. For analysis of the mitochondrial number and individ- ual mitochondrial area, ROIs of 225 μm2 were randomly selected within peripheral regions of cells from max projec- tion images and followed by manual thresholding, as previ- ously described [45]. Mitochondrial number and size in ROI were obtained using the Analyze particles plugin in Fiji with a minimum area of 0.2 μm2. At least 10 cells were analyzed for each independent experiment (n = 3, minimum of 30 cells in total per condition).i Several cells were analyzed in each independent experi- ment (as indicated). Subcellular fractionation Mitochondria-enriched fractions were obtained as previ- ously described [44]. Briefly, cells were harvested and resuspended in mitochondrial isolation buffer (250 mM sucrose, 1 mM EDTA, 5 mM HEPES, pH 7.4) supple- mented with 1% protease inhibitor cocktail and 2 mM sodium orthovanadate. Cells were lysed with 15 strokes using a 25-gauge syringe on ice and centrifuged at 1500g for 5 min (4 °C). The resulting supernatant (TCL) was centrifuged at 12,500g for 10 min (4 °C). The obtained supernatant was considered as the cytosol-enriched frac- tion, whereas the pellet was resuspended in the mitochon- drial buffer and a cycle of centrifugations at 1500g and 12,500g was repeated. The final pellet was considered as the mitochondria-enriched fraction. For assessing distribution of Src among mitochondrial subcompartments, mitochondria-enriched fractions were resuspended in 1 M HEPES–KOH buffer (pH 7.4) in the presence of 0.125, 0.25 or 0.5% of digitonin (Sigma- Aldrich, MO, USA) and incubated at room temperature (RT) with continuous shaking at 1000 rpm. After 15 min, samples were centrifuged at 12,500g at 4 °C for 10 min. The resulting pellets and supernatants were processed for SDS-PAGE. The presence of proteins from different mito- chondrial subcompartments in pellets and supernatants was analyzed using immunoblotting. For analysis of the mitochondrial morphology, cells were first presented as fragmented, tubular and elongated. The mitochondrial network was classified as fragmented when mitochondria are short and spherical; elongated when > 50% of mitochondria are longer than 5 μm and highly intercon- nected; tubular when the mitochondrial network appeared as an intermediate between fragmented and elongated. At 1 327 Page 12 of 17 O. Lurette et al. 1 3 3 Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology Page 13 of 17 327 327 Mitochondrial fission was also analyzed using live cell imaging of Src+/+ and Src−/− MEFs expressing mtGFP and treated with 10 µM carbonyl cyanide-p-trifluoromethoxy- phenylhydrazone (FCCP) at 37 °C in 5% CO2 and 95% humidity. Live cells were examined at different time points using the EVOS FL Auto2 imaging system and a 40 × objec- tive (LPLAN 40 ×, 0.65NA, EVOS), with appropriate excita- tion and emission filters. At least 40 cells were analyzed for each independent experiment (n = 4). Fig. 6 The kinase activity of mitochondrial matrix-localized Src affects mitochondrial shape. Subcellular fractionation To minimize bias and make sure the analyzed cells represented the overall population of cells, coverslips were divided in four quarters for each independ- ent experiment, from which an approximate equal number of cells were randomly selected, When appropriate, ROIs were selected within peripheral regions to ensure an appro- priate distribution of all organelles, as described previously [45], in contrast to the perinuclear region, which is known to “compact” organelles due to steric hindrance leading to changes in their morphologies. The number of mitochondrial fusion and fission events in live cells was analyzed as previously described [23]. Briefly, cells expressing mtGFP were imaged every 2.5 s for 450 s, and then analyzed using Fiji in at least 5 cells in 3–5 inde- pendent experiments (total of 15 cells for MEFs and a total of 25 cells for HeLa). Fusion and fission events were calcu- lated in ROI of 225 μm2 randomly selected within periph- eral regions of cells from max projection images. A single fission event was noted when one mitochondrion divided in two and remained separated for at least the next two time- frames. A fusion event was noted when two adjacent orga- nelles connected and remained fused for at least the next three timeframes. OPA1 oligomerization Src+/+ and Src−/− MEFs were treated with the crosslinker bismaleimidohexane (1 mM) during 30 min at 37 °C. Upon treatment, MEFs were directly processed for SDS-PAGE. SDS‑PAGE and BN‑PAGE For SDS-PAGE experiments, samples were diluted in SDS- PAGE sample buffer (62.5 mM Tris–HCl, pH 6.8; 10% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 0.5% bromophenol blue, 2.5% (v/v) β-mercapto-ethanol) and boiled at 95 °C during 5 min. Proteins were then separated at 200 V during 60 min, using 10 or 12% polyacrylamide gel containing 0.35% (V/V) of 2,2,2-trichloroethanol for total protein staining, as described [47]. Briefly, the 2,2,2-trichlo- roethanol added directly in SDS-PAGE gels interacts with tryptophan in loaded protein and induces UV light-induced fluorescence which can be visualized on a 300 nm transil- luminator. Immunolabeling can then be normalized to the Mitochondrial fusion was also analyzed using live cell imaging of Src+/+ and Src−/− MEFs co-expressing mtD- sRed and photo-activable mitochondria-targeted GFP (PA- mtGFP, Addgene, #23348). PA-mtGFP was photoactivated with 405 nm laser (at 2% laser intensity) during 1 s in a randomly selected ROI of 10 μm2. Cells were then imaged every 10 min using a 60 × oil objective (UPLAN 60 × oil, 1.35NA, Olympus). GFP and DsRed fluorescence in ROIs were analyzed using Fiji in 5–8 cells in 5 independent exper- iments (a total of 37 cells per condition). GFP fluorescence was normalized by DsRed fluorescence. 1 3 O. Lurette et al. Page 14 of 17 7 Endogenous mitochondrial matrix-localized Src does not ulate starvation-induced mitochondrial elongation. a Repre- ative micrographs of TOM20 labeled in Src+/+ and Src−/− MEFs ed with HBSS during 5 h (n = 3). b Quantitative analysis of chondrial morphology showing the percentage of cells with gated mitochondria, as shown in a. c Representative micro- hs of TOM20 and FLAG labeled in HeLa cells over-expressing chondria-targeted mtSrc-FLAG, or mitochondria-targeted kinase- mtSrc(KD)-FLAG, and treated with HBSS (5 h) as indicated 3–6). d Quantification of the number of cells with elongated mitochondria as shown in c. e Representative immunoblotting (n = 3) of pY416-Src, Src and total protein load (TPL) in total cell lysates (TCL) and mitochondria-enriched fractions (Mito) obtained from HeLa cells treated with HBSS during 5 h. f Quantification of the activity (left) and levels (right) of Src based on the immunoblotting shown in e. Protein levels were normalized by TPL. Data are shown as mean ± S.E.M. A.U.: arbitrary unit. Data with different letters are statistically different (p < 0.05), according to two-way ANOVA fol- lowed by Tukey’s post hoc test (for b and d). mitochondria as shown in c. e Representative immunoblotting (n = 3) of pY416-Src, Src and total protein load (TPL) in total cell lysates (TCL) and mitochondria-enriched fractions (Mito) obtained from HeLa cells treated with HBSS during 5 h. f Quantification of the activity (left) and levels (right) of Src based on the immunoblotting shown in e. Protein levels were normalized by TPL. Data are shown as mean ± S.E.M. A.U.: arbitrary unit. Data with different letters are statistically different (p < 0.05), according to two-way ANOVA fol- lowed by Tukey’s post hoc test (for b and d). p < 0.01 according to Student’s t test. Scale bars: 20 μm SDS‑PAGE and BN‑PAGE p < 0.01 according to Student’s t test. Scale bars: 20 μm O. Lurette et al. 327 Page 14 of 17 327 Page 14 of 17 ig. 7 Endogenous mitochondrial matrix-localized Src does not modulate starvation-induced mitochondrial elongation. a Repre- mitochondria as shown in c. e Representative immunoblotting (n of pY416-Src, Src and total protein load (TPL) in total cell lys Fig. 7 Endogenous mitochondrial matrix-localized Src does not modulate starvation-induced mitochondrial elongation. a Repre- sentative micrographs of TOM20 labeled in Src+/+ and Src−/− MEFs treated with HBSS during 5 h (n = 3). b Quantitative analysis of mitochondrial morphology showing the percentage of cells with elongated mitochondria, as shown in a. c Representative micro- graphs of TOM20 and FLAG labeled in HeLa cells over-expressing mitochondria-targeted mtSrc-FLAG, or mitochondria-targeted kinase- dead mtSrc(KD)-FLAG, and treated with HBSS (5 h) as indicated (n = 3–6). d Quantification of the number of cells with elongated mitochondria as shown in c. e Representative immunoblotting (n = 3) of pY416-Src, Src and total protein load (TPL) in total cell lysates (TCL) and mitochondria-enriched fractions (Mito) obtained from HeLa cells treated with HBSS during 5 h. f Quantification of the activity (left) and levels (right) of Src based on the immunoblotting shown in e. Protein levels were normalized by TPL. Data are shown as mean ± S.E.M. A.U.: arbitrary unit. Data with different letters are statistically different (p < 0.05), according to two-way ANOVA fol- lowed by Tukey’s post hoc test (for b and d). p < 0.01 according to Student’s t test. Scale bars: 20 μm 1 3 1 3 Page 15 of 17 327 Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology 327 total UV light-induced fluorescence (corresponding to the total protein load). respiration, and (iii) maximal uncoupled respiration induced by FCCP (0.5 μM steps with 2.5 μM final concentration) providing a measure of the maximal capacity of ETS under conditions of physiological substrate supply in the intact cells. After SDS-PAGE, proteins were transferred to polyvi- nylidene difluoride (PVDF) membranes. Membranes were blocked for 1 h in TBS-T (50 mM Tris–Cl, pH 7.6; 150 mM NaCl, 0.1% Tween) containing 5% BSA or 5% skimmed milk and incubated with primary antibodies overnight at 4 °C. SDS‑PAGE and BN‑PAGE Protein immunodetection was performed using pri- mary antibodies directed against Src (#2108S, Cell Sign- aling), NDUFA9 (ab14713, Abcam), UQCRC2 (ab14742, Abcam), COXIV (ab16056, Abcam), ATPB (ab14730, Abcam), VDAC (ab14734, Abcam), TOM20 (sc-17764, Santa Cruz), SOD2 (#13194S, Cell Signaling), cytochrome c (ab133504, Abcam), ATP5α (ab14748, Abcam), myc (#2276S, Cell Signaling), α-tubulin (#3763S, Cell Sign- aling), Drp1 (#8570S, Cell Signaling), FIS1 (#ALX-210- 1037-0100, Enzo life sciences), MiD51 (#20164-1-AP, Proteintech), OPA1 (#612607BD, Biosciences), ERp57 (#AF8219, R&D Systems), Smac-Diablo (#15108S, Cell Signaling), V5 (#13202S, Cell signaling) and FLAG (#F1804, Sigma-Aldrich). Statistical analyses Data are presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 9. Data were analyzed using Student t test, one-way or two-way ANOVA followed by Tukey post hoc test, as appropriate. For two-way ANOVA and post hoc tests, statistical differences (p < 0.05) are pre- sented with letters: datapoints with different letters are sta- tistically different (p < 0.05). For instance, a datapoint with the letter a is statistically different (p < 0.05) from datapoints with the letters b or bc, whereas it is not statistically different (p > 0.05) from datapoints with the letters a or ab. Citrate synthase activity Citrate synthase enzymatic activity was determined with a BioTek Synergy H1 microplate reader (Biotek, Montréal, QC, Canada) at 37 °C by following the reduction of 5,5′-dith- iobis (2-nitrobenzoic acid) (ε = 13.6 mL cm−1 µmol−1) at 412 nm for 8 min. The reaction medium contained 0.1 mmol L−1 DTNB, 0.1 mmol L−1 Acetyl-CoA and the reaction was started with the addition of 0.13 mmol L−1 oxaloacetic acid in 100 mmol L−1 imidazole–HCl, pH = 8. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00018-022-04325-y. Supplementary Information The online version contains supplemen- tary material available at https://doi.org/10.1007/s00018-022-04325-y. Acknowledgements Electron microscopy was performed at the Imag- ing facility of the department of biology at the University of Padova (Italy). The area of individual mitochondria (mitochondrial area, n = 131–132) and the perimeter of individual mitochondria (mitochondrial perimeter, n = 131–133) were then analyzed using Fiji. Author contributions OL, HG, NP, JP and EHC designed the study. OL, HG, JLM, RJM, JPR, GHC, ND, NP and EHC performed experi- ments and analyzed data. OL, HG, JLM, NP, JP and EHC wrote and edited the manuscript. Electron microscopy Src+/+ and Src−/− MEFs were fixed with 2.5% glutaralde- hyde in 0.1 M sodium cacodylate buffer pH 7.4 at 4 °C. Samples were then incubated with 1% osmium tetroxide and 1% potassium ferricyanide in 0.1 M sodium cacodylate buffer for 1 h at 4 °C. After three water washes, samples were dehydrated in a graded ethanol series and embedded in epoxy resin. Ultrathin sections (60–70 nm) were obtained with an Ultrotome V (LKB) ultramicrotome, counterstained with uranyl acetate and lead citrate and viewed with a Tec- nai G2 (FEI) transmission electron microscope operating at 100 kV. Images were captured with a Veleta (Olympus Soft Imaging System) digital camera at the Imaging facility of the department of biology at the University of Padova (Italy). References 19. Klinghoffer RA, Sachsenmaier C, Cooper JA, Soriano P (1999) Src family kinases are required for integrin but not PDGFR signal transduction. EMBO J 18:2459–2471. https://doi.org/10. 1093/emboj/18.9.2459 1. Pernas L, Scorrano L (2016) Mito-morphosis: mitochondrial fusion, fission, and cristae remodeling as key mediators of cel- lular function. Annu Rev Physiol 78:505–531. https://doi.org/10. 1146/annurev-physiol-021115-105011 j 20. Chang C-R, Blackstone C (2007) Cyclic AMP-dependent pro- tein kinase phosphorylation of Drp1 regulates its GTPase activ- ity and mitochondrial morphology. J Biol Chem 282:21583– 21587. https://doi.org/10.1074/jbc.C700083200 2. Benard G, Bellance N, James D et al (2007) Mitochondrial bioen- ergetics and structural network organization. J Cell Sci 120:838– 848. https://doi.org/10.1242/jcs.03381 3. Tondera D, Grandemange S, Jourdain A et al (2009) SLP-2 is required for stress-induced mitochondrial hyperfusion. EMBO J 28:1589–1600. https://doi.org/10.1038/emboj.2009.89 21. Cribbs JT, Strack S (2007) Reversible phosphorylation of Drp1 by cyclic AMP-dependent protein kinase and calcineurin regu- lates mitochondrial fission and cell death. EMBO Rep 8:939– 944. https://doi.org/10.1038/sj.embor.7401062 4. Gomes LC, Di Benedetto G, Scorrano L (2011) During autophagy mitochondria elongate, are spared from degradation and sustain cell viability. Nat Cell Biol 13:589–598. https://doi.org/10.1038/ ncb2220 22. Yu R, Liu T, Ning C et al (2019) The phosphorylation status of Ser-637 in dynamin-related protein 1 (Drp1) does not determine Drp1 recruitment to mitochondria. J Biol Chem 294:17262– 17277. https://doi.org/10.1074/jbc.RA119.008202 5. Griparic L, Kanazawa T, van der Bliek AM (2007) Regulation of the mitochondrial dynamin-like protein Opa1 by proteolytic cleavage. J Cell Biol 178:757–764. https://doi.org/10.1083/jcb. 200704112 23. Berbusse GW, Woods LC, Vohra BPS, Naylor K (2016) Mito- chondrial dynamics decrease prior to axon degeneration induced by vincristine and are partially rescued by overexpressed cyt- Nmnat1. Front Cell Neurosci 10:179. https://doi.org/10.3389/ fncel.2016.00179 6. Legros F, Lombès A, Frachon P, Rojo M (2002) Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins. Mol Biol Cell 13:4343– 4354. https://doi.org/10.1091/mbc.e02-06-0330 24. Karbowski M, Arnoult D, Chen H et al (2004) Quantitation of mitochondrial dynamics by photolabeling of individual orga- nelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis. J Cell Biol 164:493–499. https://doi.org/10.1083/jcb.200309082 7. Tilokani L, Nagashima S, Paupe V, Prudent J (2018) Mitochon- drial dynamics: overview of molecular mechanisms. Essays Bio- chem 62:341–360. https://doi.org/10.1042/EBC20170104 8. Yeatman TJ (2004) A renaissance for SRC. Nat Rev Cancer 4:470–480. https://doi.org/10.1038/nrc1366 25. Cellular respiration Funding This work was funded through grants awarded to EHC by Natural Sciences and Engineering Research Council of Canada (grant number RGPIN-2015-05880), Canadian Health Research Institute (grant number 156238), Canada Research Chair program, New Brun- swick Health Research Foundation, New Brunswick Innovation Foun- dation and to JP by Medical Research Council funding, UK (MC_ UU_00015/7). OL and GHC are the recipient of a NBHRF scholarship and JLM is a MRC-funded PhD student. Oxygen consumption was measured using the high- resolution respirometry system Oxygraph-2k Oroboros (Innsbruck, Austria). Cell respiration was measured with 1 × 106 cells mL−1 according to volume-specific flux at 37 °C in 2 mL chambers at a stirring rate of 750 rpm. Three different states of endogenous respiration with intact cells were measured: (i) basal respiration representing the endog- enous physiological coupled state, (ii) respiration with oli- gomycin (2 μg mL−1) representing the non-coupled resting Data availability Complete data or material used in the present study will be available upon reasonable request. 1 3 327 Page 16 of 17 O. Lurette et al. Consent for publication All the authors approved the manuscript. 14. Demory ML, Boerner JL, Davidson R et al (2009) Epidermal growth factor receptor translocation to the mitochondria. J Biol Chem 284:36592–36604. https://doi.org/10.1074/jbc.M109. 000760 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. 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/. 15. Guedouari H, Savoie M-C, Jean S et al (2020) Multi-omics reveal that c-Src modulates the mitochondrial phosphotyrosine proteome and metabolism according to nutrient availability. Cell Physiol Biochem 54:517–537 16. Djeungoue-Petga M-A, Lurette O, Jean S et al (2019) Intrami- tochondrial Src kinase links mitochondrial dysfunctions and aggressiveness of breast cancer cells. Cell Death Dis 10:1–15. https://doi.org/10.1038/s41419-019-2134-8 17. Guedouari H, Ould Amer Y, Pichaud N, Hebert-Chatelain E (2021) Characterization of the interactome of c-Src within the mitochondrial matrix by proximity-dependent biotin identifica- tion. Mitochondrion 57:257–269. https://doi.org/10.1016/j.mito. 2020.12.012 18. Zhao J, Zhang J, Yu M et al (2013) Mitochondrial dynamics regulates migration and invasion of breast cancer cells. Onco- gene 32:4814–4824. https://doi.org/10.1038/onc.2012.494f Conflict of interest The authors declare that there is no conflict of in- terest. Conflict of interest The authors declare that there is no conflict of in- terest. 13. Miyazaki T, Neff L, Tanaka S et al (2003) Regulation of cytochrome c oxidase activity by c-Src in osteoclasts. J Cell Biol 160:709–718. https://doi.org/10.1083/jcb.200209098 Consent for publication All the authors approved the manuscript. Declarations 12. Bard F, Patel U, Levy JB et al (2002) Molecular complexes that contain both c-Cbl and c-Src associate with Golgi membranes. Eur J Cell Biol 81:26–35. https://doi.org/10.1078/0171-9335-00217 References Cereghetti G, Costa V, Scorrano L (2010) Inhibition of Drp1- dependent mitochondrial fragmentation and apoptosis by a poly- peptide antagonist of calcineurin. Cell Death Differ 17:1785– 1794. https://doi.org/10.1038/cdd.2010.61 9. Ishizawar R, Parsons SJ (2004) c-Src and cooperating partners in human cancer. Cancer Cell 6:209–214. https://doi.org/10.1016/j. ccr.2004.09.001 26. Nakano M, Imamura H, Nagai T, Noji H (2011) Ca2+ regula- tion of mitochondrial ATP synthesis visualized at the single cell level. ACS Chem Biol 6:709–715. https://doi.org/10.1021/ cb100313n 10. Kaplan KB, Swedlow JR, Varmus HE, Morgan DO (1992) Asso- ciation of p60c-src with endosomal membranes in mammalian fibroblasts. J Cell Biol 118:321–333. https://doi.org/10.1083/jcb. 118.2.321 27. Hebert-Chatelain E (2013) Src kinases are important regulators of mitochondrial functions. Int J Biochem Cell Biol 45:90–98. https://doi.org/10.1016/j.biocel.2012.08.014 11. David-Pfeuty T, Nouvian-Dooghe Y (1990) Immunolocalization of the cellular src protein in interphase and mitotic NIH c-src overexpresser cells. J Cell Biol 111:3097–3116. https://doi.org/ 10.1083/jcb.111.6.3097 p g j 28. Salvi M, Brunati AM, Bordin L et al (2002) Characterization and location of Src-dependent tyrosine phosphorylation in rat 1 3 Page 17 of 17 327 Mitochondrial matrix‑localized Src kinase regulates mitochondrial morphology 327 brain mitochondria. Biochim Biophys Acta BBA Mol Cell Res 1589:181–195. https://doi.org/10.1016/S0167-4889(02)00174-X 39. McLelland G-L, Goiran T, Yi W et al (2018) Mfn2 ubiquitination by PINK1/parkin gates the p97-dependent release of ER from mitochondria to drive mitophagy. Elife 7:e32866. https://doi.org/ 10.7554/eLife.32866 p g 29. Demory ML, Boerner JL, Davidson R et al (2009) Epidermal growth factor receptor translocation to the mitochondria: regula- tion and effect. J Biol Chem 284:36592–36604. https://doi.org/10. 1074/jbc.M109.000760 40. Signorile A, Sgaramella G, Bellomo F, De Rasmo D (2019) Pro- hibitins: a critical role in mitochondrial functions and implication in diseases. Cells 8:71. https://doi.org/10.3390/cells8010071i j 30. Ogura M, Yamaki J, Homma MK, Homma Y (2012) Mitochon- drial c-Src regulates cell survival through phosphorylation of res- piratory chain components. Biochem J 447:281–289. https://doi. org/10.1042/BJ20120509 41. Li H, Ruan Y, Zhang K et al (2016) Mic60/Mitofilin determines MICOS assembly essential for mitochondrial dynamics and mtDNA nucleoid organization. Cell Death Differ 23:380–392. https://doi.org/10.1038/cdd.2015.102 31. Rambold AS, Kostelecky B, Elia N, Lippincott-Schwartz J (2011) Tubular network formation protects mitochondria from autophagosomal degradation during nutrient starvation. Proc Natl Acad Sci 108:10190–10195. https://doi.org/10.1073/pnas.11074 02108 p g 42. Zlatkine P, Mehul B, Magee AI (1997) Retargeting of cytosolic proteins to the plasma membrane by the Lck protein tyrosine kinase dual acylation motif. J Cell Sci 110:673–679. https://doi. org/10.1242/jcs.110.5.673 32. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References Head B, Griparic L, Amiri M et al (2009) Inducible proteolytic inactivation of OPA1 mediated by the OMA1 protease in mam- malian cells. J Cell Biol 187:959–966. https://doi.org/10.1083/jcb. 200906083 g j 43. Benediktsson AM, Schachtele SJ, Green SH, Dailey ME (2005) Ballistic labeling and dynamic imaging of astrocytes in organo- typic hippocampal slice cultures. J Neurosci Methods 141:41–53. https://doi.org/10.1016/j.jneumeth.2004.05.013 33. Hebert-Chatelain E, Jose C, Gutierrez Cortes N et al (2012) Pres- ervation of NADH ubiquinone-oxidoreductase activity by Src kinase-mediated phosphorylation of NDUFB10. Biochim Biophys Acta 1817:718–725. https://doi.org/10.1016/j.bbabio.2012.01.014 44. Guedouari H, Daigle T, Scorrano L, Hebert-Chatelain E (2017) Sirtuin 5 protects mitochondria from fragmentation and degrada- tion during starvation. Biochim Biophys Acta BBA Mol Cell Res 1864:169–176. https://doi.org/10.1016/j.bbamcr.2016.10.015 34. Livigni A, Scorziello A, Agnese S et al (2005) Mitochondrial AKAP121 links cAMP and src signaling to oxidative metabo- lism. Mol Biol Cell 17:263–271. https://doi.org/10.1091/mbc. e05-09-0827 45. Nagashima S, Tábara L-C, Tilokani L et al (2020) Golgi-derived PI(4)P-containing vesicles drive late steps of mitochondrial divi- sion. Science 367:1366–1371. https://doi.org/10.1126/science. aax6089 46. Prudent J, Zunino R, Sugiura A et al (2015) MAPL SUMOyla- tion of Drp1 stabilizes an ER/mitochondrial platform required for cell death. Mol Cell 59:941–955. https://doi.org/10.1016/j.molcel. 2015.08.001 35. Hébert Chatelain E, Dupuy J-W, Letellier T, Dachary-Prigent J (2011) Functional impact of PTP1B-mediated Src regulation on oxidative phosphorylation in rat brain mitochondria. Cell Mol Life Sci CMLS 68:2603–2613. https://doi.org/10.1007/ s00018-010-0573-6 47. Ladner CL, Yang J, Turner RJ, Edwards RA (2004) Visible fluo- rescent detection of proteins in polyacrylamide gels without stain- ing. Anal Biochem 326:13–20. https://doi.org/10.1016/j.ab.2003. 10.047 36. Yamashita S, Jin X, Furukawa K et al (2016) Mitochondrial divi- sion occurs concurrently with autophagosome formation but inde- pendently of Drp1 during mitophagy. J Cell Biol 215:649–665. https://doi.org/10.1083/jcb.201605093 Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. p g j 37. Tan AR, Cai AY, Deheshi S, Rintoul GL (2011) Elevated intra- cellular calcium causes distinct mitochondrial remodelling and calcineurin-dependent fission in astrocytes. Cell Calcium 49:108– 114. https://doi.org/10.1016/j.ceca.2010.12.002 38. Nemani N, Carvalho E, Tomar D et al (2018) MIRO-1 determines mitochondrial shape transition upon GPCR activation and Ca2+ stress. Cell Rep 23:1005–1019. https://doi.org/10.1016/j.celrep. 2018.03.098 1 3 1 3
W4317810326.txt	https://www.mdpi.com/2304-8158/12/3/503/pdf?version=1674299547	en		Effects of 6-Shogaol on Glucose Uptake and Intestinal Barrier Integrity in Caco-2 Cells	Foods	2,023	cc-by	7,205	foods Article Effects of 6-Shogaol on Glucose Uptake and Intestinal Barrier Integrity in Caco-2 Cells Wenya Jiao 1 , Yaxin Sang 1 , Xianghong Wang 1, * 1 2 * and Shuo Wang 2, * College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China Tianjin Key Laboratory of Food Science and Health, School of Medicine, Nankai University, Tianjin 300071, China Correspondence: wangshipin2017@163.com (X.W.); s.wang@tust.edu.cn (S.W.); Tel.: +86-(0312)-7528195 (X.W.); +86-022-60912490 (S.W.) Abstract: As the main bioactive component in dried ginger, 6-shogaol has potential hypoglycemic activity, but its mechanism is still unclear. The process of carbohydrate digestion and glucose absorption is closely related to the enzymatic activity of epithelial brush cells, expression of glucose transporters, and permeability of intestinal epithelial cells. Therefore, this study explored the hypoglycemic mechanism of 6-shogaol from the perspective of glucose uptake, absorption transport, and protection of intestinal barrier function. Based on molecular docking, the binding energy of 6-shogaol and α-glucosidase is −6.24 kcal/mol, showing a high binding affinity. Moreover, a-glucosidase enzymatic activity was reduced (−78.96%) when the 6-shogaol concentration was 500 µg/mL. After 6-shogaol intervention, the glucose uptake was reduced; the relative expression of glucose transporters GLUT2 and SGLT1 were down regulated; and tight junction proteins ZO-1, Occludin and Claudin were up regulated in differentiated Caco-2 cells. This study confirmed that 6-shogaol effectively inhibits the activity of α-glucosidase and has beneficial effects on glucose uptake, protection of intestinal barrier function, and promotion of intestinal material absorption. Keywords: 6-shogaol; Caco-2 cells; glucose uptake; barrier function 1. Introduction Citation: Jiao, W.; Sang, Y.; Wang, X.; Wang, S. Effects of 6-Shogaol on Glucose Uptake and Intestinal Barrier Integrity in Caco-2 Cells. Foods 2023, 12, 503. https://doi.org/10.3390/ foods12030503 Received: 13 November 2022 Revised: 10 January 2023 Accepted: 17 January 2023 Published: 21 January 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). The dried ginger (Rhizoma zingiberis) is derived from the dried rhizome of Zingiber officinale Rose. It has been reported that ginger has a variety of health benefits, such as antioxidant [1,2], anti-inflammatory [3], immune regulation [4], antiemetic [5], hypoglycemic [6,7], and lipid-lowering [8] properties, among others. Gingerols, as the main flavor and active components in ginger, are important contributors to the functional properties of ginger. The 6-shogaol belongs to a kind of gingerols, which has a higher concentration in dried ginger than in undried ginger [3]. This is because 6-gingerol is easily dehydrated and transformed into 6-shogaol under the influence of temperature during drying [9]. In recent years, studies have reported the hypoglycemic effect of 6-shogaol, including reducing blood sugar [10], improving kidney damage caused by diabetes [11], and diabetes cardiomyopathy [12]. For streptozotocininduced diabetes mice, the administration of 6-shogaol significantly reduced blood glucose; improved insulin resistance; and alleviated pancreatic, kidney, and liver damage [10,11]. Based on the cell model of diabetes cardiomyopathy induced by high glucose, 6-shogaol pretreatment significantly increased its cell viability and reduced its apoptosis, reflecting the potential protective effect of 6-shogaol on the induction of diabetes cardiomyopathy [12]. In addition, it was found that 6-shogaol could effectively regulate the glucose utilization of 3T3-L1 adipocytes and C2C12 myotube cell lines [13]. These results indicate that 6-shogaol can be used as a therapeutic agent to prevent complications in patients with diabetes. Type 2 diabetes (T2DM) is an endocrine disorder of protein, lipid, and carbohydrate metabolism, which is usually accompanied by a variety of complications [14]. Its main feature is the increase in fasting and postprandial blood glucose levels. At present, in addition Foods 2023, 12, 503. https://doi.org/10.3390/foods12030503 https://www.mdpi.com/journal/foods Foods 2023, 12, 503 2 of 11 to diet management, some chemosynthetic drugs are also often used to treat T2DM. However, it still has adverse reactions, such as hypoglycemia and gastrointestinal irritation [15]. Therefore, people pay more and more attention to reducing blood glucose and improving T2DM through active ingredients in food and drug homologous foods. In recent years, the relevant research on the hypoglycemic effect of natural active ingredients mainly focuses on the following aspects: (1) inhibiting the activity of related enzymes; (2) improving insulin resistance; (3) liver glycogen decomposition and gluconeogenesis. α-glucosidase plays a key role in improving blood glucose levels, and is a potential target for designing drugs for the treatment of diabetes [16]. At present, the research of α-glucosidase inhibitors is based on this target. It can delay the decomposition and digestion of complex carbohydrates and disaccharides by inhibiting the activity of α-glucosidase that decomposes oligosaccharides into monosaccharides [17]. Thus, delaying the absorption of glucose in the intestinal cavity and lowering the postprandial blood glucose. Chronic hyperglycemia caused by insulin resistance or insufficient insulin secretion is the characteristic of T2DM [18]. Therefore, insulin resistance plays a key role in the pathogenesis of T2DM and improving insulin resistance is an effective way to improve T2DM. In addition to insulin resistance, the increase in endogenous glucagon will promote gluconeogenesis, enhance glycogen degradation, and inhibit glycogen synthesis to stimulate liver glucose production [19]. This may be a goal of reducing blood glucose and treating diabetes. On the other hand, repairing the damaged hepatic glycogen structure is also a method to improve the abnormal hepatic glycometabolism in diabetes [20]. In addition, carbohydrate digestion and glucose absorption also play an important role in maintaining glucose homeostasis. The digestive process is affected by the activities of a variety of key digestive enzymes, while the absorption process is closely related to the expression level of glucose transporters, intestinal epithelial cell permeability, intestinal barrier function, and other factors [21,22]. Differentiated Caco-2 cells have the morphological and functional characteristics of small intestinal cells, showing brush, such as borders, tight junctions, and uptake of transporters. These transporters regulate the permeability of drugs from intestinal cavity to systemic circulation and are closely related to intestinal permeability and epithelial barrier function [23]. In recent years, there are few reports on the effects of 6-shogaol on glucose uptake and glucose transporters in the studies on its hypoglycemic efficacy. In this study, we explored the effect of 6-shogaol on the activity of α-glucosidase, glucose uptake transport of Caco-2 cells, and the protection of intestinal barrier function, thus revealing the hypoglycemic mechanism. It is expected to provide a valuable reference for the development of dried ginger bioactive substances as hypoglycemic agents. 2. Materials and Methods 2.1. Materials and Chemicals Caco-2 cells were purchased from the national experimental cell resource sharing service platform (Beijing, China). The 6-shogaol and α-glucosidase were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). The glucose test kit was purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Antibodies for Occludin, GLUT2 and Claudin-1 were purchased from Abcam Biotechnology (Cambridge, UK). ZO-1 rabbit antibody and SGLT1 rabbit antibody were obtained from Proteintech Group (Chicago, IL, USA) and ImmunoWay Biotechnology (Plano, TX, USA), respectively. 2.2. Molecular Docking The structure file of 6-shogaol (PubChem CID:5281794) was prepared from pubChem database. The prepared 6-shogaol file and the three-dimensional crystal structure of α-glucosidase (PDB ID:3W37) from the PDB database were used. The structures were modelled in Discovery Studio 2016, water molecules were removed, hydrogen atoms were added, and the structures were docked. After docking, the activity was evaluated according to docking calculation results. Foods 2023, 12, 503 3 of 11 2.3. Determination of α-Glucosidase Activity Using PNPG (4-Nitrophenyl-β-D-glucopyranoside) method, the reaction system was slightly modified according to the method of predecessors [24]. Firstly, 25 µL of 6-shogaol (500, 300, 200, 100, 50, 25, 12.5, 6.25, 0 µg/mL) and acarbose (100 µg/mL) reacted with 50 µL α-glucosidase, respectively, at 37 ◦ C for 15 min. Then, 100 µL PNPG (10 mmol/L) was added for 30 min. Finally, 50 µL Ca2 CO3 (2 mol/L) was added to terminate the reaction, and the absorbance value was measured at 405 nm. Calculations were conducted according to the following formula: Inhibition rate (%) = ( A1 − A2 ) − ( A3 − A4 ) × 100% ( A1 − A2 ) where A1 represents the control, A2 represents the blank, A3 represents the sample to be tested, and A4 represents the sample blank. 2.4. Cell Cultures Caco-2 cells were cultured in DMEM high glucose medium with 10% (v/v) fetal bovine serum added. The cells were incubated at 37 ◦ C and 5% carbon dioxide humidified atmosphere, and then subcultured when 80–90% confluence was reached. When approaching fusion, cells were digested with trypsin, counted, and inoculated onto transwell culture plate, and the culture medium was replaced every two days. After 1 week, the medium was replaced every day until 21 days. 2.5. Determination of Cell Metabolic Activity The cell concentration was adjusted to 1 × 104 cells/well (100 µL) on 96-well plate. After 24 h of culture, the old medium was replaced by 100 µL medium containing different concentrations (0, 5, 10, 20, 40, 60, 100 µg/mL) of 6-shogaol. After continuous cultivation for 24 h, 10 µL CCK8 was added to each well, and the absorbance was measured at 450 nm after incubation for 2 hours. Calculations were conducted according to the following formula: Cell Metabolic Activity (%) = A1 − A0 × 100% A2 − A0 where A0 represents the blank group; A1 represents the sample to be tested; A2 represents the control. 2.6. Measurement of Transmembrane Resistance Value TEER The cell concentration was adjusted to 2.5 × 105 cells/well (0.5 mL was added to upper chamber, 1.5 mL was added to the basal chamber) on 6-well plate, and each plate was set with a cell-free well as a control. The culture medium was changed every 2 days and changed every day after one week for 21 days. During this period, the Millicell® ERS-2 voltohm meter (EMD Millipore Corporation, USA) was used to monitor the transmembrane electrical resistance (TEER) of each well and check the integrity of cells for three consecutive weeks. Calculations were conducted according to the following formula: TEER value (Ω·cm2 )= (R1 − R0 ) × 1.1 cm2 (effective area of 12-well plate) where R1 represents the resistance value of each well and A0 represents the resistance value of the blank group. 2.7. Determination of Glucose Content The cell concentration was adjusted to 2.5 × 105 cells/well (0.5 mL was added to upper chamber, 1.5 mL was added to the basal chamber) on 6-well plate. After the 21-day process, it was replaced with serum-free DMEM culture medium for starvation overnight. The cells were divided into five groups: blank group (without cells), control group (with Foods 2023, 12, 503 4 of 11 cells; without 6-shogaol), and dose group (with cells; 5, 10, 20 µg/mL of 6-shogaol). The content of glucose in cell culture medium was detected at 12 h and 24 h, according to the manufacturer’s instructions of the glucose kit. 2.8. Western Blot Analysis After cells in each group were treated with 6-shogaol of different concentrations for 24 h, the culture medium was discarded. After PBS washing, the prepared lysis solution (mammalian protein extraction kit solution and protein inhibitor cocktail solution in a proportion of 1:99) was added and placed on ice for complete lysis. Then, cells and lysis solution were transferred to centrifuge tubes to separate supernatant. The protein concentration was determined with BSA kit and adjusted to be consistent. Protein sample and 5×SDS-PAGE loading buffer was mixed and boiled, the target protein was separated by SDS gel electrophoresis with a sample loading of 40 µg/lane. It was transferred to the membrane, sealed, incubated with antibodies, and colored under the illumination instrument [25]. 2.9. Statistical Analysis GraphPad Prism 9.1.0 software was used to process and analyze data, expressed in mean ± SEM. One-way analysis of variance was used for significant differences, p < 0.05 or p < 0.01, and it was considered to be significantly different or extremely significantly different from the control group. 3. Results 3.1. Molecular Interaction Molecular docking technology was beneficial to the discovery of new α-glucosidase inhibitors [26]. Discovery Studio 2016 (DS2016) was used for molecular simulation and the binding sites and forces were shown in Figure 1. Both 6-shogaol and acarbose can effectively bind to the active pocket of α-glucosidase protein, and their binding energies are −6.24 and −7.20 (kcal/mol), respectively, indicating that they have high affinity with α-glucosidase. The 6-shogaol interacts with α-glucosidase under the combined action of conventional hydrogen bond, carbon hydrogen bond, pi-sigma, pi-pi stacked, and other forces. Among them, the conventional hydrogen bonds were formed with the amino acid residues ASP568 (1.69 Å), ASP357 (2.02 Å), and ARG552 (2.95 Å), and the bond length formed with ASP568 (1.69 Å) was the shortest and the binding was the closest. Carbon hydrogen bond was formed with ASP469 (2.69 Å). Sulfur-X was formed with MET470 (3.20 Å). Pi-anion was formed with ASP469 (3.80 Å). Pi-sigma were formed with PHE476 (2.91 Å). Pi-stacked were formed with PHE601 (4.92 Å) and TRP329 (5.36 Å). Forming alkyl was formed with LYS506 (4.23 Å). Acarbose interacts with α-glucosidase under the combined action of van der Waals force, conventional hydrogen bond, pi-donor hydrogen bond, and pi-alkyl. The van der Waals forces were formed with the amino acid residues HIS626, TRP467, ASP597, TRP565, ARG624, ASP568, ARG552, ILE233, MET470, and PHE476. Conventional hydrogen bonds were formed with ASP357 (4.83 Å), ASP469 (5.10 Å), ALA234 (3.95 Å), and LYS506 (4.30 Å). Pi-donor hydrogen bond was formed with PHE601. The pi-alkyl bonds were formed with TRP432 (6.81 Å) and TRP432 (6.60 Å). 3.2. Inhibitory Effect on A-Glucosidase Preliminarily, we explored the inhibition of 6-shogaol on α-glucosidase. It can be seen from Figure 2 that the inhibition rate of 6-shogaol and acarbose groups was significantly increased compared with the control group. In the positive control group, the inhibition rate of acarbose with the concentration of 100 µg/mL was 98.94%. The activity of α-glucosidase was inhibited by 6-shogaol at different doses. When the concentration of 6-shogaol was 500 µg/mL, the inhibition rate was 78.96%. The results showed that 6-shogaol inhibited the activity of α-glucosidase in a dose-dependent manner. As a typical glucosidase inhibitor, acarbose has shown strong inhibition ability. Foods 2023, 12, x FOR PEER REVIEW 5 of 12 Foods 2023, 12, 503 5 of 11 Å), ALA234 (3.95 Å), and LYS506 (4.30 Å). Pi-donor hydrogen bond was formed with PHE601. The pi-alkyl bonds were formed with TRP432 (6.81 Å) and TRP432 (6.60 Å). Figure 1. Molecular models of 6-shogaol and acarbose binding to α-glucosidase protein: (A) 3D model of 6-shogaol and α-glucosidase (PDBID:3A4A) crystal structure docking. (B) 2D model of 6shogaol and α-glucosidase docking. (C) 3D model of acarbose and α-glucosidase (PDBID:3A4A) crystal structure docking. (D) 2D model of acarbose and α-glucosidase docking. 3.2. Inhibitory Effect on ɑ-Glucosidase Preliminarily, we explored the inhibition of 6-shogaol on α-glucosidase. It can be seen from Figure 2 that the inhibition rate of 6-shogaol and acarbose groups was significantly increased compared with the control group. In the positive control group, the inhibition rate of acarbose with the concentration of 100 μg/mL was 98.94%. The activity of α-glucosidase was inhibited by 6-shogaol at different doses. When the concentration of 6-shogaol Figure Molecular models and acarbose binding to α-glucosidase protein: (A) 3D Figure 1. Molecular models of of 6-shogaol 6-shogaol acarbose binding to α-glucosidase (A) 3D was 5001.μg/mL, the inhibition rate wasand 78.96%. The results showed thatprotein: 6-shogaol inhibmodel of 6-shogaol and α-glucosidase (PDBID:3A4A) crystal structure docking. (B) 2D model of 6model 6-shogaol and α-glucosidase crystal structure 2D model of ited theofactivity of α-glucosidase in a(PDBID:3A4A) dose-dependent manner. Asdocking. a typical(B)glucosidase shogaol and α-glucosidase docking. (C) 3D model of acarbose and α-glucosidase (PDBID:3A4A) 6-shogaol and α-glucosidase docking. (C) 3D model of acarbose and α-glucosidase (PDBID:3A4A) inhibitor, has shown inhibition ability. crystal acarbose structure docking. (D) 2Dstrong model of acarbose and α-glucosidase docking. crystal structure docking. (D) 2D model of acarbose and α-glucosidase docking. 3.2. Inhibitory Effect on ɑ-Glucosidase Preliminarily, we explored the inhibition of 6-shogaol on α-glucosidase. It can be seen from Figure 2 that the inhibition rate of 6-shogaol and acarbose groups was significantly increased compared with the control group. In the positive control group, the inhibition rate of acarbose with the concentration of 100 µg/mL was 98.94%. The activity of αglucosidase was inhibited by 6-shogaol at different doses. When the concentration of 6shogaol was 500 µg/mL, the inhibition rate was 78.96%. The results showed that 6-shogaol inhibited the activity of α-glucosidase in a dose-dependent manner. As a typical glucosidase inhibitor, acarbose has shown strong inhibition ability. Figure2.2.Inhibition Inhibitionofofα-glucosidase α-glucosidaseactivity activitybyby6-shogaol. 6-shogaol. Figure 3.3. Caco-2 Cell Transmembrane Resistance 3.3. Caco-2 Cell Transmembrane Resistance To evaluate the monolayer integrity of differentiated Caco-2 cells, we measured the To within evaluate the weeks. monolayer integrity differentiated Caco-2 cells,of wecell measured the TEER three As shown in of Figure 3, with the extension culture time, Foods 2023, 12, x FOR PEER REVIEW TEER within three weeks. As shown in Figure 3, with the extension of cell culture6 time, of 12 the monolayer transmembrane resistance of cells continued to increase every week. It the resistance of cells to increase everya week. has hasmonolayer been > 500transmembrane Ω·cm2 at 21 days, indicating thatcontinued Caco-2 cells have formed dense Itmono2 at 21 days, indicating that Caco-2 cells have formed a dense monolayer been > 500 Ω∙cm layer structure. structure. Figure 2. Inhibition of α-glucosidase activity by 6-shogaol. 3.3. Caco-2 Cell Transmembrane Resistance To evaluate the monolayer integrity of differentiated Caco-2 cells, we measured the TEER within three weeks. As shown in Figure 3, with the extension of cell culture time, the monolayer transmembrane resistance of cells continued to increase every week. It has Figure3.3.Change ChangeininTEER TEER value value of of Caco-2 significance, as compared Figure Caco-2 cells cellswithin within2121days. days.* represents * represents significance, as comwith the TEER value on the 7th day ( p < 0.01). pared with the TEER value on the 7th day ( p < 0.01). 3.4. Effect on the Activity of Caco-2 Cells CCK8 method was used to detect the effect of 6-shogaol on the metabolic activity of Caco-2 cells. It can be seen from Figure 4 that the activity of Caco-2 cells was significantly Foods 2023, 12, 503 Figure 3. Change in TEER value of Caco-2 cells within 21 days. * represents significance, as com6 of 11 pared with the TEER value on the 7th day (** p < 0.01). 3.4. Effect on the Activity of Caco-2 Cells 3.4. CCK8 Effect on the Activity of Caco-2 Cells the effect of 6-shogaol on the metabolic activity of method was used to detect Caco-2CCK8 cells. method It can bewas seenused fromtoFigure thateffect the activity of Caco-2 cells was significantly detect4the of 6-shogaol on the metabolic activity of reduced whenItthe 6-shogaol 40 μg/mL, while there wassignificantly no signifiCaco-2 cells. canconcentration be seen from of Figure 4 that was the activity of Caco-2 cells was cant difference the controlofgroup and was the concentration of 5,there 10, and μg/mL. reduced whenbetween the concentration 6-shogaol 40 µg/mL, while was20no signifiTherefore, it is considered thatcontrol 6-shogaol hasand no toxic effect on cells when its and concentration cant difference between the group the concentration of 5, 10, 20 µg/mL. Therefore, it is20 considered that 6-shogaol has no toxic effect cells when was 5, 10, and μg/mL, and subsequent experiments were on carried out. its concentration was 5, 10, and 20 µg/mL, and subsequent experiments were carried out. Figure4.4.Effect Effectofof6-shogaol 6-shogaolon onthe themetabolic metabolicactivity activityofofCaco-2 Caco-2cells. cells.* represents representssignificance, significance,asas Figure comparedtotocontrol controlgroup group((pp<<0.05). 0.05). compared 3.5. Effect on Glucose Consumption of Caco-2 Cells 3.5. Effect on Glucose Consumption of Caco-2 Cells Figure 5 shows the results of glucose content in culture medium after intervention of Figure 5 shows the results of glucose content in culture medium after intervention of 6-shogaol for 12 h and 24 h. It can be seen that, with the extension of time, the residual 6-shogaol for 12 h and 24 h. It can be seen that, with the extension of time, the residual glucose in the control group and 6-shogaol intervention group decreased significantly glucose in the control group and 6-shogaol intervention group decreased significantly compared with the blank group, which is due to the consumption of glucose intake energy Foods 2023, 12, x FOR PEER REVIEW compared with the blank group, which is due to the consumption of glucose intake energy 7 of 12 in the process of cell growth. Compared with the control group, the glucose content of in6-shogaol the process of cell growth. Compared with the control group, the glucose content of 6at different concentrations increased after intervention, indicating that 6-shogaol shogaol at different concentrations increased after intervention, indicating that 6-shogaol inhibited the glucose uptake of cells. inhibited the glucose uptake of cells. Figure 5. Effect of 6-shogaol on glucose consumption in Caco-2 cells. # compared with blank (### p < 0.001), Figure 5. Effect of 6-shogaol on glucose consumption in Caco-2 cells. # compared with blank (### p represents significance, as compared to control group (* pgroup < 0.001, pp << 0.001, 0.01, * ** p <p 0.05). < 0.001), * represents significance, as compared to control (*** < 0.01, * p < 0.05). 3.6. Effect on the Expression of Related Proteins in Caco-2 Cells 3.6. Effect on the Expression of Related Proteins in Caco-2 Cells Western blot results were shown in Figure 6. The expression of related proteins was Western results were showndegrees. in FigureGLUT2 6. The expression related proteinsin was regulated byblot 6-shogaol in varying and SGLT1ofare key proteins the regulated by 6-shogaol in varying degrees. GLUT2 and SGLT1 are key proteins in the glucose transport process, and ZO-1, Occludin and Claudin are tight junction proteins, glucose transport and ZO-1, and of Claudin are tight junction proteins, which are relatedprocess, to the integrity andOccludin permeability the intestinal barrier. It can be seen which related to due the integrity and permeability of the intestinal barrier. It can be from are Figure 6 that to the intervention of 6-shogaol, the relative expression of seen ZO-1, from Figure 6 that due to the intervention of 6-shogaol, the relative expression of ZO-1, Occludin, and Claudin proteins was significantly increased, and the relative expression of Occludin, and Claudin proteins was significantly increased, and the relative expression of GLUT2 and SGLT1 proteins was decreased, especially at high concentrations. This suggests that 6-shogaol can inhibit glucose transport and protect the intestinal barrier. 3.6. Effect on the Expression of Related Proteins in Caco-2 Cells Foods 2023, 12, 503 Western blot results were shown in Figure 6. The expression of related proteins was regulated by 6-shogaol in varying degrees. GLUT2 and SGLT1 are key proteins in the glucose transport process, and ZO-1, Occludin and Claudin are tight junction proteins, which are related to the integrity and permeability of the intestinal barrier. It can be seen 7 of 11 from Figure 6 that due to the intervention of 6-shogaol, the relative expression of ZO-1, Occludin, and Claudin proteins was significantly increased, and the relative expression of GLUT2 and SGLT1 proteins was decreased, especially at high concentrations. This sugGLUT2 and6-shogaol SGLT1 proteins was decreased, especially high concentrations. This suggests gests that can inhibit glucose transport andatprotect the intestinal barrier. that 6-shogaol can inhibit glucose transport and protect the intestinal barrier. Figure Figure6.6.Effect Effectof of6-shogaol 6-shogaolon onthe theexpression expressionof ofrelated relatedproteins proteinsininCaco-2 Caco-2cells. cells.(A) (A)The Theexpressions expressions ofofZO-1, Occludin, Claudin, GLUT2, and SGLT1 were analyzed by Western blot. (B–F) The quantitaZO-1, Occludin, Claudin, GLUT2, and SGLT1 were analyzed by Western blot. (B-F) The quantitive analysis of protein expressions in Caco-2 cells. * represents significance, as compared to control tative analysis of protein expressions in Caco-2 cells. * represents significance, as compared to concells p <(*0.01, p < 0.05). trol (* cells p < 0.01, p < 0.05). 4. Discussion 4. Discussion The monitoring and treatment of T2DM has been a hot topic of research and concern The monitoring and treatment of T2DM has been a hot topic of research and concern for decades. The American Diabetes Association predicts that diabetes patients will increase for decades. The American Diabetes Association predicts that diabetes patients will into 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045, which indicates that diabetes crease to 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045, which indicates that has become a global epidemic health problem [27]. The main manifestation of T2DM is the diabetes has become a global epidemic health problem [27]. The main manifestation of increase in blood glucose. The effective control of postprandial blood glucose increase is T2DM is the increase in blood glucose. The effective control of postprandial blood glucose an effective measure to reduce blood glucose. When the body ingests food, carbohydrates increase is an effective measure to reduce blood glucose. When the body ingests food, are digested into glucose under the action of digestive enzymes in the gastrointestinal tract. α-glucosidase belongs to carbohydrate digestion enzyme and is the rate-limiting enzyme for glucose production. Inhibiting its activity can reduce the production of glucose. Furthermore, glucose is absorbed by epithelial cells through the brush border membrane by the intestinal tract depending on specific glucose cotransporters. The glucose enters into the blood circulation, causing the blood glucose to rise [28]. Therefore, the inhibition of carbohydrate digestion and glucose absorption and transport in the intestine is an effective way to reduce blood glucose. Therefore, we considered the influence of 6-shogaol on the process of glucose digestion, absorption, and transport in order to clarify the mechanism of 6-shogaol’s hypoglycemic effect. α-glucosidase is a key enzyme responsible for digesting dietary carbohydrates into glucose. It hydrolyzes glycosidic bonds in various sugar compounds by means of nuclear or exonuclear cleavage to produce monosaccharides, oligosaccharides, or glycosaminoglycans [29]. Hence, reducing α-glucosidase activity could delay glucose release and further absorption. In this study, the inhibitory effect of 6-shogaol on α-glucosidase was predicted by molecular docking technology. Molecular docking is a method to predict the position and affinity of ligands at receptor binding sites, which is often used in drug design research [30]. It is noteworthy that it has been widely used in food science in recent years, such as in the study of enzyme activity and substrate [31]. The interaction process between the receptor and ligand includes hydrogen bonds, electrostatic interaction, van der Waals force, hydrophobic interaction, etc. [32]. The strength of these interactions is crucial to evaluate the affinity between the receptor and ligand. In the study of the inhibitory effect of pepper plant chemical components on α-glucosidase, luteolin showed a strong inhibitory effect on α-glucosidase, which may be related to the formation of hydrogen bonds and hydrophobic interaction of the key amino acid residues of α-glucosidase [33]. In a study on the inhibitory effect of traditional Chinese medicine ingredients on α-glucosidase, supramolec- Foods 2023, 12, 503 8 of 11 ular docking was used to simulate and clarify its mechanism [29]. The results showed that its inhibitory effect originated from bonding with the residue of α-glucosidase. Generally speaking, the lower the energy required for the combination of the ligand receptor, the easier it is to dock. When the binding energy is less than 0 kcal/mol, it is considered to be able to bind spontaneously [34]. Marilisa et al. predicted the high affinity of chromogenic acids to α-glucosidase by molecular docking, obtained the negative binding energy, and pointed out that the predicted enzyme-substrate binding was spontaneous and the affinity for α-glucosidase was higher than that of acarbose [35]. In this study, we predicted the inhibition of α-glucosidase by 6-shogaol through molecular docking technology. The 6-Shogaol and α-glucosidase are docked through conventional hydrogen bond, carbon hydrogen bond, pi-sigma, pi-pi stacked, and other forces. Additionally, the docking results of acarbose and α-glucosidase showed that the van der Waals force, conventional hydrogen bond, pi-donor hydrogen bond, and pi-alkyl were formed between them. The binding energy of 6-shogaol and acarbose with α-glucosidase protein are −6.24 and −7.20 kcal/mol, which means it is considered that they can spontaneously bind with α-glucosidase and have high binding affinity. This result suggests that 6-shogaol has potential hypoglycemic activity. Compared with 6-shogaol, the lower binding energy of acarbose and α-glucosidase may be related to the formation of more hydrogen bonds and van der Waals force. Furthermore, the enzyme activity experiment in vitro was carried out to verify the results of molecular docking to confirm the inhibitory effect of 6-shogaol on α-glucosidase. A series of concentration gradients were selected for the enzyme activity test to show the inhibition of 6-shogaol more clearly on α-glucosidase and its dose-dependent effect. Our previous study showed that the 6-shogaol content in ginger reached 531 µg/g [36], and clinical study showed that the toxicity of 2 g ginger per day on the human body was negligible. Therefore, we suggest 500 µg/g as the maximum concentration. The results showed that 6-shogaol had a strong inhibitory effect on α-glucosidase in a dosedependent manner [37]. Differentiated Caco-2 cells are mature human intestinal mucosal models, which can differentiate into brush border membranes and basolateral membranes and express a variety of enzymes and nutrient transporters in vitro [38]. TEER value indicates the resistance of the cell monolayer, which can be used to verify the integrity of the barrier. When TEER value ≥ 500 Ω, it indicates that the cells have formed a dense monolayer structure [21]. In our study, the TEER value of Caco-2 cells on the 21st day of differentiation in transwell culture plate was more than 500 Ω, indicating that a monolayer structure has been formed, which is similar to the previous results [28]. Under our test conditions, the CCK8 test showed that 5, 10, and 20 µg/mL of 6-shogaol had no toxic effect on cells, so the maximum concentration used in the test was 20 µg/mL. Furthermore, we measured the glucose content in the cell culture medium of each group and found that the glucose content increased after 6-shogaol intervention, indicating that 6-shogaol inhibited the glucose uptake of cells. Similarly, previous studies have shown that anthocyanins, coumaric acid, and quercetin can inhibit the glucose uptake of Caco-2 cells [39]. Caco-2 cells after differentiation are similar to intestinal absorption cells in morphology, with microvilli and tightly connected cell monolayers and related transporters [40]. The transport of glucose through intestinal brush border membrane plays a key role in metabolic regulation. Glucose is mainly transported by sodium-dependent glucose transporter (SGLT1) and facilitated-transporter glucose transporter (GLUT2) [41]. When the glucose concentration in the intestine cavity is low, its transport mainly depends on the active transport of SGLT1 to transfer the glucose in the intestine cavity to the epithelial cells. However, when the concentration is high, glucose is mainly transported into the blood by GLUT2 [28]. Studies have shown that flavonoids can reduce the glucose uptake of Caco-2 cells by reducing the expression of glucose transporter gene and inhibiting the binding sites of SGLT1 and GLUT2 [42]. In this study, 6-shogaol intervention also reduced the relative expression of Caco-2 cells transporters SGLT1 and GLUT2, especially at high concentrations. This suggests that 6-shogaol may reduce the glucose uptake and utilization of Caco-2 cells by inhibiting the expression of related transporters. Occludin, Claudin, Foods 2023, 12, 503 9 of 11 and ZO-1 play a crucial role in establishing cell–cell contact and maintaining cell bypass permeability. In the study on the enhancement of Caco-2 cells barrier by flavonoids in black ginger, it was shown that the increase in the expression of Occludin and Claudin-1 proteins in cells enhanced the integrity of the barrier, increased the trans epithelial resistance, and decreased the permeability of glucan [43]. In a study based on the Caco-2/HT29 coculture model, attempting to evaluate the effect of indole-3-propionic acid on intestinal barrier function, it was found that the increase in trans epithelial resistance was consistent with the increase in tight junction proteins (Claudin-1, Occludin, and ZO-1) [44]. Similarly, in our study, the relative expression of the tight junction proteins Occludin, Claudin, and ZO-1 was up-regulated in the 6-shogaol group. It shows that 6-shogaol has a certain protective effect on intestinal barrier function. 5. Conclusions We preliminarily predicted the inhibition of α-glucosidase by 6-shogaol using molecular docking technology and verified it based on the enzyme activity experiment, proving that 6-shogaol can inhibit the activity of α-glucosidase. Furthermore, based on the differentiated Caco-2 cell model, the effects of 6-shogaol on glucose absorption and transport and intestinal barrier protection were investigated. The results showed that 6-shogaol could reduce the glucose uptake of cells, inhibit the glucose transport by down regulating the expression of glucose transporters (SGLT1 and GLUT2), and enhance the intestinal barrier function by up regulating the expression of tight junction proteins (Claudin-1, Occludin, and ZO-1). This study provides a new way for 6-shogaol to treat T2DM and provides theoretical support for the development and utilization of functional products of dried ginger. Author Contributions: Conceptualization and resources, Y.S. and S.W.; methodology, data curation, and writing—original draft preparation, W.J.; supervision and project administration, X.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Hebei Province Key Research and Development Plan Project [grant number 21327314D], Industrial Innovation Team Program of Vegetables of Modern Agricultural Industry Technology System of Hebei Province [grant number HBCT2018030208], and Double FirstClass Food Processing Discipline Group [grant number 1090063-2]. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. References 1. 2. 3. 4. 5. 6. 7. 8. Osae, R.; Apaliya, M.T.; Kwaw, E.; Chisepo, M.T.R.; Antiri, E.A.; Yarley, O.P.N.; Alolga, R.N. Antioxidant activities, antiinflammatory, cytotoxic effects, quality attributes, and bioactive compounds of ghanaian ginger under varied dehydration conditions. J. Food Process. Preserv. 2021, 45, e15994. [CrossRef] Tinello, F.; Zannoni, S.; Lante, A. Antioxidant properties of soybean oil supplemented with ginger and turmeric powders. Appl. Sci. 2020, 10, 8438. [CrossRef] Mustafa, I.; Chin, N.L.; Fakurazi, S.; Palanisamy, A. Comparison of phytochemicals, antioxidant and anti-inflammatory properties of sun-, oven- and freeze-dried ginger extracts. Foods 2019, 8, 456. [CrossRef] Lee, J.S.; Kim, B.; Kim, J.H.; Jeong, M.; Lim, S.; Byun, S. Effect of differential thermal drying conditions on the immunomodulatory function of ginger. J. Microbiol. Biotechnol. 2019, 29, 1053–1060. [CrossRef] [PubMed] Chang, W.P.; Peng, Y.X. Does the oral administration of ginger reduce chemotherapy-induced nausea and vomiting? A metaanalysis of 10 randomized controlled trials. Cancer Nurs. 2019, 42, E14–E23. [CrossRef] Kumar, A.; Sundaram, K.; Teng, Y.; Mu, J.; Sriwastva, M.K.; Zhang, L.; Hood, J.L.; Yan, J.; Zhang, X.; Park, J.W. Ginger nanoparticles mediated induction of foxa2 prevents high-fat diet-induced insulin resistance. Theranostics 2022, 12, 1388. [CrossRef] Abdulrazak, A.; Tanko, Y.; Mohammed, A.; Mohammed, K.A.; Sada, N.M.; Dikko, A.A.U. Effects of clove and fermented ginger on blood glucose, leptin, insulin and insulin receptor levels in high fat dietinduced type 2 diabetic rabbits. Niger. J. Physiol. Sci. Off. Publ. Physiol. Soc. Niger. 2018, 33, 89–93. Hee, S.S.; Feng, F.; Inhae, K. Ginger (zingiber officinale) attenuates obesity and adipose tissue remodeling in high-fat diet-fed c57bl/6 mice. Int. J. Environ. Res. Public Health 2021, 18, 631. Foods 2023, 12, 503 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 10 of 11 Dalsasso, R.R.; Valencia, G.A.; Monteiro, A.R. Impact of drying and extractions processes on the recovery of gingerols and shogaols, the main bioactive compounds of ginger. Food Res. Int. 2022, 154, 111043. [CrossRef] Yi, J.K.; Ryoo, Z.Y.; Ha, J.J.; Oh, D.Y.; Kim, M.O.; Kim, S.H. Beneficial effects of 6-shogaol on hyperglycemia, islet morphology and apoptosis in some tissues of streptozotocin-induced diabetic mice. Diabetol. Metab. Syndr. 2019, 11, 15. [CrossRef] Al Malki, W.H.; Abdel-Raheem, I.T.; Dawoud, M.Z.; Abdou, R.F. 6-shogaol protects against diabetic nephropathy and cardiomyopathy via modulation of oxidative stress/nf-kappa b pathway. Pak. J. Pharm. Sci. 2018, 31, 2109–2117. [PubMed] Hassan, A.M.W.; Talat, A.R.I. Cardioprotective effect of 6-shogaol against hyperglycemia-induced toxicity in h9c2 cardiomyocytes via suppressing of nf-kappa b pathway. Pak. J. Pharm. Sci. 2019, 32, 327–332. Wei, C.-K.; Tsai, Y.-H.; Korinek, M.; Hung, P.-H.; El-Shazly, M.; Cheng, Y.-B.; Wu, Y.-C.; Hsieh, T.-J.; Chang, F.-R. 6-paradol and 6-shogaol, the pungent compounds of ginger, promote glucose utilization in adipocytes and myotubes, and 6-paradol reduces blood glucose in high-fat diet-fed mice. Int. J. Mol. Sci. 2017, 18, 168. [CrossRef] [PubMed] Patricia, M.D.C.; Herminia, V.M.E.; Zyanya, R.C. Annona muricata as possible alternative in the treatment of hyperglycemia: A systematic review. J. Med. Food 2022, 25, 219–229. Bilin, X.; Zhiliang, L.; Ting, Z.; Jianfeng, Z.; Shuzhen, W.; ChiTang, H.; Shiming, L. Bioactives of momordica charantia as potential anti-diabetic/hypoglycemic agents. Molecules 2022, 27, 2175. Ha, T.J.; Park, J.E.; Kang, B.-K.; Kim, H.-S.; Shin, S.-O.; Seo, J.-H.; Oh, E.; Kim, S.; Kwak, D. A-glucosidase inhibitory activity of isoflavones and saponins from soybean (glycine max l.) and comparisons of their constituents during heat treatments. J. Korean Soc. Food Sci. Nutr. 2019, 48, 953–960. [CrossRef] Klumperman, J.; Fransen, J.; Boekestijn, T.C.; Oude Elferink, R.; Matter, K.; Hauri, H.-P.; Tager, J.M.; Ginsel, L. Biosynthesis and transport of lysosomal alpha-glucosidase in the human colon carcinoma cell line caco-2: Secretion from the apical surface. J. Cell Sci. 1991, 100, 339–347. [CrossRef] Qian, K.; Tan, T.; Ouyang, H.; Yang, S.-L.; Zhu, W.-F.; Liu, R.-H.; Wen, Q.; Feng, Y.-L. Structural characterization of a homopolysaccharide with hypoglycemic activity from the roots of pueraria lobata. Food Funct. 2020, 11, 7104–7114. [CrossRef] Sharma, A.X.; Quittner-Strom, E.B.; Lee, Y.; Johnson, J.A.; Martin, S.A.; Yu, X.; Li, J.; Lu, J.; Cai, Z.; Chen, S. Glucagon receptor antagonism improves glucose metabolism and cardiac function by promoting amp-mediated protein kinase in diabetic mice. Cell Rep. 2018, 22, 1760–1773. [CrossRef] Liu, Y.; Yang, L.; Zhang, Y.; Liu, X.; Wu, Z.; Gilbert, R.G.; Deng, B.; Wang, K. Dendrobium officinale polysaccharide ameliorates diabetic hepatic glucose metabolism via glucagon-mediated signaling pathways and modifying liver-glycogen structure. J. Ethnopharmacol. 2020, 248, 112308. [CrossRef] Sharma, S.; Tripathi, P.; Sharma, J.; Dixit, A. Flavonoids modulate tight junction barrier functions in hyperglycemic human intestinal caco-2 cells. Nutrition 2020, 78, 110792. [CrossRef] [PubMed] Papoutsis, K.; Zhang, J.; Bowyer, M.C.; Brunton, N.; Gibney, E.R.; Lyng, J. Fruit, vegetables, and mushrooms for the preparation of extracts with alpha-amylase and alpha-glucosidase inhibition properties: A review. Food Chem 2021, 338, 128119. [CrossRef] [PubMed] Iftikhar, M.; Iftikhar, A.; Zhang, H.; Gong, L.; Wang, J. Transport, metabolism and remedial potential of functional food extracts (ffes) in caco-2 cells monolayer: A review. Food Res. Int. 2020, 136, 109240. [CrossRef] [PubMed] Li, J.W.; Luo, J.Y.; Chai, Y.Y.; Guo, Y.; Yang, T.Z.; Bao, Y.H. Hypoglycemic effect of taraxacum officinale root extract and its synergism with radix astragali extract. Food Sci. Nutr. 2021, 9, 2075–2085. [CrossRef] Jiao, W.Y.; Mi, S.; Sang, Y.X.; Jin, Q.X.; Chitrakar, B.; Wang, X.H.; Wang, S. Integrated network pharmacology and cellular assay for the investigation of an anti-obesity effect of 6-shogaol. Food Chem. 2022, 374, 131755. [CrossRef] He, M.; Zhai, Y.H.; Zhang, Y.Q.; Xu, S.; Yu, S.X.; Wei, Y.X.; Xiao, H.F.; Song, Y.D. Inhibition of alpha-glucosidase by trilobatin and its mechanism: Kinetics, interaction mechanism and molecular docking. Food Funct. 2022, 13, 857–866. [CrossRef] Saeedi, P.; Petersohn, I.; Salpea, P.; Malanda, B.; Karuranga, S.; Unwin, N.; Colagiuri, S.; Guariguata, L.; Motala, A.A.; Ogurtsova, K. Global and regional diabetes prevalence estimates for 2019 and projections for 2030 and 2045: Results from the international diabetes federation diabetes atlas. Diabetes Res. Clin. Pract. 2019, 157, 107843. [CrossRef] Xu, X.Q.; Wang, P.Y.; Wang, B.G.; Wang, M.K.; Wang, S.Y.; Liu, Z.H.; Zhang, Y.; Kang, W.Y. Glucose absorption regulation and mechanism of the compounds in lilium lancifolium thunb on caco-2 cells. Food Chem. Toxicol. 2021, 149, 112010. [CrossRef] Feng, Y.Y.; Nan, H.J.; Zhou, H.Y.; Xi, P.H.; Li, B. Mechanism of inhibition of alpha-glucosidase activity by bavachalcone. Food Sci. Technol. 2022, 42, 123421. [CrossRef] Tao, X.; Huang, Y.; Wang, C.; Chen, F.; Yang, L.; Ling, L.; Che, Z.; Chen, X. Recent developments in molecular docking technology applied in food science: A review. Int. J. Food Sci. Technol. 2020, 55, 33–45. [CrossRef] Ben-Shimon, A.; Eisenstein, M. Looking at enzymes from the inside out: The proximity of catalytic residues to the molecular centroid can be used for detection of active sites and enzyme–ligand interfaces. J. Mol. Biol. 2005, 351, 309–326. [CrossRef] [PubMed] Surovi, S.; Manobjyoti, B. Molecular docking: Challenges, advances and its use in drug discovery perspective. Curr. Drug Targets 2019, 20, 501–521. Meiqi, L.; Xi, B.; Xueting, Z.; Hongbing, R.; Shengbao, C.; Xiaosong, H.; Junjie, Y. Exploring the phytochemicals and inhibitory effects against α-glucosidase and dipeptidyl peptidase-iv in chinese pickled chili pepper: Insights into mechanisms by molecular docking analysis. LWT 2022, 162, 113467. Foods 2023, 12, 503 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 11 of 11 Na, W.; WenBo, A.; Nan, Z.; JingChun, F.; Xin, F.; WeiJie, Y. Exploring the active ingredients and mechanism of action of huanglian huazhuo capsule for the treatment of obese type-2 diabetes mellitus based on using network pharmacology and molecular docking. Evid. -Based Complement. Altern. Med. 2022, 2022, 2780647. Alongi, M.; Celayeta, J.M.F.; Vriz, R.; Kinsella, G.K.; Rulikowska, A.; Anese, M. In vitro digestion nullified the differences triggered by roasting in phenolic composition and α-glucosidase inhibitory capacity of coffee. Food Chem. 2020, 342, 128289. [PubMed] Li, T.Y.; Liu, W.H.; Liang, N.; Zhang, Q.; Wang, X.H. Quantitative assessment of gingerols and the antioxidant activity in ginger and its processed ginger. Food Ind. 2016, 37, 180–183. Singletary, K. Ginger: An overview of health benefits. Nutr. Today 2010, 45, 171–183. [CrossRef] Liu, S.Y.; Ai, Z.Y.; Meng, Y.; Chen, Y.Q.; Ni, D.J. Comparative studies on the physicochemical profile and potential hypoglycemic activity of different tea extracts: Effect on sucrase-isomaltase activity and glucose transport in caco-2 cells. Food Res. Int. 2021, 148, 110604. [CrossRef] Mieres-Castro, D.; Theoduloz, C.; Sus, N.; Burgos-Edwards, A.; Schmeda-Hirschmann, G.; Frank, J.; Jimenez-Aspee, F. Iridoids and polyphenols from chilean gaultheria spp. Berries decrease the glucose uptake in caco-2 cells after simulated gastrointestinal digestion. Food Chem. 2022, 369, 130940. [CrossRef] Borchardt, R.T.; Hidalgo, I.J.; Raub, T.J.; Borchardt, R.T. Characterization of the human colon carcinoma cell line (caco-2) as a model system for intestinal epithelial permeability, gastroenterology, 96, 736–749, 1989-the backstory. AAPS J. 2011, 13, 323–327. [CrossRef] Pico, J.; Martinez, M.M. Unraveling the inhibition of intestinal glucose transport by dietary phenolics: A review. Curr. Pharm. Des. 2019, 25, 3418–3433. [CrossRef] [PubMed] Noonong, K.; Pranweerapaiboon, K.; Chaithirayanon, K.; Surayarn, K.; Ditracha, P.; Changklungmoa, N.; Kueakhai, P.; Hiransai, P.; Bunluepuech, K. Antidiabetic potential of lysiphyllum strychnifolium (craib) a. Schmitz compounds in human intestinal epithelial caco-2 cells and molecular docking-based approaches. BMC Complement. Med. Ther. 2022, 22, 235. [CrossRef] [PubMed] Mayangsari, Y.; Okudaira, M.; Mano, C.; Tanaka, Y.; Ueda, O.; Sakuta, T.; Suzuki, Y.; Yamamoto, Y.; Suzuki, T. 5,7dimethoxyflavone enhances barrier function by increasing occludin and reducing claudin-2 in human intestinal caco-2 cells. J. Funct. Foods 2021, 85, 104641. [CrossRef] Li, J.J.; Zhang, L.; Wu, T.; Li, Y.F.; Zhou, X.J.; Ruan, Z. Indole-3-propionic acid improved the intestinal barrier by enhancing epithelial barrier and mucus barrier. J. Agric. Food Chem. 2021, 69, 1487–1495. 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https://openalex.org/W4385893510	https://zenodo.org/record/8211682/files/v.3%2C%20n.8%2C%202023-35-54.pdf	English	null	ON THE MARGINS, THE SPEECH: THE LITERATURE OF PATRÍCIA MELO AS A REPRESENTATION OF THE INVISIBLE IN LESS THAN ONE	Zenodo (CERN European Organization for Nuclear Research)	2,023	cc-by	10,829	Internationally Indexed Scientific Journal ON THE MARGINS, THE SPEECH: THE LITERATURE OF PATRÍCIA MELO AS A REPRESENTATION OF THE INVISIBLE IN LESS THAN ONE DOI: 10.5281/zenodo.8211682 DOI: 10.5281/zenodo.8211682 Alexandra Alves da Silva Graduated in Letters (Portuguese/Literatures) from the State University of Rio de Janeiro – Faculty of Teacher Training (2004). Master's student in Literary Studies (PPLIN/UERJ) is a member of GEFIS – Feminist and Intersectional Studies Groups (GEFIS/UERJ-CNPq). She has been working in basic education since 2001, as a teacher of Portuguese, Literature and Writing in primary and secondary education (private network). Her research is centered on systemic and decolonial violence by women. Lattes: https://lattes.cnpq.br/7045452830200358. ORCID: https://orcid.org/0000-0003-1287-0559. Email: prof.alexandra.ead@gmail.com. Graduated in Letters (Portuguese/Literatures) from the State University of Rio de Janeiro – Faculty of Teacher Training (2004). Master's student in Literary Studies (PPLIN/UERJ) is a member of GEFIS – Feminist and Intersectional Studies Groups (GEFIS/UERJ-CNPq). She has been working in basic education since 2001, as a teacher of Portuguese, Literature and Writing in primary and secondary education (private network). Her research is centered on systemic and decolonial violence by women. Lattes: https://lattes.cnpq.br/7045452830200358. ORCID: https://orcid.org/0000-0003-1287-0559. Email: prof.alexandra.ead@gmail.com. ABSTRACT The present work aims, from the reading of the novel Menos que um (2022), by Patrícia Melo, to promote some reflections about the importance of literature as an object of representation of reality and how issues of gender, class and race have been recurrent . in the author's works. In addition to the novel's content being aligned with decolonial and intersectional thinking, the author's incursion into verisimilar work is also significant, as she appropriates a literature whose speech space denounces the permanence of colonial practices as a strong political project. Under the theoretical contribution of authors such as Teresa de Lauretis (1994), Françoise Vergès (2022), Mbembe (2021), I emphasize the idea that the maintenance of this social ill is also linked to necropolitics. Starting from events that feedback Brazilian illnesses perpetuated by colonial behavior to recent Brazilian issues, the characters involved in the novel, mainly Glenda and Jessica, intertwine, creating a kaleidoscope composed of the excluded, whose future seems to have no prospect of change for some. p p g eywords: Intersectionality. Decolonialism. Female authority. Feminist literary criticism The Animal The Animal The Animal Yesterday I saw an animal In the filth of the patio Picking up food among the debris. When I found something, I didn't examine it or smell it: I swallowed it voraciously. The animal wasn't a dog, It wasn't a cat, It wasn't a mouse. The animal, my God , was a man. Manuel Bandeira , Belo Belo. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 35 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal The idea of the animal-man, so often explored in Brazilian literature, can be experienced again through the comparison between two objects from the fictional field. Although the poem O Bicho , by Manuel Bandeira may have been written in the 1940s and represents a critique of the social order by the poet in that period, it seems to be directly linked to the present, since the country remains immersed in poverty and hunger . In the poem, the imagery construction provokes revulsion by the characterization of a hostile, filthy, chaotic environment, in addition to causing astonishment with the breach of expectations of the readers in the tenth verse, since after the poet mentions the words animal , dog , cat and mouse , it is through a vocative that surprises us with the revelation that that man-animal, picking up food among the debris, was actually a human being. In addition to the zoomorphization of Naturalism, whose aspect is more centered on the individual's biological instincts, the idea of the animal-man brought here animalizes the human being due to degrading circumstances and absences, ranging from beans to the ceiling. In this bias, bringing the image of a person in a situation of extreme vulnerability and with a tone of denunciation, the novel Menos que um (2022), thirteenth book by Patrícia Melo, exposes the Brazilian tragedy related to people in a situation of social vulnerability. This is an ignored Brazil, since it is estimated that today there are more than 206 thousand people in this condition. With that, from plastic to plasticity, the cover and the map of hunger hurt, since the book's art itself (fig. The Animal 01) presents a lot of meanings inherent to the events of Brazilian reality, as well as already inserting readers into the indigences of the characters; imagetically, a young man-animal is represented, similar to the one in Bandeira's poem, also already punished for the sufferings to which he is subjected. He is broken, he lacks fingers, part of his face, body and the right side of his brain – responsible for musical and visual skills –, that is, he is more concerned with solving immediate physical issues: hunger, beans, limiting it to (in)existence. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 36 Figure 01: Book cover Just like in the poem O Bicho , by Manuel Bandeira, he seems to be looking among the remains of plastic for something that can satisfy that hunger, something that someone would no longer need, which is why he discarded it. This man (animalized by circumstances) is less than one, he is not whole, he has no identity, he has lost his dignity, his clothes. They are the fragments of a human being who exists, but seems invisible, because he is totally ignored by society. However, something even more remarkable is knowing that this cover was inspired by a photo (fig. 02) captured by photographer Danilo Verpa, who works for the newspaper Folha de São Paulo. In this record, taken during the pandemic period, we see not only the young Reginaldo Gonçalves da Silva, 41, but also another man, with his back turned, picking up food among the debris in the filth of a courtyard in the richest city in Brazil. Both are surrounded by dumpsters, garbage bags, dirt from discarded materials, all close to an overpass. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 37 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 Figure 02: Reginaldo, 41 years old, separates food that would go to waste in São Paulo. Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal Figure 02: Reginaldo, 41 years old, separates food that would go to waste in São Paulo. 3 https://www12.senado.leg.br/noticias/infomaterias/2022/10/retorno-do-brasil-ao-mapa-da-fome-da-onu- preocupa-senadores-e-estudiosos . Accessed on 03/03/2023. The Animal According to Eagleton: Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal the City Hall, released in January 4. Bringing the representation of such abject reality to the artistic field, considering that literature has the ability to boost reflection in order to sensitize and sensitize readers to the works, the analyzed narrative – literary and fictional – presents a plot whose complexity is faced with the difficulty of distinguishing fact from fiction. According to Eagleton: The distinction between "fact" and "fiction", therefore, does not seem to us to be very useful, and one of the reasons for this is that the distinction itself is often questionable. It has been said, for example, that the opposition we have established between "historical" and "artistic" truth (...) In late sixteenth- and early seventeenth-century English, the word "novel" was used, which it seems, both for real and fictitious events, and even newspaper news could hardly be considered factual (EAGLETON, 2006, p. 2). In this bias, such difficulty in distinguishing between fact and fiction pervades the actions and scenes narrated in the book Menos que um , because the stories intersect with the factual, revealing scenarios of chaos and deprivation. Because it is a delicate topic, it has rarely been approached so closely to reality, but a good novel is capable of transporting readers to incomprehensible experiences, even though they have never been through the situation, or been to those places. Patrícia Melo narrates a kaleidoscope of different daily struggles, inserting us in a reality as indigestible as it is ignored: a tragedy that jumps out at our eyes, because when we walk through the streets we see hunger, helplessness, the dehumanization of the people who inhabit this public space in a terrible shortage. The author talks about the physical and emotional deprivation of those rejected and the facets of the conditions that lead these individuals to become a street person, in addition to representing them with exact dimension, bringing their pain, their absence, their hunger and experiences with a fierce realism. Everyone involved in the narrative seems to compose a social gear of marginalized, rejected people and, with this assertive look, Patrícia Melo constructs the subjectivity of each character without caricatures or romanticization, consequently tracing back an imaginary timeline from slavery to the present day. , because, as already mentioned, most people in this condition are black. Current Scientific Journal – (August) v.3, n.8, 2023 39 https://www.currentscientific.com/p/current.html 4https://observatorio3setor.org.br/noticias/numero-de-pessoas-em-situacao-de-rua-cresceu-no-brasil- em2022/#:~:text=Hoje%2C%20no%20pa%C3% ADs%2C%20more%20de,in%20street%20of%20capital%20paulista . Accessed on 03/02/2023. The Animal Source: Folhapress/photography by Danilo Verpa Source: Folhapress/photography by Danilo Verpa This photographic record is confirmation of a desolate scenario of systemic violence that endorses the need for changes, since the great protagonist of this very cruel reality – including in the novel – is social inequality, which coincidentally (or not) increased during the period of Covid-19 isolation. In addition, in some moments of the novel, several characters reflect on the fact that people confuse them with garbage, as unclean, being discarded socially. In the literary field, the pains of hunger and misery intertwine in a scenario of chaos, bringing a human mosaic, whose subjectivities of the characters oscillate between (in)existence and suffering. Outside the fictional scope, according to Agência Senado, in 2022, the Second National Survey on Food Insecurity in the Context of the Covid-19 Pandemic in Brazil pointed out that 33.1 million people are not guaranteed what to eat — which represents 14 millions of new Brazilians facing hunger. According to the study, more than half (58.7%) of the Brazilian population lives with food insecurity to some degree: mild, moderate or severe 3. In the meantime, Menos que um brings together a collection of tragedies, composed of recent events and retold as remnants of a country that has not been able to resolve the issues of slavery until today, since the overwhelming majority of people living on the streets are black and the Racialized bodies are also socio-historical, as they carry the remnants of slavery. According to a survey carried out by the UFMG Brazilian Observatory of Public Policies with the Homeless Population in 2022, until May alone, 5,039 thousand people went to live on the streets of the São Paulo capital. Thus, the homeless population in the city reached 42,240 people, a number greater than the almost 32 thousand, indicated by the last census of 3 https://www12.senado.leg.br/noticias/infomaterias/2022/10/retorno-do-brasil-ao-mapa-da-fome-da-onu- preocupa-senadores-e-estudiosos . Accessed on 03/03/2023. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 38 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 the City Hall, released in January 4. Bringing the representation of such abject reality to the artistic field, considering that literature has the ability to boost reflection in order to sensitize and sensitize readers to the works, the analyzed narrative – literary and fictional – presents a plot whose complexity is faced with the difficulty of distinguishing fact from fiction. The Animal (...) Zeca, the owner of the bakery exactly at the moment when Marreco (a militiaman), with the police that Douglas already knew, raised a toast. And when asked the reason for the celebration, Marreco replied: – Here we only celebrate two things: the boss's birthday and the death of a bandit. (MELO, 2022, p.160) They killed people like João Henrique, Zélia's son, black and poor kids from the periphery, who they called bandidinhos. Marginal. Slag. and they charged for it. (...) Zeca, the owner of the bakery exactly at the moment when Marreco (a militiaman), with the police that Douglas already knew, raised a toast. And when asked the reason for the celebration, Marreco replied: p – Here we only celebrate two things: the boss's birthday and the death of a bandit. (MELO, 2022, p.160) These figures are dehumanized, one of them is Marreco, who appeared driving a black zero Honda, a good that did not match his salary pattern: “I'm a turner, I do cartwheels, I have my own business” (...) He attacked communism. Attack the Supreme. It attacked the vaccine. He attacked the quota system. Attacked the press. And he passionately defended the release of weapons: it is the effective policy to put an end to banditry, he believed. (...) “Start adding up. Put together the military police plus civil police plus members of the Armed Forces plus the entire family of each of these men. We are an army of nearly twenty million people. And we've already made a president. Do you understand what that means? (MELO, 2022, p.198) “I'm a turner, I do cartwheels, I have my own business” (...) He attacked communism. Attack the Supreme. It attacked the vaccine. He attacked the quota system. Attacked the press. And he passionately defended the release of weapons: it is the effective policy to put an end to banditry, he believed. (...) “Start adding up. Put together the military police plus civil police plus members of the Armed Forces plus the entire family of each of these men. We are an army of nearly twenty million people. And we've already made a president. Do you understand what that means? The Animal According to the Brazilian Observatory of Public Policies with the Homeless Population, 68% of homeless people are black, 87% men, most aged between 18 and 59 years old and without complete primary education. In the novel, the abandoned, the despised, the encumbrances are brought: – cracudos, unemployed, drunks, foreigners, scavengers, girls, women, street vendors, orphans... people who, by force of circumstances, had to occupy the 4https://observatorio3setor.org.br/noticias/numero-de-pessoas-em-situacao-de-rua-cresceu-no-brasil- em2022/#:~:text=Hoje%2C%20no%20pa%C3% 39 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 streets in search of survival. With this, the protagonist is social inequality itself, since all individuals are important for the construction of the human mosaic, but they seem to be less Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal streets in search of survival. With this, the protagonist is social inequality itself, since all individuals are important for the construction of the human mosaic, but they seem to be less than one, because they are erased, disappearing, losing their identities, their dignity: Your mind frees itself from small bills, the eighty cents set aside for this, two twenty for that, you get rid of the economy of small things, what if I only eat the sausage, without the bread? How much is left for tomorrow? What if I cut out the coffee? And then, the most important thing: you stop being the guardian of little things, of two shorts, four underpants, five socks, a pair of black pants, soap, toothpaste, a comb, shaving equipment, photos of your wedding, photos of ice cream shop, photos that I didn't even look at anymore. (MELO, 2022, p.322) In this passage, we managed to get a brief idea of how these people fade away, lose their dignity and identity as they leave behind their belongings and memories. In this passage, we managed to get a brief idea of how these people fade away, lose their dignity and identity as they leave behind their belongings and memories. On the other hand, I highlight something that is quite marked in the novel: the lack of humanization of the militiamen; these torture, strangle, cut penises and kill without any sensitivity or empathy towards the next: They killed people like João Henrique, Zélia's son, black and poor kids from the periphery, who they called bandidinhos. Marginal. Slag. and they charged for it. The Animal (MELO, 2022, p.198) Such characters seem to be at the service of the powerful and act by controlling places and bodies through discipline, atrocity and murder, in addition to reproducing models of genocide and massacres even with colonial characteristics, whose targets are mainly people from socially disadvantaged black skins. To support this survey, according to Achille Mbembe: Policing and discipline techniques, in addition to the choice between obedience and simulation that characterized the colonial and post-colonial Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 40 Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal potentate, are gradually being replaced by a more tragic alternative, given its extremism. Technologies of destruction have become more tactile, more anatomical and sensory, within a context in which the choice is made between life and death. If power still depends on strict control over bodies (or their concentration in fields), the new technologies of destruction are less concerned with inscribing bodies in disciplinary apparatuses than with inscribing them, at the opportune moment, in the order of maximum economy, now represented by the “massacre”. In turn, the generalization of insecurity deepened the social distinction between those who have weapons and those who do not (“weapons distribution law”). Increasingly, war is not taking place between armies of two sovereign states. It is fought by armed groups that act behind the mask of the State against armed groups that do not have a State, but that control quite different territories; both sides have as their main targets civilian populations that are unarmed or organized as militias. In cases where armed dissidents have not fully seized state power, they produce territorial partitions, achieving control over entire regions on the feudal model. (MBEMBE, 2018, pp.: 59 and 60) Thus, these militiamen are a strong indication that they are at the service of someone who wants to exterminate minority social groups in order to clean the city from the presence of the excluded (according to the concept of necropolitics, defined by Achille Mbembe, the State decides who deserves to live and who deserves to die), that's why he makes victims daily. In this bias, the control of these more susceptible bodies – to biopower, sovereignty, the state of exception and the policy of death – occurs without punishment, since it is legitimized by the State itself. The Animal (MELO, 2022, p.93) In addition, I watched the news and noticed the exponential increase in people who lost li In addition, I watched the news and noticed the exponential increase in people who lost their lives: It was at night, at home, in front of the TV, that he watched the count of the dead on the news, that he understood the reality around him. The progress was astounding. From five thousand dead to twenty thousand. From twenty thousand to eighty thousand. From eighty thousand to one hundred thousand! Hundred thousand! Two hundred thousand! And it kept growing. Everywhere. Impressive data. Deaths of the day, moving average. Number of people infected. Propagation rates. Evolution charts. The numbers made headlines daily. All of this was destroying his faith, leaving him with a feeling of impotence and dread, which suffocated him on sleepless nights. (idem) In the pandemic and isolation period, hundreds of people also lost their jobs, their homes, their families; in April 2023, there are already more than 700 thousand dead by Covid- 19 in Brazil. With that, the whole set of actions caused appalling misery and, for those who no longer had an average social condition, the street was left: a public space through which everyone had to pass. Unfortunately, our eyes seem to ignore the scenario, either because of anguish, fear, or the impossibility of doing something to help. In the fictional field, but in a hyper-realistic way, Patrícia Melo, who presents herself as a great writer of contemporary Brazilian literature – something that is confirmed, mainly in her last two published works: Mulheres stackadas (2019) and Menos que um ( 2022 ) . ) –, presents equally cutting, laconic, corrosive narratives, but mainly in these two novels, violence, which is already a recurring theme in his writing, is presented in a systemic and constant way, as if the impetuosity of the processes that make up this violence and tortures reached death as a banal and inevitable spectacle. However, far beyond these already remarkable literary characteristics, the Brazilian author has brought important decolonial themes, providing greater engagement with issues of gender, class and race, assuming and confirming a prominent position and emerging as an important voice of female authorship. The Animal Hypothetically, we relate places and facts narrated in the novel to the city and streets of São Paulo, as we can see the social hygiene actions carried out by Mayor Dória 5, the presence of the priest (referring to the figure of Father Júlio Lancelotti who, precisely during the pandemic 6, carried out and still carries out humanizing work). In addition to these factors, it brings the cartography of the largest cracolândia in Brazil – also located in São Paulo – and the occupations of uninhabited places, whose female leadership – mainly led by single mothers in reality – was metaphorized in the 7figure of the character Tula. We also assume that the chronology of the facts narrated takes place in the pandemic scenario, since the character Douglas, the gravedigger, is appalled at the number of dead he had to bury: The bodies are no longer placed on the funeral table, inside the chapel, to say goodbye to family and friends. They came packed in bags, inside sealed Current Scientific Journal – (August) v.3, n.8, 2023 41 https://www.currentscientific.com/p/current.html 5 https://portal-justificando.jusbrasil.com.br/noticias/420362999/higienismo-de-doria-ea-populacao-de-rua . Accessed on 9/2/2022. 6 https://g1.globo.com/profissao-reporter/noticia/2021/04/21/padre-julio-lancelloti-mostra-trabalho-na-pandemia- dizem-que-sou-um-padre-maloqueiro.ghtml . Accessed on 9/2/2022 7 https://oglobo.globo.com/brasil/noticia/2022/07/aqui-eu-renasci-nas-ocupacoes-de-sao-paulo-mulheres-e-maes- solo-sao-maioria.ghtml . Accessed on 9/2/2022. 41 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 coffins, which were not transported in coffins, but by tractors, in groups of eight, to be buried in mass graves. The queues for burials did not allow the gravediggers to drink even a glass of water in peace during the workday. (MELO, 2022, p.93) Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal coffins, which were not transported in coffins, but by tractors, in groups of eight, to be buried in mass graves. The queues for burials did not allow the gravediggers to drink even a glass of water in peace during the workday. (MELO, 2022, p.93) coffins, which were not transported in coffins, but by tractors, in groups of eight, to be buried in mass graves. The queues for burials did not allow the gravediggers to drink even a glass of water in peace during the workday. The Animal Most of the actions take place on the street: a space, sometimes, personified, because at the same time that it excludes – all those who were expelled from home, expatriates, denied their existence –, it shelters people that society does not want; it is also a space that brings together the entire city, from the richest to the poorest. So there was everything on the street. Model, dondoca, crazy person, bandit, housewife. The gutter welcomes everyone like a generous mother (p.48). In addition, on the street, there were also those who transited, ignoring Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 42 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 the urban tragedy, since they were only concerned with arriving, leaving the office for lunch Current Scientific Journal - ISSN 2764-1759 the urban tragedy, since they were only concerned with arriving, leaving the office for lunch and returning to their homes at the end of the day. the urban tragedy, since they were only concerned with arriving, leaving the office for lunch and returning to their homes at the end of the day. It is interesting to point out that the author allows not only the representation of these characters, but also assigns them the narration in the first person, giving greater reliability to the actions. It is also significant to realize that the book is divided into three parts of seventeen chapters each, that is, the number corresponding to a particular presidential candidate in 2018 who, due to necropolitical actions and the extermination of minorities, aggravated the scenario of hunger and of misery in the country. Thus, the legitimization of violent behavior to defend private property in the face of this intensification of inequalities is apparent when we read: “Where did you hide my gun?” He owned a hardware store. Shortly before, kids had entered the garage of his luxurious house, in the neighboring neighborhood, and stolen an old Browning (gun) from his car. (...) “it really seems that now there was a great tolerance by the State for types like that, who frequented shooting ranges and defended an eye for an eye, a tooth for a tooth”. (MELO, 2022, p.53) This possible analogy is made so that it is clear who intensified such tragedies and how our vote may be able to at least. minimize the suffering of the poorest. The Animal In addition, in some speeches reproduced by the militiamen, we perceive a similar association with the aforementioned political figure: Most of the recordings showed semi-drunk men talking about canceled CPFs, denialist theories, rifle numbers, improvised graves, beatings, capsules collected before the forensics arrived, missed payments and military uprisings as if they were employees of a factory having a rough time and obscene at fraternization parties. (...) “They feel unattainable. Their crimes, when reported, are left to rot on special sticks. (ditto, p.293) With this, we realized that the militiamen knew that they would get out of any situation with impunity, since the State legitimized this violent and corrupt behavior. A human mosaic: non-persons? It would be impossible to address all the characters in this work, but some stand out, such as Chilves: a black man, coming from the dump, lost a brother suffocated in the avalanche of garbage. He was Jessica's partner, with whom he had a son and, after being arrested, he woke up to political awareness: he died dreaming. Current Scientific Journal – (August) v.3, n.8, 2023 43 Seno Chacoy: Venezuelan, felt superior to homeless people. When he was employed, he would throw water on those individuals, wet the cardboard that served as their bed, calling Seno Chacoy: Venezuelan, felt superior to homeless people. When he was employed, he would throw water on those individuals, wet the cardboard that served as their bed, calling Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 43 Current Scientific Journal - ISSN 2764-1759 them riffraff. He was clean, shaved and dressed with dignity. With the death of his wife, the stepson – a drug addict, who stole household items to bet on games of chance – returned to his mother's house and changed the locks so that his stepfather would not enter. After going to a shelter and complaining about the public services provided, he was unable to stay in the accommodation and ended up becoming a homeless person. Post: professional pimp, abuser of Glenda. In the end, our desire for revenge comes true: he was beaten so badly that he seems to have actually died. Rita: she was not homeless, had more space to speak, was a journalist and a friend of Glenda. Showed empathy towards Jessica when Chilves seemed interested in her. “– Think with me, Humble Teacher of Truth. I never had to sleep on the street. No one snatched a child from my arms. Be fucking nice to your girlfriend." (p. 312). Rita's attitude indicates the possibility of us interpreting it as sisterhood towards Jessica. Douglas: gravedigger, found it strange that a lady was living in the cemetery, but discovered that her son was buried there and had been killed by militiamen who wanted to “sanitize” the region. Adherent to the religion of good, he buried countless bodies, which represents the moment of the sanitary tragedy in Brazil. He also witnessed the collective mourning of people who, sometimes, could not even afford a coffin. A human mosaic: non-persons? Representing people in situations of extreme vulnerability would already be very impactful from the point of view of violence experienced daily by these marginalized people. However, considering the analysis focus of the project I am part of under the guidance of Professor Maximiliano Torres: What about feminisms, what are they intended for? , I will raise, in a little more detail, questions pertaining to two characters in particular, they are Glenda and Jessica. Less than One 's most intersectional and empathetic character To begin studies on this character, who is the most intersectional in the novel, we turn to the definition by Patricia Hill Collins and Sirma Bilge when they state: Intersectionality investigates how intersectional power relations influence social relations in societies marked by diversity, as well as everyday individual experiences. As an analytical tool, intersectionality considers that the categories of race, class, gender, sexuality, nationality, ability, ethnicity and age group – among others – are interrelated and mutually affect each other. Intersectionality is a way of understanding and explaining the complexity of the world, people and human experiences. (COLLINS and BILGE, 2021, pp.: 15-6) Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 44 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 With such information about intersectionality, we bring Glenda, who is representative of a strong transvestite, rejected by her family – something unfortunately commonplace for this Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal With such information about intersectionality, we bring Glenda, who is representative of a strong transvestite, rejected by her family – something unfortunately commonplace for this part of the population. He met Jessica under the following circumstances: Glenda distributed hygiene kits, along with Rita and the staff from the batbanho, a trailer with a water tank where two showers were installed and which drove around the city, hitched to an old pickup truck, offering free showers for people who lived on the street. One day, after Jessica had taken a bath and received new clothes, Glenda pulled her aside and said, look, daughter, you're like that, clean, you're very good. I organize some houses around the square and I need a helper. Now the two of them also cleaned a kitchenette that Glenda said belonged to “a classy businessman”, but who, in reality, was a sickly waiter. (...) “I've lived on the street, Glenda had told him, “I know very well what it's like to be let go, we get used to the bagaceira, the lice, the bodum, but to serve my fine clientele you have to smell good, clean poor style, did you?” (MELO, 2022, p.41) In the literary representation of the novel, we perceive the need to deconstruct prejudice in relation to this community, since Patrícia Melo brings Glenda as the most human, most empathetic character in the entire plot. When she was still in a situation of lesser poverty, that is, when she at least had a place to live in the boarding house, she was the one who offered – within her financial possibilities – food and hygiene kits for women living on the streets. Unfortunately, in a sequence of routine actions in relation to this part of the LGBTQIAP+ community , Glenda ended up suffering a series of physical and emotional violence. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html (4) Paradoxically, therefore, the construction of gender is also done through its deconstruction, that is, in any discourse, feminist or not, that sees gender as just a false ideological representation. Gender, like the real, is not only the effect of representation, but also its excess, that which remains outside discourse as a potential trauma that, if/when not contained, can disrupt or destabilize any representation. (LAURETIS, 1994, p.209) (1) Gender is (a) representation – which does not mean that it does not have concrete or real implications, both social and subjective, in people's material lives. Quite the opposite. (2) The representation of genre is its construction – and in a more common sense it can be said that all Western art and high culture is a record of the history of this construction. (3) The construction of the genre is taking place today at the same pace as in the past, such as the Victorian era, for example. And it continues to occur not only where it is expected to happen – in the media, in public and private schools, in the courts, in the nuclear, extended or single-parent family – in short, what Louis Althusser called "the ideological apparatuses of the State". The construction of gender is also (1) Gender is (a) representation – which does not mean that it does not have concrete or real implications, both social and subjective, in people's material lives. Quite the opposite. (2) The representation of genre is its construction – and in a more common sense it can be said that all Western art and high culture is a record of the history of this construction. (3) The construction of the genre is taking place today at the same pace as in the past, such as the Victorian era, for example. And it continues to occur not only where it is expected to happen – in the media, in public and private schools, in the courts, in the nuclear, extended or single-parent family – in short, what Louis Althusser called "the ideological apparatuses of the State". The construction of gender is also carried out, although in a less obvious way, in the academy, in the intellectual community, in avant-garde artistic practices, in radical theories, and even, in a very marked way, in feminism. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html However, before the age of twelve, when discovering herself a woman, readers are faced with this moving account of the character: It was as if that dress were a female soul looking for a body and, as she felt it slipping over her head and molding itself to her torso, as if it had been made to measure, she almost heard the perfect fit click between that pink beauty and her true feminine identity. (MELO, 2022, p.139) In this sense, it should be noted that the very identification of this person with the female gender could generate misogyny, lack of acceptance towards her, in addition to revealing open prejudice by a large part of society, as in this scene: “– Travecão! – shouted the mason hanging from a scaffolding at the work Central Park do Brasil” (p.237). In order to be reliable in relation to what the most recent studies on feminism theorize, it is essential for our reflections to highlight this character, since the discussion about gender is a key point for us to raise this hypothesis of violence towards Glenda. Reinforcing this survey in a theoretical way, we resort to what Teresa de Lauretis says: Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 45 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal (1) Gender is (a) representation – which does not mean that it does not have concrete or real implications, both social and subjective, in people's material lives. Quite the opposite. (2) The representation of genre is its construction – and in a more common sense it can be said that all Western art and high culture is a record of the history of this construction. (3) The construction of the genre is taking place today at the same pace as in the past, such as the Victorian era, for example. And it continues to occur not only where it is expected to happen – in the media, in public and private schools, in the courts, in the nuclear, extended or single-parent family – in short, what Louis Althusser called "the ideological apparatuses of the State". The construction of gender is also carried out, although in a less obvious way, in the academy, in the intellectual community, in avant-garde artistic practices, in radical theories, and even, in a very marked way, in feminism. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html Thus, Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 46 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 according to Vergès, we infer that plural feminisms need to encompass and account for all types f i l t ti i t d f t t d b l d Current Scientific Journal - ISSN 2764-1759 according to Vergès, we infer that plural feminisms need to encompass and account for all types of violent actions against women as gender factors, aggravated by class and race. Following real-life statistics, Glenda had already prostituted herself and Poste, her former pimp, constantly exploited her. One night, he entered the simple pension where she lived, violently assaulted her and took her wallet. For this, she received eighty-seven stitches on her face and was told that she would change her name to Lady Frankenstein. When she was in the hospital, she was humiliated by doctors and other employees, since she was admitted to the male ward and, although she had informed her social name, she was only called Weverton Freitas: “She said, indignant, that they did not accept her social name (. ..) They didn't allow her to go to the female ward (Cf. MELO, p.70). After the episode, many began to call her a “monster”. This scene makes us reflect on the violence committed against these bodies. Glenda was in a hospital environment in which she should have been well cared for, respected, welcomed, however, she was humiliated, her social name was not respected, she was rejected. After a mess on the street, Glenda was approached by militiaman Cleber, who said: “– You are under arrest!” (Melo, p.280). In this speech, Cleber refers to her as a man, demarcating the prejudice and exclusion of people with this sexual orientation. Such lack of respect for this group of the LGBTQIAP+ community reveals an even more violent condition in relation to black and poor people, that is, racial, class and identification with the female gender factors intensify the trivialization of practices of violence . Due to these factors, we also infer that there is an extreme trivialization of the murders of people belonging to the aforementioned community. “He's a transvestite,” added Marreco. And there's a reporter (Rita) who's making a hell of a fuss about this shit. You know, these perverts these days are worth more than me, than you, than anybody. “And if it was black, it was worth twice as much,” added Cleber. (ditto, p.295) Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html (4) Paradoxically, therefore, the construction of gender is also done through its deconstruction, that is, in any discourse, feminist or not, that sees gender as just a false ideological representation. Gender, like the real, is not only the effect of representation, but also its excess, that which remains outside discourse as a potential trauma that, if/when not contained, can disrupt or destabilize any representation. (LAURETIS, 1994, p.209) By bringing the theory of this construction, or even the recognition of gender defined by Lauretis to the fictional field of the novel Menos que um , the definition would represent the need for identification in the life of the character Glenda, who abandons the nickname that the family chose, assumes her social name and begins to live with his true identity. But some questions remain unanswered: Why do the aggressors in this community act with such cruelty? Why so much hatred towards the feminine elements present in these bodies? Could it be the fact that they also carry characteristics of women and, therefore, such aversion? Although attempts are made to raise these questions, there is no justification for so much violence. From this perspective, Françoise Vergès, in the work A Feminist Theory of Violence , emphasizes the need to consider broad factors to cover protection for all women, which is why she points out: Trans people and those who refuse to identify with a gender are the most targeted by violence, but, as not all attacks on women – cis, trans, lesbians – and non-binary people, within a year, are reported, and considering that non- white women are underrepresented in these numbers – as the violence committed against them, including murders, do not receive the same attention – it would be necessary to include race, class, gender and sexuality discrimination in these estimates to address systemic violence against women. (VERGÈS, 2021, p.83) From this perspective, Vergès endorses what was problematized by the author, representing the murder of a trans woman as something trivialized and, worse, due to the fact that Glenda was killed by militiamen, we realize that they still placed themselves in a position of moral saviors and good morals, exterminating a person with that sexual orientation. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html “The T population in Brazil has a life expectancy of less than 35 years, which is comparable to the life expectancy of the Middle Ages, when there was no penicillin or basic sanitation.” Still according to the specialist, data from the National Association of Transvestites and Transsexuals (ANTRA) that 90% of the T population is in a situation of prostitution. According to her, the number reports the lack of opportunities in the job market for this group. The scenario, however, is in the process of changing. (CNN Brazil) This reality is as cruel as it is ignored, since the violence of unemployment, exclusion, lack of opportunities, prejudice and abomination towards the T population reflects a behavior that goes beyond misogyny; it is gratuitous hatred, persecution, transphobia and, above all, the lack of empathy for human beings. In this sense, readers are faced with indignation in the face of these murders and, with the rawness and representation of Patrícia Melo's writing, they feel in tune with the characters of the novel. Glenda was missing for some time, but when her body was found, there was a simple burial so that readers could say goodbye symbolically to the character: At that moment, Jessica took a plastic bag full of sequins from her pocket that she had found in one of the drawers in the closets of Glenda's apartment and spread them over the grave. The place was filled with colored dots that, in contrast to the dark earth, sparkled in the sun. (MELO, 2022, p.317) At that moment, Jessica took a plastic bag full of sequins from her pocket that she had found in one of the drawers in the closets of Glenda's apartment and spread them over the grave. The place was filled with colored dots that, in contrast to the dark earth, sparkled in the sun. (MELO, 2022, p.317) It is a poetic scene whose description is emotional, however, in addition to literary experimentation, it is possible to reflect on issues that are not commonly discussed because of prejudice, hypocrisy and false moralism. Unfortunately, in 2023 there is still a lot of neglect and exclusion of this group, but one of the roles of literature is to bring any subject, hitherto virtually ignored, as a form of reflection and analysis. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html Such trivialization occurs mainly in relation to transvestites, whose bodies – which preserve the identification with the feminine – are even more vulnerable; most of these people die young, without the right to a decent burial. Scars and sequins were nearly all that was left of Glenda. (idem, p.314), who died tragically; she was murdered by the militiamen, but the most shocking thing is knowing that she was castrated and, even after being killed, she was humiliated by the assassins. “He's a transvestite,” added Marreco. And there's a reporter (Rita) who's making a hell of a fuss about this shit. You know, these perverts these days are worth more than me, than you, than anybody. “And if it was black, it was worth twice as much,” added Cleber. (ditto, p.295) Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 47 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 Still in this scenario of cruelty towards the trans community, in an interview with CNN8, lawyer Marina Ganzarolli, president of the OAB-SP Sexual and Gender Diversity Commission, stated that: Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal Still in this scenario of cruelty towards the trans community, in an interview with CNN8, lawyer Marina Ganzarolli, president of the OAB-SP Sexual and Gender Diversity Commission, stated that: Still in this scenario of cruelty towards the trans community, in an interview with CNN8, lawyer Marina Ganzarolli, president of the OAB-SP Sexual and Gender Diversity Commission, stated that: The life expectancy of this group is comparable to figures from the Middle Ages. “The T population in Brazil has a life expectancy of less than 35 years, which is comparable to the life expectancy of the Middle Ages, when there was no penicillin or basic sanitation.” Still according to the specialist, data from the National Association of Transvestites and Transsexuals (ANTRA) that 90% of the T population is in a situation of prostitution. According to her, the number reports the lack of opportunities in the job market for this group. The scenario, however, is in the process of changing. (CNN Brazil) The life expectancy of this group is comparable to figures from the Middle Ages. 8 https://www.cnnbrasil.com.br/nacional/expectativa-de-vida-de-trans-no-brasil-se-equipara-com-idade-media- diz-advogada/ . Accessed on 03/04/2023. Jessica: a teenager in (re)construction The truth is that, on the street, there is not a single woman who does not have the same or worse story. A Husband Of My Mother. A stranger you have to call Uncle just because he crawled into Aunt Broken Glass's Heart's bed. And who suddenly wants to put the dick in his mouth. Even today she had nightmares about that boyfriend of her aunt's, that man who smelled like Milk of Roses and who was loved in the neighborhood, a respected taxi driver. (MELO, 2022, p.73) Jessica's situation does not happen in an isolated or rare way, since most aggressions against girls and women occur inside the victims' homes and, in most cases, by a parental member of the victim, or by someone known to the family. . Following this statistic, in the work A feminist theory of violence , Françoise Vergès highlights: Every day, on average, 137 women are murdered in the world by someone close to them. Among these, more than a third are murdered by their spouse or ex-spouse. Around 15 million adolescents (aged fifteen to nineteen) worldwide have been subjected to forced sexual intercourse (penetration or forcibly imposed sexual acts) at some point in their lives. (VERGÈS, 2021, p.83) He got involved with Chilves, another homeless person, but when he was arrested, Jessica became vulnerable, which is why she ended up triggering her addiction to crack and started to prostitute herself to support her addiction – even though she was pregnant with a baby. Even though she was aware that she was offering her own body in exchange for little money, here we are faced with the subjectivity of the teenager's behavior, who kept her thoughts confidential: Guys could fuck your body. For a market price, they could squeeze, kiss, suck, lick and mount. Everything included in the package. But, inside her mind, there was a little sign hung by herself: No strangers allowed. Her thoughts, her ideas, her projects, everything there was like a room she kept all to herself. Braided. (MELO, 2022, p.151) She was enticed by the character Poste, the same as Glenda's aggressor, who ended up managing, as a pimp, her earnings from working as a prostitute. Jessica: a teenager in (re)construction This character is a fifteen-year-old girl who ran away from home after being abused numerous times by her uncle (her aunt's husband) and, in a sequence of tragedies, ended up becoming a homeless person. When Jessica went to visit Glenda, hospitalized because of Poste's Current Scientific Journal – (August) v.3, n.8, 2023 48 https://www.currentscientific.com/p/current.html 8 https://www.cnnbrasil.com.br/nacional/expectativa-de-vida-de-trans-no-brasil-se-equipara-com-idade-media- diz-advogada/ . Accessed on 03/04/2023. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 48 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 aggression, we found out why she had lost the ability to express herself, as the revelation to readers began when a teacher argued with her: Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal aggression, we found out why she had lost the ability to express herself, as the revelation to readers began when a teacher argued with her: aggression, we found out why she had lost the ability to express herself, as the revelation to readers began when a teacher argued with her: aggression, we found out why she had lost the ability to express herself, as the revelation to readers began when a teacher argued with her: “Are you having a problem at home?” But she didn't know how to respond. He hated every word in that story: chu-pa... the... dick... of... your... fake little uncle. Lick lick. He was going to tell Glenda that she too had a Pole in her life. The truth is that, on the street, there is not a single woman who does not have the same or worse story. A Husband Of My Mother. A stranger you have to call Uncle just because he crawled into Aunt Broken Glass's Heart's bed. And who suddenly wants to put the dick in his mouth. Even today she had nightmares about that boyfriend of her aunt's, that man who smelled like Milk of Roses and who was loved in the neighborhood, a respected taxi driver. (MELO, 2022, p.73) “Are you having a problem at home?” But she didn't know how to respond. He hated every word in that story: chu-pa... the... dick... of... your... fake little uncle. Lick lick. He was going to tell Glenda that she too had a Pole in her life. Jessica: a teenager in (re)construction So, with the false idea that he was protecting her from street violence, all Poste had to do was drink or snort and the conversation was already different: my wife this-and-that, he said: “Next time, I’ll cut your throat, threaten .” (Melo, p.152). Being a woman is already a perilous factor in relation to the Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 49 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 objectification of bodies, vulnerability to aggression, harassment and rape. Thinking about all these systemic violence is already very distressing, however, if we add such issues to the susceptibility of women living on the street, everything becomes even more tragic. In the novel, some women found themselves in a degrading situation, because when they were not under the command of an exploitative pimp, violence continued to come from all sides, so they tried to protect themselves in every way: Internationally Indexed Scientific Journal objectification of bodies, vulnerability to aggression, harassment and rape. Thinking about all these systemic violence is already very distressing, however, if we add such issues to the susceptibility of women living on the street, everything becomes even more tragic. In the novel, some women found themselves in a degrading situation, because when they were not under the command of an exploitative pimp, violence continued to come from all sides, so they tried to protect themselves in every way: Many women on the street had no owners. They carried stilettos. They slept on the blade. Some went crazy. Or they dressed like grown men. There were also those who avoided any type of hygiene to cause disgust. They reeked of shit. Still, many were attacked at night when they slept. (ditto, p.153) Here we perceive the degradation of these vulnerable female bodies that need proper hygiene, especially during the menstrual period, but which, in addition to not having such dignity, were kept fetid to avoid rape. This reflection by Jessica gives us a brief idea of how painful the lives of these people must be, especially that of women. In addition, thinking about the cartography of cities, some spaces are even more dangerous, because they are less crowded and lit, or because they are hostile towards the marginalized. Jessica: a teenager in (re)construction (MELO, 2022, p.204) The girls were assaulted, physically and emotionally tortured, as they were kept away from family life and from the possibility of denouncing irregularities: The girls were assaulted, physically and emotionally tortured, as they were kept away from family life and from the possibility of denouncing irregularities: “Demon whore, do you think you can fight against the power of God?”, asked the monitor after finding, under Jessica's T-shirt, a sheet of paper which read: “They beat the inmates. Get me out of here.” Jessica, on that occasion, was in the room where she would receive the weekly video call from Glenda and Chilves, which ended up being suspended as a punishment. For the next few days, completely isolated in the contrition house, defecating and urinating into a hundred-gallon oil can, she thought she would go crazy. Who would die of despair. (ditto, p.208) Many interns were assigned to manufacture sausages with the aim of being sold, further increasing the clinic's earnings. When Jessica managed to escape, she tried to communicate and report the irregularities to a journalist, who reported her, which is why she was forced to return to prison. Only after complaints from Rita, another journalist and friend of Glenda, did Jessica manage to be freed. Upon arriving at Casa do Esplendor Divino, Rita asked “What are those purple spots on your shins?” and Jessica replied: “Broomed. Here everyone gets caught. For any reason." (Melo, p.240). Upon being released from prison, she found Chilves – fresh out of the penitentiary system; It is interesting to reflect that at least when I was in prison, in jail, I had a roof over my head and food, so was it such a great privilege to win freedom? What does it mean to be free in this environment of extreme hardship? So, even though Jessica was already at an advanced stage of pregnancy, she went to sleep with Chilves in the street on the cardboard. A short time later, the girl went into labor and went to the hospital, where she experienced obstetric violence, when the tears returned to her face and she began to express herself again. Jessica: a teenager in (re)construction Thus, according to Vergès: City architecture was not designed to encourage a relaxed social life; she is hostile to women, particularly women of color, homeless, refugees, elderly, immigrants, poor, disabled, black and Arab people. The city is organized to obstruct the circulation of these people, it has “invisible walls” that racialized women and children must learn to identify in order to get around and avoid. (VERGÈS, 2021, p.82) With this metaphor of “invisible walls” by Vergès, we realize that the streets offer even more hostility and risks to girls like Jessica, whose life was a succession of sufferings. In which places could she and so many other marginalized people in the same situation remain without disturbing others? Even when she was pregnant, Jessica was taken to an evangelical recovery clinic for addicts, whose nickname was Casa do Esplendor Divino. This mean and corrupt clinic received funding from the State, but kept the inmates in private prison so that they would work in a forced way: “The monitors call it therapy, but the name is different: slave labor” (Melo, p.207). The inmates had to wash stones, paint walls with whitewash and were subjected to penance every time they broke some internal regulation, which almost always changed: For eating out of hours. For having had an epileptic seizure. For having “stolen” a bag of biscuits in the kitchen. For being caught naked, admiring her seven-month pregnant belly in front of the mirror. For having gone into the woods at Bible reading time. For refusing to take the medicine. For trying to Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 50 Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html https://www.currentscientific.com/p/current.html Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 kill himself with tranquilizers. For listening to funk. For not singing hymns in church services. For having answered the monitor. Or stopped responding. For trying to run away. The scope of punishments wasn't much different from a game without rules. What was allowed yesterday is now forbidden. (MELO, 2022, p.204) Current Scientific Journal - ISSN 2764-1759 kill himself with tranquilizers. For listening to funk. For not singing hymns in church services. For having answered the monitor. Or stopped responding. For trying to run away. The scope of punishments wasn't much different from a game without rules. What was allowed yesterday is now forbidden. Jessica: a teenager in (re)construction However, after a few days of hospitalization, she received the news that she could not keep custody of her own daughter because she was addicted to crack and because she did not have the financial and emotional conditions to offer the child a dignified life: The judge advised that Lorraine would be sent to a shelter. And that Jessica should undergo drug recovery treatment before getting custody of her daughter. Jessica couldn't believe her ears. Until days ago, Lorraine was inside her belly. It was as if they were saying to him: let's take your lung to the shelter. We're going to get you out of your brain for a while. (...) he only handed over Lorraine after the judge threatened to suspend her right to visit the girl at the shelter. In the elevator, held in Chilves' arms, Jessica was Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 51 Internationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 sobbing. He vomited on the sidewalk. Then he sat in front of the courthouse door, refusing to leave. (MELO, 2022, p.289) Internationally Indexed Scientific Journal sobbing. He vomited on the sidewalk. Then he sat in front of the courthouse door, refusing to leave. (MELO, 2022, p.289) After that first cry, the tears came, it is not known if for Lorraine (the baby), for Glenda (her missing friend), or if for herself (Melo, p.315); in fact, she wept because of life, because of the miserable circumstances, because of the violence she had suffered, because of the whole tragedy that her existence had become. At the end of her trajectory, she got custody of her daughter after being reunited with her mother and, thus, was able to offer a home, food and care for the baby. In addition, she moved in with her mother, Zélia, at Douglas's house, returned to her studies and was very dedicated to her lessons. Douglas once thought: I wish Danny (Douglas' daughter) was like Jessica, so devoted to school. When Danny was born, he promised himself: this one will study, this one will go to university. His dream was for Danny to break the mold of his lineage. First to graduate. But, judging from the pace of the carriage, it was Zélia who would experience this joy first. (MELO, 2022, p.360) Therefore, when we think about the VII SEPPLIN proposal – projects for a new time and solutions for the future , education is the only mechanism capable of symbolizing the possibility of change in people's lives. Symbolically, education and class consciousness are addressed in the novel in order to lead to the most victorious outcomes of the characters who had the opportunity to follow such paths. Final considerations There are countless themes that can be analyzed in Menos que um (2022). In this excerpt, I tried to show how the author makes use of fiction that emphasizes the invisibility of people in situations of vulnerability and violence to which they are subjected on a daily basis. This, through the rawness of Patrícia Melo's writing as a way of decolonizing the thinking of those who maintain this structure of class, race and even gender, since everyone has a voice. From the perspective of Glenda and Jessica's eyes and actions, so many people were represented – for suffering countless negative consequences of a toxic patriarchal structure towards people like them. These two characters provide us with rich material for analysis, so there is still much to be explored in relation to them. It is interesting how the author manages to deconstruct the narratives that may have already addressed similar issues, bringing the speech of each of these characters as a strong element of social denunciation. In this way, the writer also deconstructs the narratives with a single point of view, demonstrating not only the malleability to create fictions, but also Current Scientific Journal – (August) v.3, n.8, 2023 Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 52 Current Scientific Journal - ISSN 2764-1759 implementing an engaged literature, since it maintains direct dialogues with decolonial factors, race, gender and class. The novel is sensitive from the point of view of the transformation that education is capable of promoting, since, in addition to the character Jessica, the Iraquitan writer also represents this possibility of change, since at the end of the author's text, he publishes a homonymous book , bringing metalanguage as a way of representing the invisible under the gaze of an invisible. The narrative spaces represent the cartography of violence and Patrícia Melo brings this structure ingrained in Brazilian culture, therefore, the novel even resembles a dystopia, however it tells part of the Brazilian reality, since they are similar to announcements of imminent tragedies. Unlike the canonical themes, in relation to interpersonal relationships, this author's literature by itself already represents the adversities in different social strata and, therefore, there is still much to be explored in terms of writing and theoretical analysis, especially in this work. Final considerations Therefore, with a laconic writing – with her cutting literature, corrosive humor and the presence of constant violence, sometimes bringing death as a spectacle for those who read the works – the writer confirms her consolidation as a great author of Brazilian literature contemporary, representing characters of different classes, races, ethnicities, or sexual orientations. Thus, Patrícia Melo occupies a prominent place in Brazil, being translated to numerous countries as well. Thinking about these issues makes us understand the importance of this great author's writing, as it makes the country's marginalized and invisible peoples visible to the world, the animal-men, showing not only what is most beautiful, attractive and touristic, but revealing the true face behind what is sometimes deliberately hidden, ignored, that is, the speeches from the margins, by the margins. EAGLETON, Terry. Theory of Literature: An Introduction / Terry Eagleton - Waltensir translation Other; [revision of the translation of João Azenha Jr]. 6th ed. São Paulo: Martins Fontes, 2006; BANDEIRA, Manuel. Beautiful beautiful . 1st ed. São Paulo. Global Editora, 2014; COLLINS, Patricia Hill and BILGE, Sirma. Intersectionality. Trans. Rane Souza. São Paulo: Boitempo, 2021; EAGLETON, Terry. Theory of Literature: An Introduction / Terry Eagleton - Waltensir translation Other; [revision of the translation of João Azenha Jr]. 6th ed. São Paulo: Martins Fontes, 2006; References BANDEIRA, Manuel. Beautiful beautiful . 1st ed. São Paulo. Global Editora, 2014; EAGLETON, Terry. Theory of Literature: An Introduction / Terry Eagleton - Waltensir translation Other; [revision of the translation of João Azenha Jr]. 6th ed. São Paulo: Martins Fontes, 2006; Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 53 nternationally Indexed Scientific Journal Current Scientific Journal - ISSN 2764-1759 LAURETIS, Teresa de. The technology of gender . Trans. Susana B. Funck. In: HOLLANDA, Heloísa Buarque de; org. Trends and impasses; Feminism as a critique of culture. Rio de Janeiro, Rocco, 1994. p. 206-42; Current Scientific Journal - ISSN 2764-1759 Internationally Indexed Scientific Journal LAURETIS, Teresa de. The technology of gender . Trans. Susana B. Funck. In: HOLLANDA, Heloísa Buarque de; org. Trends and impasses; Feminism as a critique of culture. Rio de Janeiro, Rocco, 1994. p. 206-42; LAURETIS, Teresa de. The technology of gender . Trans. Susana B. Funck. In: HOLLANDA, Heloísa Buarque de; org. Trends and impasses; Feminism as a critique of culture. Rio de Janeiro, Rocco, 1994. p. 206-42; MBEMBE, Achilles. Necropolitics . Publisher #1, 2020; MBEMBE, Achilles. Necropolitics . Publisher #1, 2020; MBEMBE, Achilles. Necropolitics . Publisher #1, 2020; MELO, Patricia. Stacked women. 1st ed. São Paulo: LeYa, 2019; ______. Less than one. 1st ed. São Paulo: LeYa, 2022; ______. Less than one. 1st ed. São Paulo: LeYa, 2022; VERGÈS, Françoise. A decolonial feminism . São Paulo. Ubu Publisher, 2020; ______. A feminist theory of violence . São Paulo. Ubu Publisher, 2021. ______. A feminist theory of violence . São Paulo. Ubu Publisher, 2021. Current Scientific Journal – (August) v.3, n.8, 2023 https://www.currentscientific.com/p/current.html 54
https://openalex.org/W4225152622	https://www.auctoresonline.org/uploads/articles/1647678719Galley_Proof-IJCCR-22-RA-212_pddf.pdf	English	null	Head and Neck Neurothekeoma of Lower Lip with Aggressive Reconstruction using Bengt-Johanson’s Step Technique: Case Presentation and Systematic Review of Literature	International journal of clinical case reports and reviews	2,022	cc-by	10,885	Presentation and Systematic Review of Literature Romano Antonio MD1, Committeri Umberto MD1, Troise Stefania MD1, Maglitto Fabio1, Dell’Aversana Orabona Giovanni PhD 1, Norino Giovanna MD 1, Sani Lorenzo MD 1, Arena Antonio1, Barone Simona1, Iaconetta Giorgio MD PhD2, Califano Lugi MD PhD1 1Maxillofacial Surgery Unit, University of Naples Federico II, Via Pansini, Naples, Italy. 2Neurosurgery Department, University of Salerno, Via Giovanni Paolo II, Fisciano, SA, Italy. Corresponding Author: Stefania Troise, Maxillofacial Surgery Unit, University of Naples, Federico II, Naples, Italy. Corresponding Author: Stefania Troise, Maxillofacial Surgery Unit, University of Naples, Federico II, Naples, Italy. e: March 08, 2022 \| Accepted Date: March 16, 2022 \| Published Date: March 21, 2 Received Date: March 08, 2022 \| Accepted Date: March 16, 2022 \| Published Date Citation: Romano A, Committeri U, Troise S, Maglitto F, Dell'Aversana Orabona G, Norino G, Sani L, Arena A, Barone S, Iaconetta G, Califano L. (2022). Head and Neck Neurothekeoma: Systematic Review of Literature and Presentation of a Lower Lip Aggressive Case Reconstructed with Bengt-Johanson’s Step Technique. International Journal of Clinical Case Reports and Reviews. 11(1); DOI: 10.31579/2690-4861/212 Citation: Romano A, Committeri U, Troise S, Maglitto F, Dell'Aversana Orabona G, Norino G, Sani L, Arena A, Barone S, Iaconetta G, Califano L. (2022). Head and Neck Neurothekeoma: Systematic Review of Literature and Presentation of a Lower Lip Aggressive Case Reconstructed with Bengt-Johanson’s Step Technique. International Journal of Clinical Case Reports and Reviews. 11(1); DOI: 10.31579/2690-4861/212 Copyright: © 2022 Troise Stefania, 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: © 2022 Troise Stefania, 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 Aim: Neurothekeoma is a benign lesion of superficial tissue mainly localized in the head-neck area. The aim of the article is to provide a systematic literature review on the tumor’s main characteristics and its correct surgical treatment. We support our theory with the report of an aggressive case of the lower lip region which required a large excision and reconstruction with local flaps. Materials and Methods: The literature review is based on the scientific materials produced from December 1980 to March 2020 on the head and neck neurothekeomas. In total, 76 papers were included in the study. We presented a classic neurothekeoma S-100 protein positive case that required wide local excision with healthy margins and reconstruction with Bengt-Johanson’s step technique. Results: Most of the papers were case reports (47,4 %) and reviews (19,7 %) with 721 evaluated neurothekeomas. The male to female ratio was 1:1,8 with a mean age of 26,4 years. The most frequent site of lesions was the head (36,1%), and the three subtypes were divided in cellular (65,7%) mixed (17,5%) and classic (16,8%). The classic neurothekeoma resulted more immunoreactive for S-100 Protein while cellular neurothekeomas for NKI/C3. Our case was successful without recurrence at 1 year follow-up. Conclusion: Our review highlights that neurothekeoma mainly occurs in young women in the superficial planes of the head and neck. The most frequent type is the cellular one, but the most aggressive is the classic type due to a high local recurrence of protein S-100. A local excision is sufficient for the cellular neurothekeoma, while for the classic type a wide local excision with healthy tissue margins is necessary. The treatment of our case demonstrates that by following this guide, relapse can be avoided. Keywords: facial neurothekeoma; head and neck; lower lip; Bengt-Johanson’s step technique, S-100 prote tional Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. Open Access Research Article International Journal of Clinical Case Reports and Reviews Stefania Troise AUCTORES Globalize your Research tional Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. Open Access Research Article International Journal of Clinical Case Reports and Reviews Stefania Troise * AUCTORES Globalize your Research ws Copy rights@ Stefania Troise et.al. International Journal of Clinical Case Reports and Reviews Stefania Troise * Globalize your Research Globalize your Research Case Report Case Report On February 2020, a 64 years old patient was admitted at the Maxillo- Facial Surgery Unit of Federico II University of Naples. The patient was affected by a neoplasia of the lower lip. The Magnetic resonance imaging (MRI) with contrast showed a non-homogeneous captive mass of 2 cm in diameter, hypointense in T1 and hyperintense in T2 in the lower lip that was scarcely differentiated between the skin and the deep muscle planes (Figure 1). Further histological examinations based on incisional biopsy showed a neoplasm with appreciable mitotic activity, with prevalent lobulated areas alternating with diffuse areas, composed of fascicles of fusate or epithelioid cells immersed in a myxoid matrix. The neoplasm resulted immunoreactive to S-100 protein but not to CD31, CD34, p63, CK, Mart1, and HMB45. Generally, the neurothekeoma tumor manifests itself as an asymptomatic, solitary, slow-growing nodule that involves the skin and superficial subcutis of the head and neck region, or of the extremities. It usually requires surgical excision that, based on histopathological and immune histochemical features, can be less or more extended. In this paper, an extensive review of the literature related to head and neck neurothekeomas has been conducted. Furthermore, a case of lower lip classic neurothekeoma, treated by our team, is presented. The aim of the study is to show the clinical, histopathological and immunohistochemical features of these tumors, and to offer indications for the different surgical procedures according to the characteristics of the neoplasms. An elective surgery to remove the tumor and reconstruct the region was performed. A) Cutaneous Frontal View of the tumor B) Mucosal view of the tumor C) Axial D) Coronal and E) Sagittal plan of the tumor. Figure 1: Pre surgical evaluation A) Cutaneous Frontal View of the tumor B) Mucosal view of the tumor C) Axial D) Coronal and E) Sagittal plan of the tumor. Figure 1: Pre surgical evaluation A) Cutaneous Frontal View of the tumor B) Mucosal view of the tumor C) Axial D) Coronal and E) Sagittal plan of the tumor. Materials and Methods We report the case report and the literature review process. by dense collagen septae and classically S-100- negative but NKIC3 positive [5]. Surgical Procedure The tumor excision was planned with a margin of 1 cm, the corners of healthy tissue were tensioned by n. 2 silk sutures in order to reduce the traumatism of healthy tissue, and to allow a better removal of the tumor. The surgical procedure consisted of 2 steps. Firstly, the tumor removal and after the use of a stairs flap (Bengt-Johanson’s Step Flap [8]) to reconstruct the defect. Antisepsis procedures were performed with iodine- povidone solution and administration of 1g of intravenous Ceftriaxone. Local anesthesia was realized by infiltration of a 2% carbocaine solution and adrenaline to obtain hemostasis and flap hydrodissection. The tumor was removed at full thickness, was oriented with nylon threads and sent for histopathological examination. The residual defect measured about 4 cm, >50% of lower lip (Figure 2). Introduction The term “classic neurothekeoma” (classic NTK) will be used henceforth when referring to a tumor also known as a nerve sheath myxoma characterized by an abundant myxoid matrix, and scattered collections of epithelioid schwann cells in corded, nested or syncytial- like patterns, and typically S-100 immunoreactive [4]. Neurothekeoma is a benign tumor first described by Harkin and Reed in 1969 and classified as “nerve sheath myxoma” [1]. The term neurothekeoma was coined in 1980 by Gallagher and Helwig in their report on 53 dermal tumors with similar features. [2] Argenyi et al [3] further classified neurothekeoma as classic, cellular and mixed type according to cellularity, mucin content, and growth pattern. The “cellular neurothekeoma” (cellular NTK) is a tumor composed of nests and bundles of variably epithelioid-to-spindled cells often separated Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 1 of 16 Copy rights@ Stefania Troise et.al. International Journal of Clinical Case Reports and Reviews Materials and Methods We report the case report and the literature review process. The so-called “mixed-type” of neurothekeomas shows overlapping features of both variants [6]. The tumors have been subclassified as cellular neurothekeomas when they have <10% myxoid matrix, mixed- type neurothekeomas when they have >10% and <50% myxoid matrix, and myxoid neurothekeomas when they have >50% myxoid matrix [7]. Step 2: Bengt-Johanson’s Step Flap Reconstruction [8] In our case the defect was paramedian and greater than 2 cm in width. Therefore, 2 asymmetric flaps with bilateral staircase technique were performed (Figure 3). Two incisions were made bilaterally from the lateral sides of the defect, extended horizontally and vertically downward, toward to the chin for 2 cm. The incisions ended with the Burow triangles, with an inferiorly located apex, and a base that approximately corresponds to 2/3 of the last horizontal incision. The dissection was performed above the mimic muscles, by setting up a flap of skin and subcutaneous, with A) Reconstruction surgical planning B) Final result after cutaneous suture in Nylon. Figure 3: Bengt-Johanson’s step technique for lower lip reconstruction. A) Reconstruction surgical planning B) Final result after cutaneous suture in Nylon. Figure 3: Bengt-Johanson’s step technique for lower lip reconstruction. A) Reconstruction surgical planning B) Final result after cutaneous suture in Nylon. Figure 3: Bengt-Johanson’s step technique for lower lip reconstruction. Step 1: Tumor excision Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 2 of 16 nternational Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. A) Surgical Planning B) Tumor delimitation C) Removed tumor with a final defect of 3 cm. Figure 2: Tumor Removal International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. A) Surgical Planning B) Tumor delimitation C) Removed tumor with a final defect of 3 cm. A) Surgical Planning C) Figure 2: Tumor Removal preservation of the orbicularis oris, depressor anguli oris, and depressor labii inferioris. The rectangles below the steps have been removed and the Burow triangles were then excised. In the last stage, the flaps are advanced and approximated using a layered closure. The two extremities of the flap were transposed medially one towards the other to ensure a coverage of the surgical gap without tension. The closure of the orbicularis muscle and of the vestibular fornix mucosa was performed with vicryl 4.0 sutures. The skin was closed with nylon 5.0 mattress sutures. At the end of the case, the gastric nose tube was positioned to reduce the opening of the mouth in the first days of post-surgery. Step 2: Bengt-Johanson’s Step Flap Reconstruction [8] Post-operative outcome C) Cutaneous view and D) Final buccal opening at 1 year. A) Cutaneous and B) mucosal final result at 1 month. C) Cutaneous view and D) Final buccal opening at 1 year. A) Low power magnification showed a non-capsulated neoplasm, with prevalent lobulated areas (star) alternating with diffuse areas (arrow), composed of fascicles of fusate or epithelioid cells immersed in a myxoid matrix (haematoxylin and eosin, original magnification, x2); B) Higher magnification highlighted appreciable mitotic activity (haematoxylin and eosin, original magnification, x10) B) Higher magnification highlighted appreciable mitotic activity (haematoxylin and eosin, original magnification, x10) C) and D) Positivity for vimentin and S100 (protein s100 and vimentin, original magnification, x4). Figure 5: Histology of the case of Classic Neurothekeoma. B) Higher magnification highlighted appreciable mitotic activity (haematoxylin and eosin, original magnification, x10 C) and D) Positivity for vimentin and S100 (protein s100 and vimentin, original magnification, x4). Figure 5: Histology of the case of Classic Neurothekeoma C) and D) Positivity for vimentin and S100 (protein s100 and vimentin, original magnification, x4). Post-operative outcome Outpatient follow-up controls were performed at 15 days, one month, two months, and up to one year. The local examination showed a result of lip competence, symmetry and absence of microstomy (Figure 4). Definitive histopathological examination revealed a classic neurothekeoma with fascicles of spindle cells dipped in a myxoid stroma. Tumor cells were positive for vimentin and s-100 protein with high mitosis (ki67 40%) (Figure 5). At one year of follow-up, recurrence was absent. Outpatient follow-up controls were performed at 15 days, one month, two months, and up to one year. The local examination showed a result of lip competence, symmetry and absence of microstomy (Figure 4). Definitive histopathological examination revealed a classic neurothekeoma with fascicles of spindle cells dipped in a myxoid stroma. Tumor cells were positive for vimentin and s-100 protein with high mitosis (ki67 40%) (Figure 5). At one year of follow-up, recurrence was absent. During the postoperative period, the patient continued the antibiotic therapy with ceftriaxone 1 gr x1/die for 7 days and daily local disinfection. Sutures were removed after 15 days. Oral and dental hygiene was required combined with diet through gastric-nose tube for 15 days. Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 3 of 16 International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. A) Cutaneous and B) mucosal final result at 1 month. C) Cutaneous view and D) Final buccal opening at 1 year. Figure 4: Post-surgical follow-up A) Low power magnification showed a non-capsulated neoplasm, with prevalent lobulated areas (star) alternating with diffuse areas (arrow), composed of fascicles of fusate or epithelioid cells immersed in a myxoid matrix (haematoxylin and eosin, original magnification, x2); B) Higher magnification highlighted appreciable mitotic activity (haematoxylin and eosin, original magnification, x10) C) and D) Positivity for vimentin and S100 (protein s100 and vimentin, original magnification, x4). Figure 5: Histology of the case of Classic Neurothekeoma. Review Process Study Design Data Research The data were collected from December 1980 to March 2020, and the primary source was the online database of the U S Library of Medicine Copy rights@ Stefania Troise et.al. International Journal of Clinical Case Reports and Reviews A) Cutaneous and B) mucosal final result at 1 month. C) Cutaneous view and D) Final buccal opening at 1 year. A) Cutaneous and B) mucosal final result at 1 month. Data Research Review Process Study Design Review Process Study Design The data were collected from December 1980 to March 2020, and the primary source was the online database of the U. S. Library of Medicine (PubMed). The Medical Subject Headings (MeSH) used were: Neurothekeoma, Face, Head, Neck, Treatment. For the literature search, keyword Neurothekeoma was paired sequentially with the other 4 The literature review on head and neck neurothekeomas was realized adhering to recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) [9]. Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 4 of 16 Page 4 of 16 Copy rights@ Stefania Troise et.al. International Journal of Clinical Case Reports and Reviews research and 283 in the second research. Then the PubMed search results were screened by a carefully reading, and 220 papers were excluded for title not inherent or the duplicates; 48 papers were excluded for the absence of abstract. Moreover, among the 118 remaining PubMed studies, 10 papers not in English, 16 not referring to head and neck, and 1 not referred to humans were excluded (elegibility criteria). At the end, among the 91 studies remaining, 10 papers were excluded because of the presence of only abstract and 5 papers were excluded after a complete reading because not interesting for the topic. keywords, having this final formula: (neurothekeoma AND face OR facial) OR (neurothekeoma AND head) OR (neurothekeoma AND neck) OR (neurothekeoma AND therapy OR treatment OR therapeutics). To avoid the loss of interesting articles, accepted by March 2020, a second research was always carried out on Pubmed using only the keyword neurothekeoma and, later on, the results were crossed. Results All the 76 analyzed papers and their features are shown in Table 1. Data extracted from each article included the type of study, total cases, sex and age of cases, sites of lesions, diameter (if reported) of lesions, invaded tissue, histological and cytological features, expressed immunohistochemical markers, type of treatment performed, subtypes of neurothekeoma. For the purposes of this study, the primary outcome was to identify the characteristics of the different neurothekeoma subtypes and to choose the correct surgical treatment according to these features. The types of study were divided in 36 case reports (47,4 %), 15 reviews (19,7 %), 12 case series (15,8 %), 12 retrospective observational studies (15,8 %) and 1 letter to editor (1,3 %). In total, the evaluated neurothekeomas cases were 721. Author/ Year Type of Study N° Cases Sex Side of Lesion Ø Local Invasion Cytological Markers Hystological Features Surgical Treatment Final Diagnosis Gallager 2 1980 observatio nal retrospecti ve 53 44 F 9 M 15 up ext, 15 face, 1 oral cavity, 3 neck, 5 shoulder, 3 trunk, 5 lower ext , 2 back 1 cm 37 subcutis, 26 dermis / nests of spindle cells between bundles of dermal collagen with eosinophilic cytoplasm. Frequent atypical hyperchromatic nuclei and mitotic figures varying from none to 5/10 high-power fields. Excision / Barnhill 5 1991 case series 11 8 f 3m 2 scalp, 2 back, 2 nose, 1 chin, 1 shoulder, 1 up extremities, 1 Forehead, 1 ear / dermis S-100, Vimentin and SMA fascicular pattern (cellular neurotek); prominent myxoid stromal change (classic neurotek) Excision 8 cellular 3 myxoid Fetsch 7 2007 observatio nal retrospecti ve 176 112 F 64 M 17 nose, 15 scalp, 31 face, 4 neck, 70 up ext, 17 trunk, 20 lower ext, 2 not reported 1,1 cm 120 subcutis, 5 muscles plane, 51 dermis Vimentin, NKI/C3, MiTF, PGP9.5, CD10, NSE, CD68, CD99, alfaSMA, Collagen IV, HMB-45 nests and bundles of epithelioid cells and spindle cells; eosinophilic cytoplasm; dense hyaline collagen. mild cytological atypia; mitotic rate 3/10 high power fields 133 Excision, 33 enucleoresec tion 63 cellular neurotek, 66 mixed, 47 classic neurotek Almeida 11 2018 case report and review 1 f multiple localized oral cavity 2 cm submucosal vimentin, CD63, CD56, whereas AE1/AE3, S100, CD34, α-SMA, GFAP, EMA spindle and epithelioid cells, forming nests and bundles, supported by fibrous stroma. Rare presence of giant cells. Eligibility Criteria The PubMed search results were screened, and duplicate articles were eliminated. Studies conducted on humans and published in English were considered. The titles and abstracts of the records were screened by one author using the inclusion criteria. We found 103 occurrences in the first The total number of articles included in our review were 76. The selection process was summarized in Figure 6. Page 5 of 16 International Journal of Clinical Case Reports and Reviews International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. Results Enucleoresec tion classic neurotek Misago 13 2004 case report 1 F scalp 1,5 cm subcutis S-100A6 protein, PGP9.5, CD10, CD68 (KP1), PG-M1, Vimentin Nests of epithelioid cells with abundant and pale eosinophilic cytoplasm, surrounded by spirally arranged stellate cells associated with a moderate amount of mucin. The mitosis rate was 2-3 / 10 high-power fields, without atypical mitotic figures The stroma was collagen and often associated with dense, sclerotic, or hyalinized collagen Excision cellular neurotek Maktabi 14 2019 case report and review 1 m lateral canthus 1,5 cm dermis CD68, Vimentin, D2–40, SMA nests of epitheli-oid/spindle cells separated by fibrous septae within a myxoid background Excision Mixed Safadi 15 2010 case report and review 1 F Oral cavity 2 cm S-100, NSE fusiform cells with eosinophilic cytoplasm, myxoid stroma enucleoresec tion classic neurotek Park 16 2016 case report 1 f Scalp 6 cm dermis vimentin, CD68, CD10 spindled and epithelioid cells arranged in fascicles Excision cellular neurotek Page 6 of 16 International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. A P bli hi LLC V l 11(1) 212 li and nodules separated by a collagen-rich stroma Benbenist y 17 2006 case report and review 1 F nasal wing 4 mm dermis NSE, MiTF, NKI / C3, PGP9.5, SMA, CD10 vimentin, CD68 nests of epithelioid cells with abundant vacuolated eosinophilic cytoplasm; nuclei with moderate atypia round or ovoid. frequent mitotic figures (4/10 high power fields) but absence of atypical mitoses. Large enucleoresec tion neurotekeom a cellulare ATIPICO Campanat i 18 2006 case report and review 1 f chin 6 cm skeletal muscle PGP, Ki-67 diffusely infiltrative borders, vascular invasion, high mitotic Large Enucleoresec tion Atypical neurotek Wilson 19 2008 case report 1 F nasal wing 1 cm NKI / C3, PGP9.5. spindle cells, focal atypia, high mitotic activity Large enucleoresec tion Atypical neurotek cellular Papadopo ulos 2004 20 case report and review 1 M neck 1 cm dermis Splindle in a myxoid stroma and separated by strands of collagen. Tumor cells nuclei were fused and rare mitoses were present. Results Excision neurotek mixed Hornick 21 2007 observatio nal retrospecti ve 133 83 F 45 M 27 up ext, 20 face, 10 nose, 4 lip, 3 scalp, 5 neck, 23lower ext, 16 trunk, 13 shoulder, 12 back 1.1 cm 69 dermis, 63 subcutis, 1 not reported NKI / C3, SMA, NSE nests and bundles of epithelioid cells and spindle cells with pale eosinophilic cytoplasm; dense hyaline collagen. mild cytological atypia; mitotic rate 3/10 high power fields enucleoresec tion cellular neurotek Rodriguez 23 2015 case report 1 F orbit 2 cm CD34, S-100 protein myxoid nodules with spindle-shaped or stellate cells; abundant myxoid matrix; no atypia were observed enucleoresec tion classic neurotek Sanchez- Orgaz 2011 24 case report 1 M orbit 1,5 cm S-100, vimentin, CD68, CD34 e CD10, EMA. spindle and stellate cells; abundant myxoid matrix. Ki-67 was less than 1%. enucleoresec tion classic neurotek Jaffer 25 2009 observatio nal retrospecti ve 43 24 f 19 m 9 upper and 4 lower ext, 7 scalp, 5 face, 5 thorax, 5 shoulder, 1 back 7 not available / / S- 100, Vimentin, CD68, NSE, CD56 plexiform, multinodular, and diffuse, osteoclast-like giant cells, and little or no myxoid stroma, Excision and Enucleoresec tion 8 Myxoid, 15 Mixed, 20 Cellular See 26 2019 case series and review 2 2 M orbit 1 cm aSMA, MITF, CD10, CD68 spindle cells with fusiform to oval nuclei with prominent nucleoli, rare mitotic figures; vascular channels Excision cellular neurotek Murphrey 27 2020 case series and review 7 4 M 3 F 3 trunk, 3 upper ext 1 nose / CD68, NKI/C3 nests of epithelioid cells with abundant eosinophilic pale colored cytoplasm, fascicular growth. The cellular stroma has been described as collagenic and dense or fibrous Excision cellular neurotek Barnhill 29 1990 case series 5 3 F 2 M 5 head-neck (1 frontal, 2 scalp, 1 neck, 1 chin) 1 cm dermis / fascicles of spindle and epithelioid cells with nuclear atypia, mitotic figures (2/10 per field) and abundant eosinophilic cytoplasm, myxoid stroma and sclerotic collagen with the presence of giant cells Excision cellular neurotek International Journal of Clinical Case Reports and Reviews Page 7 of 16 International Journal of Clinical Case Reports and Reviews International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. Results Suh 30 1992 case report 1 F scalp / dermis S-100 fusiform and stellate cells; abundant myxoid stroma, eosinophilic cytoplasm, vacuolated nuclei Excision neurotek classic Tiffee 31 1996 case report and review 1 F Lower lip / dermis S-100 fusiform and stellate cells; abundant myxoid stroma enucleoresec tion neurotek classico Tomasini 32 1996 case series 2 1 F 1 M 1 trunk, 1 frontal / dermis Vimentin, actin, XIIIa Factor epithelioid or spindle cells organized in plexuses, large vacuolated eosinophilic cytoplasm; myxoid stroma Excision neurotek cellular Breuer 33 1999 case report 1 F tongue 2 cm Muscles plan vimentin spindle cells with few mitoses; hyaline collagen enucleoresec tion cellular neurotek Yee Hang 34 Wong 2001 case report 1 M Maxillary and ethmoidal sinuses / / S-100 spindle cells in sclerotic stroma enucleoresec tion neurotek classic Cohen 35 2004 case report 1 M Superior Alveolar crest 3,5 cm Maxillary bone plane vimentin, NKIC3 irregular cell nests in a densely fibrotic stroma; vesicular, ovoid or irregularly shaped nuclei; abundant mitotic activity, 15 mitoses for 10 high-power fields Excision cellular neurotek Ward 36 2005 case series 13 9 F 4 M 3 scalp, 2 neck, 1 face, 3 up and 1 lower ext, 2 trunk, 1 not reported 1 cm Vimentin, actina (1 cases + S-100 like malignant tumor) fusiform cells with eosinophilic cytoplasm, myxoid stroma enucleoresec tion Classic neurotek Mahaling am37 2006 case report and review 1 M face 3 cm vimentin, NKI/C3, PGP 9.5, factor XIIIa CD68 / Excision cellular neurotek lopez capeda38 2007 case report 1 F nose 1,5 cm vimentin, mucina, neurofilament spindle cells arranged in nests with mitosis and collagen Excision cellular neurotek Koumanis 39 2007 case report 1 M nose 1,5 cm vimentin / enucleoresec tion classic neurotek Mathew40 2008 case report 1 M Lower eyelid 2 cm S-100 fusiform and stellate cells; myxoid stroma with collagen vortices, Schwann cells and elongated mast cells. very low mitotic activity enucleoresez ion classic neurotek Zedek41 2009 observatio nal retrospecti ve 12 9 F 3 M 2 face, 1 scalp, 4 up and 2 lower ext, 3 trunk 2 cm NKI/C3, laminin, CD68, CD10 absence of atypia, 0.67 / 10 mitosis, large vacuolated eosinophilic cytoplasm, stromal sclerosis Excision neurotek cellular Wartchow 42 2009 case series 1 M Mandibular gum (reg. Results 31) 1.7 cm actina, S-100 / CD1a, CD4 e CD68 16/10 mitosis per field, myofibroblastic characteristics, ki67 <2% Excision neurotek cellular Muller43 2009 case report 1 F Medial canthus (left eye) 1 cm NKI / C3, SMA, S100 protein / Excision Neurotek mixed vered 44 2010 observatio nal retrospecti ve 4 2 F 2 M Oral cavity 8 mm cellular (S100A6, NSE, PGP9.5, vimentin, NKI ⁄ C3); classic / Excision Cellular neurotek Page 8 of 16 International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. (S100A6, NSE, PGP9.5, vimentin, S- 100) Papalas45 2010 case series 3 3 F 2upper eyelid, 1 lower eyelid 6 mm 2 casi NKI.C3 CD34 positivi, 1 caso S-100, GFPA, Vimentin positivo. / Excision 2 cellular neurotek, 1 classic neurotek Sheth46 2011 observatio nal retrospecti ve 14 11 F 3 M face 2, art sup 7, art inf 3, tronco 2 / classic/myxoi d: S100B, aSMA; cellular: NSE, aSMA, F13A, NKIC3, podoplanin D2-40, / Enucleoresec tion 6 cellular neurotek, 8 classico neurotek Pan47 2011 case report 1 M Upper lip 8 mm NSE, alfaSMA, cd34 hypercellular epithelioid cells with diffuse and fascicular growth patterns. Casually focal mitosis. No atypia was found. Excision cellular neurotek Yamada48 2013 letter to editor 1 F nose Vimentin, MiTF, NKI/C3, Glut- 1, PGP9.5, CD10, NSE, CD68, CD99. subcutaneous multi-lobular nodular lesion spindle cells separated by a sclerotic stroma and with eosinophilic cytoplasm, ki67 <1% Excision neurotek cellular Suarez 49 2013 observatio nal retrospecti ve 18 17 F 1 M 5 nose, 4 upper and 3 lower ext 3 trunk , 2 face, 1 scalp KBA.62, CD10 epithelioid cells intercalated between bundles of fibrotic collagen, sclerotic large, pale and eosinophilic cytoplasm with mild pleomorphism Excision 1 neurotek classic; 17 neurotek cellular Stratton 50 2013 observatio nal retrospecti ve 37 21 F 16 M 13 face, 3 scalp, 11 upper ext 4 trunk 4 shoulders 2 lower ext NKI/C3, MiTF, CD68, CD10 epithelioid or spindle cells; large vacuolated eosinophilic cytoplasm; myxoid stroma. 4 cases of perineural invasion. Results 19 cases of cytological atypia, 2/10 mitosis per field Excision 33 neurotek cellulari 4 neurotek classici yun 51 2014 case report 1 F Left eyebrow skin CD68, Vimentin thin spindle-shaped and stellate cells within an abundant myxoid stroma Excision Neurotek mixed Wang 52 2016 observatio nal retrospecti ve 7 4 M 3 F not avaible NKI / C3, PGP9.5, CD68 / nerotek cellulare Tham 53 2016 case report 1 M Oral cavity S-100 fusiform and stellate cells; abundant myxoid stroma Enucleoresec tion neurotek classic Boukoval as 54 2016 case report 1 F Nasal wing 8 mm subcutis MiTF nests and fascicles of spindle and epithelioid cells with pale eosinophilic cytoplasm and vesicular nuclei; background of dense collagen, Excision cellular neurotek Bartake 55 2017 case report 1 F hard palate 1,5 cm submucosal S100 myxomatous tissue, stellate and spindle-shaped cells and nerve fibers. Enucleoresec tion classic neurotek Mora- Cantallop s 56 2020 case report 1 M Medial rectus muscle 1 cm muscolo retto mediale S-100, CD34, CD56 myxoid matrix with fusocellular and stellate cells. enucleoresec tion classic neurotek Page 9 of 16 International Journal of Clinical Case Reports and Reviews International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. Results Massimo 57 2020 case series and review 2 1 M 1 F 1 wrist, 1 upper lip 8 mm dermis CD10, CD68, SMA, and vimentin; spindle and epithelioid cells with eosinophilic cytoplasm and mild atypia Excision 2 cellular neurotek Aronson 58 1985 case report 1 f scalp 2,5 cm dermis S 100, CEA, DAKO Polygonal cells; elongated eytoplasmic processes Excision classic neurotek Henmi 59 1986 case report 1 f right nostril 1 cm dermis S- 100 cell nests consisting of atypical epithelial-like cells Enucleoresec tion classic neurotek Mason 60 1986 case report 1 f lip 1 cm dermis / nests and whorls of spindle- shaped cells with abundant myxoid cytoplasm Excision Cellular neurotek Pepine 61 1992 case report 1 m nose 1 cm dermis / epithelioid and stellate cells in a myxoid stroma Enucleoresec tion classic neurotek Husain 62 1994 case report and review 14 8 f 6 m 3lower and 3 up extremities, 2 thorax, 2 scalp, 1 shoulder, 1 lip, 1 face, 1 forhead / dermis S- 100, stellate and spindle-shaped cells; Nuclear pleomorphism Excision 3 myxoid 11 cellular Peñarroch a 63 2000 case report 1 F tongue 3 cm muscles plane S-100, protein, NSE, and vimentin fusiform cells with wavy cytoplasm, abundant capillary neovascularization; myxoid stroma Enucleoresec tion classic neurotek Barrett 64 2001 case report 1 m buccal vestibular sulcus 1 cm submucosal NKI/C3, NSE, SMA PGP, XIIIa, S100 pale epitheliod cells separated by fasciles of spindle cells Excision nerotek cellular Laskin 65 2000 case series 11 6 f 5m 2 head, 2neck, 4 lower and 3 up extremities / dermis S100, CIV, SMA, XIIIa multinodular or lobulated architecture of spinde cells Excision classic neurotek Schorting huis 2001 66 case report 1 m tongue 0.8 cm muscles plane S- 100, EMA stellate and spindle-shaped cells with basophilic ovoid vesicular nuclei scattered in a myxoid and avascular stroma Enucleoresec tion classic neurotek Makino 67 2002 case report and review 1 m tongue 1 cm muscles plane S100, NSE, VM spindle- or stellate-shaped cells with a myxoid background. Enucleoresec tion classic neurotek Levin 68 2002 case report 1 m nose 4 cm dermis S- 100, desmin, vimentin spindled and ovoid, cells separated by thick collagen bands. Results Enucleoresec tion classic neurotek Page 69 2004 case series 11 8 f 3m 5 nose, 2 neck, 2 lower and 1 upper extremities, 1 shoulder / dermis Mitf, NKI/C3 spindled and epithelioid cells, Excision 2 Cellular 9 Mixed Kim 70 2006 case report 1 f tongue 2 cm muscles plane CD56, CD68 (clone PG- M1), and desmin lobules of well- circumscribed oval-to- spindle neoplastic cells in a poor myxoid stroma fibrous connective tissue, Excision cellular neurotek Nishioka 71 2009 case series 3 2 f 1 m oral cavity 2 cm submucosal S-100 protein, NSE, NGFR pindle cells admixed with varying amount of myxoid matrix Excision cellular neurotek Plaza 72 2009 observatio nal retrospecti ve 31 23 f 8 m 10 upper and 5 lower extremities, 4 nose, 4 scalp, 3 thorax, 3 face, 2 shoulder / dermis S100A6, SMA nests and bundles of epithelioid cells Excision cellular neurotek Garcia - Gutiérrez 73 2010 case report 1 m multiple localized to the face 2-3cm dermis S100A6, CD63 (NKI/C3), CD10, and PGP 9.5 (Figs. 5A–D), XIIIa and vimentin. spindled to epithelioid cells embedded in a sclerotic stroma with focal areas of stromal hyalinization Enucleoresec tion classic neurotek Page 10 of 16 International Journal of Clinical Case Reports and Reviews International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. Results Kah 74 2011 case report 1 f palpebral 1,5 cm dermis CD63 spindle to polygonal cells within a myxoid stroma; eosinophilic cytoplasm Enucleoresec tion classic neurotek Fox 75 2012 case series 14 6 f 8 m 3 neck, 4 upper and 2 lower extremities, 2 back, 1 thorax, 1 scalp, 1 shoulder / dermis PAX2, NKI/C3, CD10,MiTF nests and fascicles of histiocytoid to spindled cells; of nests by collagen bands Excision cellular neurotek Emami 76 2013 case report 1 f oral floor 0,8 cm submucosal CD63, NKI- C3, XIIIa spindle and epitheloid cells Excision cellular neurotek Requena 77 2013 case series 9 4 f 5 m lip / dermis S100A6, MiTF, NKI/C3, PGP9.5, EMA, and NSE plexiform pattern of nests of spindle cells embedded in a slightly myxoid stroma Excision cellular neurotek Rozza De Menezes78 2013 case report and review 1 f right buccal mucosa 1,5 cm submucosal anti-S-100, NSE, EMA spindle and stellate cells with ovoid vesicular nuclei; myxoid stroma with sparse collagen fibers Enucleoresec tion classic neurotek Bashline 79 2014 case report and review 1 f scalp 0,5 cm dermis NK1C3 spindled and epithelioid cells; fascicular growth pattern Excision cellular neurotek Fried 80 2014 observatio nal retrospecti ve 34 20 f 14 m 7 nose, 11 upper and 5 lower extremities, 3 neck, 3 face, 3 shoulder, 2 thorax, / dermis SOX-10, S100, NKI/C3, SMA, MiTF spindled and/or epithelioid cells arranged in a fascicular and/or nested pattern with sparse (cellular) or abundant (classic) myxoid component Excision and Enucleoresec tion 25 Cellular 8 mixed 1 NSM Navarrete - Dechent 81 2015 case report 1 m forehead 4 mm dermis CD10 spindle cell tumor, including an eosinophilic cytoplasm with mild cellular pleomorphism and moderately dense fibrous stroma Enucleoresec tion classic neurotek Bhat 82 2015 case report 1 f neck 4 mm dermis S100 stellate cells with cytoplasmic processes, round to spindle lacking the nuclear atypia and occasional giant cells Excision cellular neurotek Gray 83 2016 case report 1 m eyelid 4mm dermis PGP 9.5, CD68, XIII nests and bundles of epithelioid to spindled cells with abundant eosinophilic cytoplasm, separated by sclerotic collagen Excision cellular neurotek Frydrych 84 2017 case report 1 f tongue 6 mm muscles plane S100, vimentin, stellate and spindle-shaped cells. Results Rare Nuclear pleomorphism and mytotic figures Enucleoresec tion classic neurotek Cavacchin i 85 2018 Case series 2 1 f 1 m forehead, thing 7 mm dermis EMA, NKI/C3 Spindle cells; abundant eosinophilic cytoplasm with vesicular nuclei and mild atypia Excision cellular neurotek Gallo 86 2019 case series 2 1 f 1 m 1 wrist, 1 upper lip 7 mm dermis CD10, CD68, vimentin, and SMA plexiform and multinodular pattern of spindle and epithelioid cells; multinucleated cells and scattered mitotic figures Excision cellular neurotek Table 1: Paper’s check list Clinical features In most cases the female sex was involved (462 F – 64,1%) with a male of cases were aged between 6 months and 25 years, 35.6% between 26 and 50 years, 12.3% between 51 and 88 years. Neurothekeomas typically t d t ti lit l i l i ith of cases were aged between 6 months and 25 years, 35.6% between 26 and 50 years, 12.3% between 51 and 88 years. Neurothekeomas typically presented as asymptomatic, solitary, slow-growing lesions with a mean diameter of 1,5 cm (the smaller lesion measured 0,4 cm while the larger Histological and Cytological features Histologically and cytologically Neurothekeomas were divided in three subtypes: cellular (65,7%) mixed (17,5%) and classic – myxoid (16,8%). All the subtypes presented as dome-shaped masses that most frequently involved the subcutaneous/submucosal plane (55,1% of the total cases) and dermas (41,5%). Skeletal muscle involvement was uncommon (2,9%) and largely restricted to the facial region (mimic muscles and rectus medial muscle). Bone plane involvement was rare (0,5%) and limited to maxillary and mandibular bones. The differences among the 3 subtypes of tumors lied in histology and cytology: 1) Cellular neurothekeoma was characterized by nests of spindle or epithelioid cells immersed in a fibrotic stroma with the presence of sclerotic collagen fibers, and non-tumor multinucleated and osteoclastic giant cells. Tumor cells had abundant eosinophilic cytoplasm with vesicular nuclei, mild atypia and low mitotic rates (ranged from 0 to 16 mitotic figures for mm2 with a mean mitotic rate of 4/mm2). Perineural invasion was uncommon, while vascular invasion was completely absent. Three cases of cellular neurothekeomas were described as atypical because of diffusely infiltrated borders, vascular invasion, severe cellular atypia, and frequent mitosis (until 15/ mm2). 2) Classic neurothekeoma was characterized by bundles of stellate cells immersed in a myxoid stroma without collagen fibers. Tumor cells had abundant eosinophilic cytoplasm with vesicular nuclei, mild atypia d l i i ( i i f 3/ 2) 3) Mi d Clinical features In most cases the female sex was involved (462 F – 64,1%) with a male to female ratio of 1:1,8. The average age of 721 cases was 26,4 years, with the youngest case of 6 months old, and the oldest of 88 years old. 52.1% Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 11 of 16 International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. neurothekeoma was characterized by features of both other subtypes, in particular both myxoid stroma and sclerotic collagen fibers. 6 cm). The sites of lesions were: 260 head (36,1%), 187 upper extremities (25,9%), 85 trunks (11,8%), 84 lower extremities (11,6%), 36 shoulder (5%), 23 neck (3,2%) and 46 not reported (6,4%). The 260 head cases were divided in 116 face (44,6%), 59 nose (22,7%), 49 scalp (18,8%), 11 oral cavity (4,2%), 10 lip (3,9%), 7 forehead (2,7%), 6 tongue (2,3%), 2 others (1 medial rectus muscle and 1 paranasal sinuses – 0,8%). Immunohistochemical features 2) Classic neurothekeoma was characte immersed in a myxoid stroma without co bundant eosinophilic cytoplasm with ve low mitotic rates (mean mitotic rate and Clinical features Histol ar %) -asymptomatic, solitary, slow- growing lesion -deep involv -<10% Histological and Cytological features Immunohistochemical features 116 face (44,6%), 59 nose ( %), 10 lip (3,9%), 7 forehea rectus muscle and 1 parana and Cytological fea nd cytologically Neurothek r (65,7%) mixed (17,5%) a presented as dome-shaped cutaneous/submucosal plan %). Skeletal muscle involv ricted to the facial region Bone plane involvement w andibular bones. The diffe histology and cytology: 1) nests of spindle or epithelio presence of sclerotic col and osteoclastic giant cells oplasm with vesicular nucle m 0 to 16 mitotic figures f Perineural invasion was mpletely absent. Three case as atypical because of d n, severe cellular atypia, an c neurothekeoma was charac n a myxoid stroma without sinophilic cytoplasm with c rates (mean mitotic r linical features Hist symptomatic, olitary, slow- rowing lesion -dee invo -<10 In order of frequency, the neoplastic cells of classic neurothekeomas were immunoreactive for S-100 Protein (86,9% of all cases), vimentin (47,8%), muscle-specific actin, a-smooth muscle actin and EMA (31,8%), neuron- specific enolase (NSE), GFAP, CD10, CD34 and XIIa Factor (18,2%) while NKI/C3 and PGP9.5 in a minor percentage of cases. In order of frequency, the neoplastic cells of cellular neurothekeomas were immunoreactive for NKI/C3 (62,1%), CD68 (55,2%), Vimentin and a- smooth muscle actin (41,4%), CD10 (37,9%), PGP9.5 (34,5%), NSE and microphthalmia transcription factor (MITF) (31,1%), S-100 Protein and XIIa Factor (20,7%) while CD99, collagen IV, HMB45, CD34, NGFR, PAX2, EMA and Podoplanin D2-40 in a minor percentage of cases. In the mixed neurothekeomas, in addition to the markers already mentioned, also CD56 and SOX10 were found. s (1 medial rectus muscle and 1 paranasal ological and Cytological featu logically and cytologically Neurothekeo pes: cellular (65,7%) mixed (17,5%) and he subtypes presented as dome-shaped m ved the subcutaneous/submucosal plane ermas (41,5%). Skeletal muscle involvem argely restricted to the facial region (m al muscle). Bone plane involvement was lary and mandibular bones. The differen mors lied in histology and cytology: 1) C cterized by nests of spindle or epithelioid ma with the presence of sclerotic collag nucleated and osteoclastic giant cells. T ophilic cytoplasm with vesicular nuclei, m (ranged from 0 to 16 mitotic figures for of 4/mm2). Perineural invasion was un ion was completely absent. Three cases o described as atypical because of diff lar invasion, severe cellular atypia, and ). Discussion Neurothekeoma is a slow-growing benign tumor that interests mainly superficial tissues and has been studied since the 1980s. The first author who coined this term was Gallager [2] who, in a retrospective observational study on 53 patients, described a benign tumor of the dermis having a neural origin and a relationship to the Schwann sheath cells of peripheral nerves. The interest in the study of this type of lesion has grown over the years, and several authors dedicated to defining the origin, the etiopathogenesis and the clinical, histological, and immunohistochemical characteristics. Thus, the data obtained from our review were compared with the international literature on this topic. [10] First of all, Neurothekeomas tends to occur in younger age, around the second or third decades with a mild female predominance [10]. Our review, according to literature analysed, highlights a male to female ratio of 1:1,8 and a mean age of 26,4 years [11]. The tumors have been classified in three subtypes based on etiopathogenetic, cyto-histological, and immunohistochemical characteristics: cellular neurothekeomas with <10% of myxoid matrix, mixed-type with 10– <50% of myxoid matrix and myxoid neurothekeomas with >50% of myxoid matrix7. The term ‘‘cellular neurothekeoma’’ was first used by Rosati et al. in 1986 to distinguish it from the myxoid variant which was defined as “classic neurothekeoma” [12]. In terms of immunohistochemical features, our review showed that the neoplastic cells of classic neurothekeomas were mainly immunoreactive for S-100 Protein (86,9% of all cases), vimentin (47,8%), muscle-specific actin, a-smooth muscle actin and EMA (31,8%) and neuron-specific enolase (NSE), GFAP, CD10, CD34 and XIIa Factor (18,2%). The expression of these factors confirms the neuronal or perineuronal origin of the classical neurothekeoma. Moreover, the expression of the S-100 protein is connected with a high local recurrence. [3 - 7] In their observational study, Fetsch et al [22] documented 16 on 34 (47%) recurrent disease on follow-up in classic neurothekeomas S-100 protein positive locally excised. Considering a relatively high local recurrence rate, a complete local excision with a margin of healthy tissue should generally be considered an optimal treatment of the disease. Hence, as the analysis of the literature [15,23,24] revealed, classic neurothekeoma with S-100 protein positivity should be excised with safety margins to prevent local recurrences. This result justifies our aggressive surgical approach with safety margins of 1 cm of healthy tissue. Discussion The cell of origin of NSM is controversial: Fetsch et al [7] postulated that SMA and EMA positivity suggests some similarity to histiocytic cells and fibroblasts, while S100 protein and NSE to Schwann cells or other perineural cells. So cellular NTK can origin by histiocytic cells and fibroblasts, while classical NTK by Schwann cells and other perineural cells. Moreover, Misago et al [13] distinguished a histiocytic origin from a fibroblastic or nervous one, based on the expression of different genes: Cellular neurothekeoma expressed genes involved in macrophage differentiation, Cell migration, cytoskeleton organization, Fibroblast growth, tissue remodeling, ECM mineralization such as ADAM12, DPT, FAP, PDPL, MMP1 and TNFAIP6. Classic neurothekeoma expressed genes involved in neural crest development, myelin and axonal growth and neuronal adhesion such as SOX10, MPZ, NTM, SOX2, PMP2, NCAM1, MBP and SORBS1. KP-1 and PG-M1 expression is associated to histiocytic differentiation [13]. The neoplastic cells of cellular neurothekeomas were mainly immunoreactive for NKI/C3 (62,1%), CD68 (55,2%), Vimentin and a- smooth muscle actin (41,4%), CD10 (37,9%), PGP9.5 (34,5%), NSE and microphthalmia transcription factor (MITF) (31,1%) confirming the histiocytic and fibroblastic origin. [13,25] The analysis of literature [26,27] showed that in these cases the recurrence rate is low, so the chosen treatment is a local excision with a few millimeters of healthy tissue. Our review, according with the literature [14,15], highlights that the head and neck are the most affected sites (39,3%) with the face and the nose at the highest occurrences. Cases of the oral cavity are rare and mainly concentrated on the area of the lips (3,9%). The most common intraoral site is the tongue. Our case was a classic neurothekeoma with immunoreactivity to the s- 100 protein. For this reason, we opted for a surgical treatment of complete excision with margins of 1 cm of healthy tissue. Considering the position and the size of the tumor, this excision required a reconstruction with local flaps. Based on data for the selected case in the literature, the Bengt- Johanson’s step flap was considered the best option. [8,28] This technique has proved to be effective both in terms of functionality and aesthetics for our patient. Furthermore, the large excision with safety margins avoided relapses at the one-year follow-up. Several authors [7,14,15] reported that tumors were non-capsulated, located in dermal tissue with a subcutaneous involvement in 85% of the cases, and typically organized in multiple small nodules. Surgery and Follow-up In all cases, the tumors were treated by surgery. In particular, cellular neurothekeomas were treated by a simple excision, while classic and mixed neurothekeomas as well as the atypical cellular forms were treated by enucleoresection with healthy margins and, if necessary, reconstruction. The reported percentage of recurrence was 7,5% without cases of metastasized tumor. Main features of the three neurothekeomas types are summarized in Table 2. and low mitotic rates (mean mitotic rate of 3/mm2). 3) Mixed Types and rates Clinical features Histological features Cytological features Immunohistochemical features Treatment Cellular NTK (65,7%) -asymptomatic, solitary, slow- growing lesion -mean diameter of 1,5 cm -deep planes not involved -<10% of myxoid matrix - nests of spindle with the presence of sclerotic collagen fibers -eosinophilic cytoplasm - vesicular nuclei - mild atypia - mean mitotic rate of 4/mm2 In order of frequency: NKI/C3, CD68, Vimentin, a-smooth muscle actin, CD10, PGP9.5, NSE and microphthalmia transcription factor (MITF) Simply Excision Classic NTK (16,8%) -asymptomatic, solitary, slow- growing lesion -mean diameter of 1,5 cm -deep planes not involved >50% of myxoid matrix - bundles of stellate cells immersed in a myxoid stroma without collagen fibers -eosinophilic cytoplasm - vesicular nuclei - mild atypia - mean mitotic rate of 4/mm2 In order of frequency: S-100 Protein, vimentin, muscle- specific actin, a-smooth muscle actin and EMA, neuron-specific enolase (NSE), GFAP, CD10, CD34 and XIIa Factor Enucleoresection with healthy margin Mixed NTK (17,5%) -asymptomatic, solitary, slow- growing lesion -mean diameter of 1,5 cm -deep planes not involved ->10– <50% of myxoid matrix - features of both other subtypes, in particular both myxoid stroma and sclerotic collagen fibers. -eosinophilic cytoplasm - vesicular nuclei - mild atypia - mean mitotic rate of 4/mm2 Both the markers of other subtypes If S-100 +: Enucleoresection with healthy margin Table 2: Summarized features of the three neurothekeomas types Page 12 of 16 International Journal of Clinical Case Reports and Reviews International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. In terms of cytomorphology, in accordance with literature [21] cellular neurothekeomas were composed of spindle cells (28%), of epithelioid cells (14%), and of cells with variably epithelioid to spindled features (58%). Classic neurothekeomas were composed of spindle cells (54%), of stellate shaped cells (49%) and of epithelioid cells (7%). Surgery and Follow-up The tumor cells contained pale vacuolized eosinophilic cytoplasm and in 70% showed mild atypia in terms of abundant vesicular, ovoid or irregularly shaped nuclei with prominent nucleoli. Cellular neurothekeomas in 10% of cases showed giant cells. The mean mitotic rate was 4 per 10 high power fields (HPF) (range, 0 to 16) for cellular neurothekeomas and 3 per 10 HPF for classic neurothekeomas. Only 4% of total tumors showed perineural invasion, and 3% showed vascular invasion.[ 3 - 7] Discussion Our research confirms that the involvement of the deep planes is rare (3,4%). Mean diameter was 1,5 cm, 90% of lesions between 0,4 cm and 2 cm, but tumor size of 6 cm was described and defined as atypical [16-18]. Wilson et al [19] affirmed that it is characterized by large size of up to 6 cm, penetration into subcutaneous fat or muscle, diffusely infiltrating borders, vascular invasion, a high mitotic rate, and marked cytological pleomorphism. Based on our data, the most common subtype is Cellular Neurothekeoma (65,7%). All the three subtypes of tumors were associated with some sclerotic collagen that was most present in cellular neurothekeomas and least evident in the myxoid examples. Moderate or marked collagen deposition around individual tumor nodules was noted predominantly in cellular neurothekeomas. Osteoclast giant cells are also present, but they are generally sparse and do not appear to be neoplastic [7]. They are identified predominantly in cellular neurothekeomas. The myxoid stroma is more abundant in the classical subtype [20]. Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 References 22. Fetsch JF, Laskin WB, Miettinen M. (2005). Nerve sheath myxoma: a clinicopathologic and immunohistochemical analysis of 57 morphologically distinctive, S-100 protein- and GFAP- positive, myxoid peripheral nerve sheath tumors with a predilection for the extremities and a high local recurrence rate. Am J Surg Pathol. 29(12):1615-1624. 1. Harkin JC, Reed RJ. (1969). Tumors of the peripheral nervous system. Atlas of tumor pathology, 2nd series, fascicle 3. Washington DC: Armed Forces Institute of Pathology. 60-64. 1. Harkin JC, Reed RJ. (1969). Tumors of the peripheral nervous system. Atlas of tumor pathology, 2nd series, fascicle 3. Washington DC: Armed Forces Institute of Pathology. 60-64. 2. Gallager RL, Helwig EB. (1980). Neurothekeomaea benign cutaneous tumor of neural origin. Am J Clin Pathol. 74:759-764. 2. Gallager RL, Helwig EB. (1980). Neurothekeomaea benign cutaneous tumor of neural origin. Am J Clin Pathol. 74:759-764. 3. Argenyi ZB, LeBoit PE, Santa Cruz D, Swanson PE, Kutzner H. (1993). Nerve sheath myxoma (neurothekeoma) of the skin: light microscopic and immunohis- tochemical reappraisal of the cellular variant. J Cutan Pathol. 20:294-303. 23. Rodríguez-Uña I, Troyano-Rivas JA, González-García C, Chícharo-de-Freitas R, Ortiz-Zapata JJ, Ortega-Medina L, Toledano-Fernández N, García-Feijoo J. (2015). Orbital nerve seath myxoma with extraocular muscle involvement: a rare case. Semin Ophthalmol. 30(4):316-320. 4. Blumberg AK, Kay S, Adelaar RS. (1989). Nerve sheath myxoma of digital nerve. Cancer; 63:1215-1218. 24. Sánchez-Orgaz M, Grabowska A, Arbizu-Duralde A, Romero- Martín R, Patrón M, Diéguez E, García-Cabezas MA. (2011). Orbital nerve sheath myxoma: a case report. Ophthalmic Plast Reconstr Surg. 27(4):106-108. 5. Barnhill RL, Dickersin GR, Nickeleit V, Nickeleit V, Bhan AK, Muhlbauer JE, Phillips ME, Mihm MC Jr. (1991). Studies on the cellular origin of neurothekeoma: clinical, ligh microscopic, immunohistochemical, and ultrastructural observations. J Am Acad Dermatol. 25:80-88. g 25. Jaffer S, Ambrosini-Spaltro A, Mancini AM, Eusebi V, Rosai J. (2009). Neurothekeoma and plexiform fibrohistiocytic tumor: mere histologic resemblance or histogenetic relationship? Am J Surg Pathol. 33(6):905-913. 6. MentzelT. (1999). Cutaneous neural neoplasms-an update. Pathologe. 20:98-109. 7. Fetsch JF, Laskin WB, Hallman JR, Lupton GP, Miettinen M. (2007). Neurothekeoma: An Analysis of 178 Tumors with Detailed Immunohistochemical Data and Long-term Patient Follow-up Information. Am J Surg Pathol. 31:1103–1114. 26. See TRO, Stålhammar G, Grossniklaus HE. (2019). Neurothekeoma of the eye, conjunctiva, and periorbital adnexa: A report of two cases and brief review. Surv Ophthalmol. 64(6):852- 857. 8. Blomgren I, Blomqvist G, Lauritzen C, Lilja J, Peterson LE, Holmström H. (1988). Financial Disclosure Statement: The authors have no financial interest to declare regarding the content of this article. Financial Disclosure Statement: The authors have no financial interest to declare regarding the content of this article. literature. J Am Acad Dermatol. 50(1):129-134. 21. Hornick JL, Fletcher CD. (2007). Cellular neurothekeoma: detailed characterization in a series of 133 cases. Am J Surg Pathol. 31(3):329-340. Conclusion Our review highlights that neurothekeoma is a benign tumor that mainly afflicts young women and mainly occurs in the superficial planes of the head and neck. Among the three types, the cellular type is the most frequent, but the most aggressive is the classic one because the expression of the S-100 protein determines a high local recurrence. For this reason, a local excision treatment is sufficient for the cellular neurothekeoma while in the classic type with the presence of this protein, a wide local excision with healthy tissue margins is required. The treatment of our case demonstrates that, by following this guide, relapse can be avoided. Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 13 of 16 Copy rights@ Stefania Troise et.al. International Journal of Clinical Case Reports and Reviews Declarations Funding: Not applicable Conflicts of interest: The authors report no conflicts of interest Availability of data and material: Not applicable Code availability: Not applicable Ethics approval: Only the patient’s consent was requested. Consent to participate: Patient’s consent was obtained. Consent for publication: Patient’s consent was obtained. Financial Disclosure Statement: The authors have no financial 16. Park MC, Seung WB. (2016). Cellular Neurothekeoma of the Scalp in the Elderly. Brain Tumor Res Treat. 4(1):17-20. 17. Benbenisty KM, Andea A, Metcalf J, Cook J. (2006). Atypical cellular neurothekeoma treated with Mohs micrographic surgery. Dermatol Surg. 32(4):582-587. Conflicts of interest: The authors report no conflicts of interest Availability of data and material: Not applicable 18. Campanati A, Brandozzi G, Sisti S, Bernardini ML, Offidani AM. (2007). Atypical neurothekeoma: a new case and review of the literature. J Cutan Pathol. 34(5):435-437. Code availability: Not applicable Ethics approval: Only the patient’s consent was requested. 19. Wilson AD, Rigby H, Orlando A. (2008). Atypical cellular neurothekeoma-a diagnosis to be aware of. J Plast Reconstr Aesthet Surg. 61(2):186-188. Consent to participate: Patient’s consent was obtained. Consent for publication: Patient’s consent was obtained. 20. Papadopoulos EJ, Cohen PR, Hebert AA. (2004). Neurothekeoma: report of a case in an infant and review of the literature. J Am Acad Dermatol. 50(1):129-134. Financial Disclosure Statement: The authors have no financial interest to declare regarding the content of this article. References Pepine M, Flowers F, Ramos-Caro FA. (1992). Neurothekeoma in a 15-year-old boy: case report. Pediatr Dermatol. 9(3):272-274. 43. Müller CS, Tilgen W, Kutzner H, Pföhler C. (2009). Recurring mixed-type neurothekeoma of the face. Dermatoendocrinol. 1(4):220-222. 62. Husain S, Silvers DN, Halperin AJ, McNutt NS. (1994). Histologic spectrum of neurothekeoma and the value of immunoperoxidase staining for S-100 protein in distinguishing it from melanoma. Am J Dermatopathol. 16(5):496-503. 44. Vered M, Fridman E, Carpenter WM, Buchner A. (2011). Classic neurothekeoma (nerve sheath myxoma) and cellular neurothekeoma of the oral mucosa: immunohistochemical profiles. J Oral Pathol Med. 40(2):174-180. 63. Peñarrocha M, Bonet J, Minguez JM, Vera F. (2000). Nerve sheath myxoma (neurothekeoma) in the tongue of a newborn. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 90(1):74-77. p 45. Papalas JA, Proia AD, Hitchcock M, Gandhi P, Cummings TJ. (2010). Neurothekeoma palpebrae: A report of 3 cases. Am J Dermatopathol. 32(4):374-379. 64. Barrett AW, Suhr M. (2001). Cellular neurothekeoma of the oral mucosa. Oral Oncol. 37(8):660-664. 46. Sheth S, Li X, Binder S, Dry SM. (2011). Differential gene expression profiles of neurothekeomas and nerve sheath myxomas by microarray analysis. Mod Pathol. 24(3):343-354. 65. Laskin WB, Fetsch JF, Miettinen M. (2000). The neurothekeoma: immune-histochemical analysis distinguishes the true nerve sheath myxoma from its mimics. Hum Pathol. 31(10):1230-1241. 47. Pan HY, Tseng SH, Weng CC, Chen Y. (2014). Cellular neurothekeoma of the upper lip in an infant. Pediatr Neonatol. 55(1):71-74. 66. Schortinghuis J, Hille JJ, Singh S. (2001). Intraoral myxoid nerve sheath tumour. Oral Dis. 7(3):196-199. 67. Makino T, Utsunomiya T, Kamino Y, Kobayashi R, Fukumoto M, Yamamoto H, Nagura H. (2002). Nerve sheath myxoma of the tongue in a child. Int J Oral Maxillofac Surg.; 31(4):451-454. 48. Yamada S, Kitada S, Nabeshima A, Noguchi H, Sasaguri Y, Hisaoka M. (2013). Benign cutaneous plexiform hybrid tumor of perineurioma and cellular neurothekeoma arising from the nose. Diagn Pathol. 8:165. 68. Levin KA, Hayden R, Hanau CA, Galindo LM. (2002). Cytologic findings of myxoid neurothekeoma: case report based on fine- needle aspiration cytology, immunohistochemistry, and correlating histopathology. Diagn Cytopathol. 27(3):173-176. 49. Suarez A, High WA. (2014). Immunohistochemical analysis of KBA.62 in 18 neurothekeomas: a potential marker for differentiating neurothekeoma, but a marker that may lead to confusion with melanocytic tumors. J Cutan Pathol. 41(1):36-41. 69. Page RN, King R, Mihm MC Jr, Googe PB. (2004). Microphthalmia transcription factor and NKI/C3 expression in cellular neurothekeoma. References Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 14 of 16 International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. 35. Cohen NA, Samadi DS, Pawel BR, Kazahaya K. (2004). Cellular neurothekeoma of the maxilla. Ann Otol Rhinol Laryngol. 113(5):384-387. 54. Boukovalas S, Rogers H, Boroumand N, Cole EL. (2016). Cellular Neurothekeoma: A Rare Tumor with a Common Clinical Presentation. Plast Reconstr Surg Glob Open. 4(8):1006. ( ) 36. Ward JL, Prieto VG, Joseph A, Chevray P, Kronowitz S, Sturgis EM. (2005). Neurothekeoma. Otolaryngol Head Neck Surg. 132(1):86-89. g p 55. Bartake A, Palaskar SJ, Narang B, Kathuriya P. (2019). Nerve sheath myxoma of the oral cavity: a distinct entity. Br J Neurosurg. 33(2):213-214. 37. Mahalingam M, Alter JN, Bhawan J. (2006). Multiple cellular neurothekeomas-a case report and review on the role of immunohistochemistry as a histologic adjunct. J Cutan Pathol. 33(1):51-56. 56. Mora-Cantallops A, Won-Kim HR, Navarro-Cantero A, Martínez-San-Millán J, Sales-Sanz M. (2021). Nerve Sheath Myxoma Involving the Medial Rectus Muscle: A Case Report. Ophthalmic Plast Reconstr Surg. 37(2):60-62. 38. López-Cepeda LD, Navarrete-Franco G, Novales-Santacoloma J, Enriquez-Merino J. (2007). Scar-like lesion on dorsal nose (cellular neurothekeoma). Head Face Med. 3:39. 57. Massimo JA, Gasibe M, Massimo I, Damilano CP, De Matteo E, Fiorentino J. (2020). Neurothekeoma: Report of two cases in children and review of the literature. Pediatr Dermatol. 37(1):187- 189. 39. Koumanis DJ, Glickman LT. (2007). Facial neurothekeoma in a 10-year-old child. Can J Plast Surg. 15(3):175-177. 58. Aronson PJ, Fretzin DF, Potter BS. (1985). Neurothekeoma of Gallager and Helwig (dermal nerve sheath myxoma variant): report of a case with electron microscopic and immunohistochemical studies. J Cutan Pathol. 12(6):506-519. 40. Mathew S, Nandeesh BN, Vasu U, Michael SG. (2008). Neurothekeoma of the eyelid: a case report. Indian J Ophthalmol. 56(4):334-336. 41. Zedek DC, White WL, McCalmont TH. (2009). Desmoplastic cellular neurothekeoma: Clinicopathological analysis of twelve cases. J Cutan Pathol. 36(11):1185-1190. 59. Henmi A, Sato H, Wataya T, Inaniwa Y, Mori Y. (1986). Neurothekeoma. Report of a case with immunohistochemical and ultrastructural studies. Acta Pathol Jpn. 36(12):1911-1919. 42. Wartchow EP, Goin L, Schreiber J, Mierau GW, Terella A, Allen GC. (2009). Plexiform fibrohistiocytic tumor: ultrastructural studies may aid in discrimination from cellular neurothekeoma. Ultrastruct Pathol. 33(6):286-292. 60. Mason MR, Gnepp DR, Herbold DR. (1986). Nerve sheath myxoma (neurothekeoma): a case involving the lip. Oral Surg Oral Med Oral Pathol. 62(2):185-186. 61. References The Step Technique for the Reconstruction of Lower Lip Defects after Cancer Resection: A Follow-up Study of 165 Cases. Scandinavian Journal of Plastic and Reconstructive Surgery. 22(1):103-111. 27. Murphrey M, Huy Nguyen A, White KP, Krol A, Bernert R, Yarbrough K. (2020). Pediatric cellular neurothekeoma: Seven cases and systematic review of the literature. Pediatr Dermatol. 37(2):320-325. 28. Roldán JC, Schulte-Mattler W. (2016). Stein's Double Cross-Lip Flaps Combined with Johanson's Step Technique for Subtotal Lower Lip Reconstruction. Plast Reconstr Surg Glob Open. 4(2):615. 9. Moher D, Liberati A, Tetzlaff J, Altman DG, PRISMA Group. (2009). Preferred reporting items for systematic reviews and meta- analyses: the PRISMA statement. Ann Intern Med. 151:264-269. y 10. Kao EY, Kernig ML. (2021). Neurothekeoma. In: StatPearls. Treasure Island (FL): StatPearls Publishing. 29. Barnhill RL, Mihm MC Jr. (1990). Cellular neurothekeoma. A distinctive variant of neurothekeoma mimicking nevomelanocytic tumors. Am J Surg Pathol. 14(2):113-120. 11. Almeida TFA, Verli FD, Dos Santos CRR, Falci SGM, Almeida LY, Almeida LKY, Mesquita ATM, León JE. (2018). Multiple Desmoplastic Cellular Neurothekeomas in Child: Report of the First Oral Case and Review of the Literature. Head Neck Pathol. 12(1):75-81. tumors. Am J Surg Pathol. 14(2):113-120. 30. Suh YL, Song KY, Kim JM. (1992). Nerve sheath myxoma (neurothekeoma)--a case report. J Korean Med Sci. 7(1):85-89. 31. Tiffee JC, Pulitzer DR. (1996). Nerve sheath myxoma of the oral cavity: case report and review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 82(4):423-425. 12. Rosati LA, Fratamico FC, Eusebi V. (1986). Cellular neurothekeoma. Appl Pathol. 4:186-191. 32. Tomasini C, Aloi F, Pippione M. (1996). Cellular neurothekeoma. Dermatology. 192(2):160-163. 13. Misago N, Satoh T, Narisawa Y. (2004). Cellular neurothekeoma with histiocytic differentiation. J Cutan Pathol. 31(8):568-572. 33. Breuer T, Koester M, Weidenbecher M, Steininger H. (1999). Neurothekeoma, a rare tumour of the tongue. ORL J Otorhinolaryngol Relat Spec. 61(3):161-164. 14. Maktabi AMY, Al-Hussain H, Khoja H, Alkatan HM. (2019). Unusual Recurrent Lateral Canthus Mass in a 16-Year-Old Male Patient: Neurothekeoma. Case Rep Oncol. 12(3):693-697. 34. Wong BY, Hui Y, Lam KY, Wei WI. (2002). Neurothekeoma of the paranasal sinuses in a 3-year-old boy. Int J Pediatr Otorhinolaryngol. 62(1):69-73. 15. Safadi RA, Hellstein JW, Diab MM, Hammad HM. (2010). Nerve sheath myxoma (neurothekeoma) of the gingiva, a case report and review of the literature. Head Neck Pathol. 4(3):242-245. References Mod Pathol. 17(2):230-234. y 50. Stratton J, Billings SD. (2014). Cellular neurothekeoma: analysis of 37 cases emphasizing atypical histologic features. Mod Pathol. 27(5):701-710. 70. Kim HJ, Baek CH, Ko YH, Choi JY. (2006). Neurothekeoma of the tongue: CT, MR, and FDG PET imaging findings. AJNR Am J Neuroradiol.; 27(9):1823-1825. 51. Yun SJ, Park HS, Lee JB, Kim SJ, Lee SC, Won YH. (2014). Myxoid Cellular Neurothekeoma: A New Entity of S100- Negative, CD68-Positive Myxoid Neurothekeoma. Ann Dermatol. 26(4):510-513. 71. Nishioka M, Aguirre RL, Ishikawa A, Nagumo K, Wang LH, Okada N. (2009). Nerve sheath myxoma (neurothekeoma) arising in the oral cavity: histological and immunohistochemical features of 3 cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 107(5):28-33. 52. Wang GY, Nazarian RM, Zhao L, Hristov AC, Patel RM, Fullen DR, Chan MP. (2017). Protein gene product 9.5 (PGP9.5) expression in benign cutaneous mesenchymal, histiocytic, and melanocytic lesions: comparison with cellular neurothekeoma. Pathology. 49(1):44-49. 72. Plaza JA, Torres-Cabala C, Evans H, Diwan AH, Prieto VG. (2009). Immunohistochemical expression of S100A6 in cellular neurothekeoma: clinicopathologic and immunohistochemical analysis of 31 cases. Am J Dermatopathol. 31(5):419-422. 53. Tham AC, Chilagondanahalli NL, Bundele MM, Kanagalingam J. (2016). Oral Neurothekeoma of the Right Buccal Mucosa. Case Rep Otolaryngol. 4709753. 73. García-Gutiérrez M, Toussaint-Caire S, González-Sánchez P, Ortiz-Hidalgo C. (2010). Multiple desmoplastic cellular Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 15 of 16 International Journal of Clinical Case Reports and Reviews Copy rights@ Stefania Troise et.al. neurothekeomas localized to the face of a 16-year-old boy. Am J Dermatopathol. 32(8):841-845. 79. Bashline B, Morrison M, Cha HC, Ramirez J. (2014). A Case of a Cellular Neurothekeoma Presenting with Headaches and Review of the Literature. Dermatopathology (Basel). 1(1):29-34. 74. Kah TA, Yong KC, Annuar FH. (2011). Neurothekeoma palpebrae in association with multiple superficial angiomyxomas: Tegumental Angiomyxoma-Neurothekeoma syndrome (TAN syndrome). Clin Pract. 1(3):67. p gy ( ) ( ) 80. Fried I, Sitthinamsuwan P, Muangsomboon S, Kaddu S, Cerroni L, McCalmont TH. (2014). SOX-10 and MiTF expression in cellular and 'mixed' neurothekeoma. J Cutan Pathol. 41(8):640- 645. 75. Fox MD, Billings SD, Gleason BC, Moore J, Thomas AB, Shea CR, Victor TA, Cibull TL. (2012). Expression of MiTF may be helpful in differentiating cellular neurothekeoma from plexiform fibrohistiocytic tumor (histiocytoid predominant) in a partial biopsy specimen. Am J Dermatopathol. 34(2):157-160. 81. Navarrete-Dechent C, Curi-Tuma M, Marín C, González S, Sandoval-Osses M. References Cellular neurothekeoma: case report and its (un) relation with nerve sheath myxoma. An Bras Dermatol. 90(3(S1)):156-159. 82. Bhat A, Narasimha A, C V, Vk S. (2015). Nerve sheath myxoma: report of a rare case. J Clin Diagn Res. 9(4):7-9. 76. Emami N, Zawawi F, Ywakim R, Nahal A, Daniel SJ. (2013). Oral cellular neurothekeoma. Case Rep Otolaryngol. 2013:935435. 83. Gray ME, Palileo CM, Sheridan RM. (2016). Cellular neurothekeoma of the eyelid in a 6-year-old boy. J AAPOS. 20(4):374-376. 77. Requena L, Sitthinamsuwan P, Fried I, Kaddu S, Schirren CG, Schärer L, Hantschke M, Cerroni L, McCalmont TH, Kutzner H. (2013). A benign cutaneous plexiform hybrid tumor of perineurioma and cellular neurothekeoma. Am J Surg Pathol. 37(6):845-852. 84. Frydrych AM, Firth NA. (2017). Oral nerve sheath myxoma: a rare and unusual intraoral neoplasm. Clin Case Rep. 6(2):302-305. 85. Cavicchini S, Guanziroli E, Del Gobbo A, Scaparro M, Gianotti R. (2018). Neurothekeoma, a hard to diagnose neoplasm among red nodules. Australas J Dermatol. 59(4):280-282. 78. Rozza-de-Menezes RE, Andrade RM, Israel MS, Gonçalves Cunha KS. (2013). Intraoral nerve sheath myxoma: case report and systematic review of the literature. Head Neck. 35(12):397- 404. 86. Gallo G, Kutzner H, Mentzel T, Cesinaro AM. (2019). Cellular neurothekeoma: Report of two cases with unusual immunohistochemical features. J Cutan Pathol. 46(1):80-83. Ready to submit your research? Choose Auctores and benefit from: ➢ fast, convenient online submission ➢ rigorous peer review by experienced research in your field ➢ rapid publication on acceptance ➢ authors retain copyrights ➢ unique DOI for all articles ➢ immediate, unrestricted online access At Auctores, research is always in progress. Learn more https://auctoresonline.org/journals/international-journal-of- clinical-case-reports-and-reviews- This work is licensed under Creative Commons Attribution 4.0 License To Submit Your Article Click Here: Submit Manuscript To Submit Your Article Click Here: DOI: 10.31579/2690-4861/212 Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Auctores Publishing LLC – Volume 11(1)-212 www.auctoresonline.org ISSN: 2690-4861 Page 16 of 16
https://openalex.org/W2070709624	https://www.redalyc.org/pdf/3074/307428857005.pdf	Portuguese	null	Lógica luciferina: argumentação em Paradise Lost, de John Milton	Acta Scientiarum. Language and Culture	2,013	cc-by	10,615	Introdução p. 379) — que informará seus outros dois poemas bíblicos de maior porte, Samson Agonistes e Paradise regain’d. De fato, Paradise Lost é pouco bélico; há apenas duas passagens de batalha, sendo uma delas frustrada. A primeira é o momento em que Satã e Morte se preparam para um embate frente a frente que não ocorre, já que Pecado lhes informa dos laços de sangue que os impedem de duelar (II. 666-734, p. 248-249). A segunda é a narração pelo anjo Rafael da batalha travada nos Céus entre as forças de Lúcifer e os anjos fiéis a Deus (VI. 59-866, p. 327- 344); ainda aqui, a maravilha e a ironia derrotam a crua descrição de embates, à moda da Ilíada. A invenção do canhão por parte de Lúcifer (VI. 470- 491, p. 334-335), o levantamento das montanhas por parte dos anjos (VI. 633-669, p. 338-339) e a luminosa glória do Filho (VI. 746-772, p. 341-342) têm maior espaço, e exercem maior fascínio. O presente artigo apresenta um estudo argumentativo de uma passagem crucial do épico Paradise Lost, do poeta inglês John Milton. Para tal, conta com o arcabouço teórico da Lógica Pragmática. O presente artigo apresenta um estudo argumentativo de uma passagem crucial do épico Paradise Lost, do poeta inglês John Milton. Para tal, conta com o arcabouço teórico da Lógica Pragmática. O presente artigo apresenta um estudo argumentativo de uma passagem crucial do épico Paradise Lost, do poeta inglês John Milton. Para tal, conta com o arcabouço teórico da Lógica Pragmática. O primeiro questionamento que essa proposta poderia suscitar é o de sua relevância. Por que empreender um estudo argumentativo de um poema épico? Quais benefícios resultariam de tal estudo? A resposta mais simples (e, espero, também a mais eficaz) é a de que minha curiosidade acerca das práticas argumentativas das personagens do poema é oriunda de sua própria estrutura. Como aponta Milton 1, as guerras e os feitos heroicos de cavaleiros foram sempre a matéria épica por excelência (IX. 28-40, p. 3792); Milton se declara avesso e inepto a semelhantes temas (IX. 40-41, p. 379), preferindo talvez “[...] the better fortitude / Of Patience and Heroic Martyrdom / Unsung” (“[...] a superior fortaleza da paciência ou do martírio heroico, ainda por ninguém cantados”, IX. 3Cito alguns exemplos: no Inferno, os anjos rebeldes se reúnem em concílio e deliberam acerca do melhor modo de enfrentar o Altíssimo, se pela guerra ou pelo uso da ardis (II. 01-416, p. 232-236); Deus justifica que o homem não cairá por Sua escolha, mas por vontade e demérito próprios (III. 80-134, p. 260-261); Satã, ao ver a beleza do novo mundo, delibera se não seria melhor se reconciliar com Deus (IV. 32-113, p. 278-280); quando Deus declara a supremacia do Filho, Lúcifer reúne os anjos sob sua liderança e os convence a se rebelar, no que é interpelado pelo anjo Abdiel (V. 772-907, p. 320-323). 1Milton alude à própria cegueira e à insônia (III. 22-55, p. 258-259; IX. 20-24, p. 378-379; a respeito da insônia, ver a edição de Milton e Teskey [2005, p. 57, nota 38.]), havendo, portanto, forte identificação entre o autor e o ‘eu’ que narra e se manifesta e o poeta. Podemos, portanto, conceber ‘Milton’ como uma personagem autobiográfica criada por John Milton para narrar o poema. 2Em todas as referências a Paradise Lost, ofereço inicialmente o livro (em numerais romanos) e os versos (em arábicos); a seguir, o número de página na edição de Hughes (1957). Fabiano Seixas Fernandes sidade Federal do Ceará, Av. da Universidade, 2853, 60020-181, Benfica, Fortaleza, Ceará, Brazil. E-mail: fbnfnds@gmail.com Universidade Federal do Ceará, Av. da Universidade, 2853, 60020-181, Benfica, Fortaleza, Ceará, Brazil. E-m RESUMO. O presente artigo realiza um estudo dos componentes argumentativos de uma passagem do épico Paradise Lost, do poeta inglês John Milton. Objetiva, através disso, compreender alguns aspectos da composição do protagonista do épico, Lúcifer/Satã. Foi analisado o diálogo entre Satã e Eva, no qual aquele a convence a comer o fruto da árvore do bem e do mal. Para a análise da situação argumentativa, foi empregada uma versão adaptada do instrumental oferecido por Douglas Walton (2008). Palavras-chave: John Milton, Paraíso perdido, argumentação. Palavras-chave: John Milton, Paraíso perdido, argumentação. Lucifer’s logic: argumentation in John Milton’s Paradise Lost ABSTRACT. The present article aims at undertaking an analysis of the argumentative component of a passage from John Milton’s Paradise Lost, which is intended to shed light on some compositional aspects of the epic’s protagonist, Lucifer/Satan. The passage selected for analysis is the one in which Satan convinces Eve to eat of the fruit of the Tree of Knowledge of Good and Evil. Its analysis was undertaken using an adapted version of the theoretical framework proposed by Douglas Walton (2008). Keywords: John Milton, Paradise Lost, argumentation. Acta Scientiarum. Language and Culture ISSN: 1983-4675 eduem@uem.br Universidade Estadual de Maringá Brasil Seixas Fernandes, Fabiano Lógica luciferina: argumentação em Paradise Lost, de John Milton Acta Scientiarum. Language and Culture, vol. 35, núm. 3, julio-septiembre, 2013, pp. 233-244 Universidade Estadual de Maringá .jpg, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=307428857005 Acta Scientiarum. Language and Culture ISSN: 1983-4675 eduem@uem.br Universidade Estadual de Maringá Brasil Seixas Fernandes, Fabiano Lógica luciferina: argumentação em Paradise Lost, de John Milton Acta Scientiarum. Language and Culture, vol. 35, núm. 3, julio-septiembre, 2013, pp. 233-244 Universidade Estadual de Maringá .jpg, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=307428857005 Acta Scientiarum. Language and Culture ISSN: 1983-4675 eduem@uem.br Universidade Estadual de Maringá Brasil Acta Scientiarum. Language and Culture ISSN: 1983-4675 eduem@uem.br Universidade Estadual de Maringá Brasil Seixas Fernandes, Fabiano Lógica luciferina: argumentação em Paradise Lost, de John Milton Acta Scientiarum. Language and Culture, vol. 35, núm. 3, julio-septiembre, 2013, pp. 233-244 Universidade Estadual de Maringá .jpg, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=307428857005 Como citar este artigo Número completo Mais artigos Home da revista no Redalyc Sistema de Informação Científica Rede de Revistas Científicas da América Latina, Caribe , Espanha e Portugal Projeto acadêmico sem fins lucrativos desenvolvido no âmbito da iniciativa Acesso Aberto Sistema de Informação Científica Rede de Revistas Científicas da América Latina, Caribe , Espanha e Portugal Projeto acadêmico sem fins lucrativos desenvolvido no âmbito da iniciativa Acesso Aberto http://www.uem.br/acta ISSN printed: 1983-4675 ISSN on-line: 1983-4683 Acta Scientiarum Doi: 10.4025/actascilangcult.v35i3.15467 http://www.uem.br/act ISSN printed: 1983-46 ISSN on-line: 1983-46 Acta Scientiarum Doi: 10.4025/actascila http://www.uem.br/acta ISSN printed: 1983-4675 ISSN on-line: 1983-4683 Doi: 10.4025/actascilangcult.v35i3.15467 Keywords: John Milton, Paradise Lost, argumentation. Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture Argumento e argumentação Entendo o conceito de ‘argumento’ como o entende a lógica: um conjunto de asserções inter- relacionadas de tal modo que uma delas (a conclusão) é tornada necessariamente verdadeira caso também o sejam as antecedentes (as premissas). ‘Argumentar’, portanto, é provar a verdade de uma asserção pela montagem de ao menos um argumento. As asserções que compõem um argumento podem ser ‘verdadeiras’ ou ‘falsas’; o argumento a que compõem pode ser ‘válido’ (ou seja, estruturalmente bem-formado, de modo a haver transferência do valor de verdade das premissas à conclusão) ou ‘inválido’5. p p ) Além da abundância de passagens dialogadas e do marcado prazer na conversa, o épico ainda conta com uma ênfase na razão. A palavra reason aparece 38 vezes ao longo do poema, aparecendo ainda outras vezes em formas derivadas (reasons, reasoned, reasoning[s] e reasonless)4; seus usos marcam o desejo das personagens em fazer o que é racional, e em justificar racionalmente os próprios atos. Para Milton, é de suma importância que suas personagens tenham ciência de que agem como agem porque seu poder de raciocínio e sua liberdade para a ação as inclinam a tal, tornando-se imprescindível lhes dar ampla voz e espaço para chegarem a suas prescindíveis e fatais conclusões. Isso reforça a importância de que haja, ao longo do poema, diálogos (ou solilóquios) que forneçam arrazoados das posturas e condutas das personagens. Cabe notar que o termo ‘verdade’ pode soar um pouco forte, e inúmeras vezes inadequado: nem sempre temos como verificar de modo satisfatório o valor de verdade de nossas asserções (pense-se, por exemplo, em quando falamos do futuro, ou quando raciocinamos hipoteticamente). Melhor seria modalizá-lo e dizer que um argumento válido pode ser ‘verdadeiro’, ‘provável’ ou ‘possível’. Quanto maior o grau de certeza que carrega o argumento — ou seja: quanto mais fortemente se aproximar de um raciocínio necessário, não contingente — , tanto mais força argumentativa terá. Toda essa ênfase no debate e na racionalidade não deveria, contudo, convencer-nos imediatamente de que as personagens estão sendo racionais: o próprio Milton alertará seus leitores da falsidade de “[...] words clothed in reason’s garb” (“[...] palavras vestidas com os atavios da razão”, II. 226, p. 237). Como leitores, devemos suspeitar não só da racionalidade pura das personagens, mas da própria capacidade do poeta (que, afinal, tem seu parti pris) em reconhecê-la. 6Talvez os termos ‘tese’ e ‘conclusão’ possam causar confusão, já que se trata, em um ou outro caso, de asserções que desejamos estabelecer como verdadeiras, prováveis ou possíveis. Emprego ‘conclusão’ para falar especificamente da asserção final de um argumento — aquela cujo valor de verdade deve ser garantido por uma estrutura que a une às premissas, a fim de garantir a transferência do valor de verdade destas. Embora as teses sejam, sem dúvida, conclusões de argumentos, são mormente defendidas por uma cadeia argumentativa. A ‘verdade’ da tese defendida por Satã (abaixo explicitada) não é fruto de um único argumento, mas de um conjunto progressivo e inter- relacionado de argumentos que vai estabelecendo asserções progressivamente, até que essa conclusão em particular possa ser provada e encerrar o debate. Assim, uma tese é, digamos, uma conclusão-alvo, fruto de uma cadeia argumentativa, e não de um argumento isolado. 4Segundo a Concordance de John Bradshaw (1894) para a obra poética de Milton. Introdução 31-33, Às batalhas, Milton parece preferir o diálogo: parte substancial dos 12 cantos de sua sacra epopeia é despendida em conversas; destas, inúmeras tomam a forma de argumentações3. O próprio prazer do ato de conversar — ou, mais especificamente, deliberar Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture 234 Fernandes por meio do diálogo aberto — é enfatizado tanto pelos anjos rebeldes (Belial defende que o Inferno, que permite aos anjos se reunirem em conselho, não é, por essa mesma razão, tão infernal) quanto por Adão (que busca subterfúgios para estender sua conversa com o anjo Rafael) e Eva (que prefere ouvir em conluio íntimo do esposo o que dissera o anjo, para que o diálogo aumente o prazer da intimidade) (respectivamente: II. 164-165, p. 236; VIII. 206-216, p. 367-368; VIII. 40-57, p. 363-364). de Walton, embora não suas traduções diretas). Esse arcabouço será usado para comentar uma cena crucial de Paradise Lost, cujo cerne é uma situação argumentativa. 5O exposto até aqui é standard, e qualquer manual de lógica provavelmente trará exposição que cubra todos esses pontos; posso, contudo, referendar o leitor mais especificamente a Margutti Pinto (2001). Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture Lógica luciferina ‘convencimento’: um arguidor que defende a tese A deseja que seu interlocutor também creia que A é verdadeira; seus esforços serão no sentido de montar uma cadeia argumentativa que demonstre ser A necessária ou altamente provável. Para tal, terá de interferir nas crenças do seu interlocutor (WALTON, 2008, p. 13). Walton chama esse sistema de crenças de commitment-set (baseando-se em HAMBLIN, 1970); poderíamos, em situações de debate estritamente abstrato, também chamá-lo ‘arcabouço teórico’. Práticas arrazoadas (ou teórico-práticas). Intermediárias; visam ao convencimento, porém com vistas à ação. Não podem ser consideradas estritamente teóricas, pois há certos tipos de argumentos e certas práticas argumentativas que seriam absolutamente incabíveis no caso de uma discussão estritamente teórica, que podem ser bastante pertinentes no caso de uma discussão acerca de como agir. Volitivas. Menos racionais, chegando à irracionalidade; visam ao alcance de objetivos pessoais, sendo a (aparência de) verdade meramente instrumental. A tese em si pode ser um mero instrumento para a satisfação dos desejos de seu proponente, que não lhe deve sequer anuência. É importante notar que a diferença entre os tipos de tese não está na estrutura interna dos argumentos que as têm como conclusões, tampouco na estrutura da cadeia argumentativa, mas na ‘atitude’ do arguidor. No caso de teses teóricas e práticas arrazoadas, os participantes abrem mão de seus desejos e interesses pessoais em prol das teses melhor estabelecidas, ou do plano de ação considerado mais racional. No caso de teses volitivas, estão inteiramente a serviço da vontade de quem as defende. Isso pode interferir no modo como serão defendidas. Volitivas. Menos racionais, chegando à irracionalidade; visam ao alcance de objetivos pessoais, sendo a (aparência de) verdade meramente instrumental. A tese em si pode ser um mero instrumento para a satisfação dos desejos de seu proponente, que não lhe deve sequer anuência. ç Inicialmente, cada interlocutor visa a fazer o outro abandonar a própria tese em favor (de uma) da(s) contrária(s); pode também, como objetivo secundário ou intermediário, visar a alterar o arcabouço teórico do outro. Cada interlocutor terá, assim, de recorrer a modos válidos de interferência, que atuem eficazmente sobre as crenças do outro. O único modo de fazer isso é empregar, na montagem de nossos argumentos, premissas que façam parte do arcabouço teórico de nosso interlocutor (WALTON, 2008, p. 5): é necessário demonstrar- lhe que, se crê em A, então deverá necessariamente crer também em B. Argumento e argumentação Assim, parece-me que empreender um estudo que avalie o grau de anuência a práticas puramente racionais em situações argumentativas dentro do poema seria uma valiosa forma de avaliar sua construção, suas ênfases filosóficas (como a do livre-arbítrio) e mesmo a constituição psicológica de seu arguidor: Satã. O ato de argumentar implica a existência de ao menos uma ‘tese’: uma asserção cujo valor de verdade é controverso, e que, portanto, precisa ser estabelecido por meio de investigação 6 . Essa controvérsia pode ser uma dúvida pessoal ou coletiva: mesmo quando é pessoal, estabelecem-se dentro do investigador ao menos dois lados, e, portanto, suas ideias vão entrar em contraste. Assim, toda argumentação é dialógica, pressupondo um estado inicial de dúvida ou desacordo; também, toda argumentação se voltará (idealmente) ao O artigo está organizado como segue: inicialmente, explicito brevemente o que entendo por ‘argumento’ e ‘argumentar’, bem como quais são e como se estruturam as situações argumentativas. Neste ponto, baseio-me em Walton (2008), embora discorde dele em alguns pontos e opte por reorganizar e complementar sua descrição do reasoned dialogue (também assinalo que os termos abaixo empregados são muitas vezes equivalentes aos O artigo está organizado como segue: inicialmente, explicito brevemente o que entendo por ‘argumento’ e ‘argumentar’, bem como quais são e como se estruturam as situações argumentativas. Neste ponto, baseio-me em Walton (2008), embora discorde dele em alguns pontos e opte por reorganizar e complementar sua descrição do reasoned dialogue (também assinalo que os termos abaixo empregados são muitas vezes equivalentes aos Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture 235 Lógica luciferina Ser ‘racional’, portanto, em uma situação argumentativa, é tentar angariar convencimento através de estratégias eficazes. Idealmente, argumentar é uma atividade estritamente teórica, que visa ao acordo de crenças. É importante notar que a diferença entre os tipos de tese não está na estrutura interna dos argumentos que as têm como conclusões, tampouco na estrutura da cadeia argumentativa, mas na ‘atitude’ do arguidor. No caso de teses teóricas e práticas arrazoadas, os participantes abrem mão de seus desejos e interesses pessoais em prol das teses melhor estabelecidas, ou do plano de ação considerado mais racional. No caso de teses volitivas, estão inteiramente a serviço da vontade de quem as defende. Isso pode interferir no modo como serão defendidas. É importante notar que a diferença entre os tipos de tese não está na estrutura interna dos argumentos que as têm como conclusões, tampouco na estrutura da cadeia argumentativa, mas na ‘atitude’ do arguidor. No caso de teses teóricas e práticas arrazoadas, os participantes abrem mão de seus desejos e interesses pessoais em prol das teses melhor estabelecidas, ou do plano de ação considerado mais racional. No caso de teses volitivas, estão inteiramente a serviço da vontade de quem as defende. Isso pode interferir no modo como serão defendidas. Também podem ser dois os tipos de objetivos: Acordo. Os participantes chegam a um consenso, saindo ambos satisfeitos, ao menos em certa medida. Note-se que, dada a natureza de certas práticas investigativas, o acordo pode ser interno a um investigador solitário. Acta Scientiarum. Language and Culture Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Tipologia das situações argumentativas Investigação. Envolve exame de evidências para se chegar a uma conclusão racional. Seu objetivo é esclarecer algum ponto controverso pela coleta e contraste de dados. As teses não precisam ser estritamente teóricas, tampouco estarem estabelecidas de antemão. Pode ser levada a termo por um único participante; quando é desenvolvida coletivamente, os participantes não se opõem a si, mas cooperam para chegar à mais racional conclusão possível, pautada nos mais sólidos e incontroversos dados que conseguirem coletar. Investigação. Envolve exame de evidências para se chegar a uma conclusão racional. Seu objetivo é esclarecer algum ponto controverso pela coleta e contraste de dados. As teses não precisam ser estritamente teóricas, tampouco estarem estabelecidas de antemão. Pode ser levada a termo por um único participante; quando é desenvolvida coletivamente, os participantes não se opõem a si, mas cooperam para chegar à mais racional conclusão possível, pautada nos mais sólidos e incontroversos dados que conseguirem coletar. Como foi dito acima, nem todas as situações argumentativas são teóricas e, portanto, nem todas colocam a verdade e seus meios legítimos de busca em primeiro plano. Passo a descrever as possíveis situações argumentativas; a relação está ordenada progressivamente, das mais marcadamente racionais até as mais marcadamente irracionais. As duas primeiras podem ser consideradas modelares7. Diálogo filosófico. Visa ao acordo por convencimento entre os debatedores, através do contraste entre teses teóricas, contrárias e mutuamente excludentes. Não tem consequências práticas. As teses podem assumir duas formas: Deliberação. Objetiva o acordo entre os interlocutores, que desejam ou necessitam decidir como agir em dada situação. As teses defendidas, em princípio, são práticas arrazoadas, mas certas teses volitivas podem ser relevantes. Pode ser levada a termo por um único participante, que oscile entre conclusões distintas e as contraste. Se as teses volitivas assumirem força em demasia, pode degenerar (aberta ou secretamente) em negociação. (1) (A w A); (2) ((A  B) w (B  A))8. No primeiro caso, debate-se o valor de verdade de uma única tese; no segundo, há (ao menos) duas teses, de modo que a verdade de (cada) uma implica a falsidade da(s) outra(s). Apesar do termo ‘diálogo’, pode também ser levado a termo por um único participante, que oscila entre opiniões contrárias ou tenta antever argumentos contrários à sua posição. O objetivo é, como vimos, atuar sobre o arcabouço teórico do interlocutor. Neste processo, também as teses originais podem ser reformuladas. Negociação. 7A tipologia das situações é derivada de Walton (2008, p. 3-8). As explicações, contudo, foram adaptadas para dar conta das categorias propostas na seção 1.2. Walton considera apenas o que chama de reasoned dialogue (e que optei por chamar de ‘diálogo filosófico’) como modelar. Creio ser a investigação mais racional, justamente pela ausência absoluta de compromisso prévio com qualquer tese; é guiada pela curiosidade, não pela convicção. Não obstante, dado o caráter necessariamente dialógico de toda inquietação racional, a ênfase na contrariedade de teses presente no diálogo filosófico torna-o modelo igualmente indispensável de situação argumentativa. 8Leia-se: 1) Ou é o caso que A, ou não é o caso que A; 2) ou é o caso que A e, portanto, não é o caso que B, ou é o caso que B e, portanto, não é o caso que A. Componentes das situações argumentativas Vimos, até aqui, que, em um debate, há (1) ‘interlocutores’ sustentando argumentativamente (2) ‘teses’ em relação às quais têm um (3) ‘objetivo’, alcançado através de certos (4) ‘meios’. Cada um desses itens pode se manifestar de mais de um modo. Vejamos. Vitória. Cada participante deseja prevalecer sobre o oponente. Aqui, há necessidade de mais de um participante, mas não exatamente de uma tese. O uso da razão pode ser meramente instrumental, não estando vetado, mas não sendo preferencial sobre outros métodos. Enquanto o acordo aceita fazer concessões (nada impede que ambos os interlocutores saiam satisfeitos), no caso da vitória, um dos interlocutores deve sair em desvantagem (derrota em competição, perda de algo que lhe seja caro, humilhação etc.). Quanto ao número de interlocutores, podemos ter as seguintes situações: Solilóquio. Um único investigador pode oscilar entre duas ou mais teses, contrastando-as e as avaliando mentalmente. Pode, também, simular diálogo com um ouvinte/leitor ideal, prevendo seus contra-argumentos para defender a própria tese. Finalmente, esses objetivos podem ser alcançados através dos seguintes meios: Diálogo. Quando há dois ou mais interlocutores anuindo a teses mutuamente excludentes, ou atribuindo valores de verdade distintos para a mesma tese. Convencimento. Os participantes buscam interferir no sistema de crenças uns dos outros, gerando crenças novas ou alterando os valores de verdade de crenças antigas. Ao final, devem concordar quanto ao valor de verdade da(s) tese(s) original(ais); estão dispostos, portanto, a expor francamente seus sistemas de crenças, oferecendo-os ao escrutínio dos demais debatedores e aceitando modificá-los caso sejam considerados inconsistentes ou errôneos. Esse convencimento pode satisfazer Em segundo lugar, as teses defendidas podem ser de três tipos: Teóricas. Mais racionais; visam ao estabelecimento da verdade sem vistas à ação. A situação de debate na qual as teses são teóricas deve ser considerada parâmetro de racionalidade, embora de modo algum a única, e talvez sequer a mais comum. Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 236 Fernandes O acordo final pode assumir quatro formas: 1) Idealmente, descobre-se a ‘verdade’ acerca do ponto controverso; 2) Já que não é possível termos certeza acerca de havermos alcançado ou não a verdade no tangente a assuntos teóricos, haveria ‘concórdia plena’: ambos passariam a sustentar a mesma tese e o mesmo arcabouço teórico, sendo este mais firmemente constituído e mais resistente a ataques que os sistemas anteriores a que anuíam; 3) Também pode-se aceitar como situação final que os arcabouços teóricos e/ou as teses se modifiquem para se tornarem não iguais, mas mais próximos; 4) Finalmente, pode-se chegar a um ‘impasse’: a impossibilidade de se alcançar uma tese conclusiva acerca do assunto debatido. Mesmo, porém, quando há impasse, pela interferência mesma nos arcabouços teóricos, há um fortalecimento destes, que devem se tornar mais resistentes a ataques, embora não se tornem imbatíveis. curiosidade puramente teórica, ou ser a base de ações futuras arrazoadas. Só é possível acordo por convencimento; não é, contudo, refratário à vitória. curiosidade puramente teórica, ou ser a base de ações futuras arrazoadas. Só é possível acordo por convencimento; não é, contudo, refratário à vitória. Anuência forçada. Simulação de convencimento por um dos participantes, que se autossilencia e finge concordar com o oponente. É específico da vitória, mas neste caso o participante derrotado ou desistente admite apenas para si a derrota ou a impossibilidade de prosseguir a discussão. Anuência forçada. Simulação de convencimento por um dos participantes, que se autossilencia e finge concordar com o oponente. É específico da vitória, mas neste caso o participante derrotado ou desistente admite apenas para si a derrota ou a impossibilidade de prosseguir a discussão. Silenciamento. Interrupção do diálogo, na qual um dos interlocutores consegue coagir o outro a parar de se manifestar. É específico da vitória, exercendo violência sobre o interlocutor derrotado. Silenciamento. Interrupção do diálogo, na qual um dos interlocutores consegue coagir o outro a parar de se manifestar. É específico da vitória, exercendo violência sobre o interlocutor derrotado. Coação. Um dos participantes obriga outro a executar ações contrárias a seus desejos ou crenças. É específico da vitória, exercendo violência sobre o interlocutor derrotado. Ocorre em debates versando sobre como agir em dada situação. Coação. Um dos participantes obriga outro a executar ações contrárias a seus desejos ou crenças. É específico da vitória, exercendo violência sobre o interlocutor derrotado. Ocorre em debates versando sobre como agir em dada situação. Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture Tipologia das situações argumentativas Objetiva o acordo entre negociantes que desejam o avanço de seus interesses e objetivos. Como se trata de garantir a satisfação de desejos, e não de estabelecer a verdade, estratégias não racionais podem ser relevantes. Não é refratária à vitória de apenas um dos negociantes. Embora o convencimento possa ser o meio preferível de se alcançar o objetivo, a possibilidade de satisfação unilateral pode convidar os demais meios a tomarem parte. Debate. Visa à vitória sobre ao menos um oponente. Cada participante defende uma tese, mas seu objetivo não é exatamente o convencimento teórico dos oponentes; basta silenciá-los, de modo a fazer com que a própria tese pareça a mais solidamente estabelecida. Normalmente, há árbitros que deliberam acerca da pertinência das colocações Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture 237 Lógica luciferina dos oponentes e de sua conformidade a um conjunto preestabelecido de regras. Desse modo, a vitória só é legítima se dada dentro de limites acordados de antemão. Não é, porém, absolutamente necessário que a vitória se dê pelo uso de estratégias puramente racionais. Embora a argumentação racional possa ser considerada a estratégia principal para se alcançar a vitória, a mera aparência de verdade ou o silenciamento dos oponentes podem ser igualmente eficazes. O debate poderia ser considerado o equivalente linguístico do ‘combate’ — o enfrentamento regrado e físico entre oponentes. dos oponentes e de sua conformidade a um conjunto preestabelecido de regras. Desse modo, a vitória só é legítima se dada dentro de limites acordados de antemão. Não é, porém, absolutamente necessário que a vitória se dê pelo uso de estratégias puramente racionais. Embora a argumentação racional possa ser considerada a estratégia principal para se alcançar a vitória, a mera aparência de verdade ou o silenciamento dos oponentes podem ser igualmente eficazes. O debate poderia ser considerado o equivalente linguístico do ‘combate’ — o enfrentamento regrado e físico entre oponentes. Honestidade: parece natural que, em uma situação de busca conjunta pela verdade, os participantes não omitam ou distorçam dados. Devem, portanto: definir claramente de que tipo de situação argumentativa se trata; definir claramente as teses defendidas; ser sinceros e explícitos em relação a seus interesses e seus sistemas de crenças. Por outro lado, não devem alterar secretamente o tipo de situação argumentativa (converter um diálogo filosófico acerca de teologia em uma negociação acerca de cargos episcopais, por exemplo). Tipologia das situações argumentativas Cooperação: a sinceridade é a mais importante prova de cooperação entre os participantes, mas há outras, dentre as quais: oferecer provas, definições e esclarecimentos sempre que solicitado; perguntar e responder de modo relevante; evitar desvios, tais como defender a tese errada, afastar-se do foco da discussão, etc.; não forçar o final do debate até que se chegue a um acordo, ou, ao menos, até que todos concordem explicitamente em suspendê-lo. Briga 9. Envolve enfrentamento (normalmente feroz e passional) entre ao menos dois participantes. Visa à vitória. Para tal, não é de modo algum necessária a recorrência a estratégias racionais. Também a briga pode ser levada para fora do estritamente linguístico e os oponentes podem se tornar fisicamente agressivos. A Figura 1 apresenta um quadro comparativo que resume as diferenças entre essas situações, utilizando os componentes da situação argumentativa anteriormente elencados: Situações argumentativas em Paradise Lost: análise do diálogo entre Satã e Eva Passo agora a empregar esse quadro descritivo para compreender o diálogo entre Eva e Satã, no qual é finalmente convencida a comer do fruto (IX. 532-781, p. 391-396). O contexto em que isso se dá é o seguinte: Satã possui a Serpente e sai à procura de Adão e Eva. Estes estão prestes a iniciar seu trabalho matinal, quando Eva propõe que cuidem do jardim separadamente. Adão a aconselha a ficarem próximos, devido ao inimigo acerca do qual foram alertados pelo anjo Rafael. Eva insiste e os dois se separam. Satã encontra-a sozinha, e a convence de que a Serpente adquiriu inteligência ao comer certo fruto. Guia-a ao fruto; reconhecendo tratar-se do fruto da árvore que lhes fora interdita, em princípio Eva se recusa a comê-lo, mas Satã argumenta pesadamente em favor de que o coma. Eva, após repassar mentalmente os argumentos de Satã, convence-se e come do fruto. Situação Interlocutores Teses Objetivos Meios Distinções adicionais Diálogo d s t a c Investigaçã o d s t a c Deliberação d s p v a c As diferenças entre o diálogo e a investigação são, nesta, o caráter mais marcadamente cooperativo e a ausência de teses previamente defendidas. Negociação d v a vit c af sil coa Debate d v vit c af sil Briga d v vit af sil coa O debate é uma competição organizada, visando à vitória — daí suas teses serem volitivas, embora possam parecer teóricas ou práticas —; pode ser acirrado e violento, embora não passional. Figura 1. Quadro comparativo das situações argumentativas. d: diálogo; s: solilóquio; t: tese teórica; p: tese prática arrazoada; v: tese volitiva; a: acordo; vit: vitória; c: convencimento; af: anuência forçada; sil: silenciamento; coa: coação. Figura 1. Quadro comparativo das situações argumentativas. d: diálogo; s: solilóquio; t: tese teórica; p: tese prática arrazoada; v: tese volitiva; a: acordo; vit: vitória; c: convencimento; af: anuência forçada; sil: silenciamento; coa: coação. Interessa-nos, desta seção do nono livro, apenas a fala de Satã na qual defende que Eva coma do fruto (IX. 679-731, p. 394-395). Como uma análise minuciosa do trecho seria demasiado extensa, destinei a explicitação dos argumentos ao Anexo; abaixo, faço um apanhado sintético, referendando os comentários aos argumentos montados no Anexo através das letras maiúsculas que identificam cada um. 9Walton utiliza o termo quarrel, que parece se restringir a uma alteração verbal. Prefiro o termo ‘briga’ para abarcar o que me pareceria o máximo da irracionalidade, o combate físico guiado pelo impulso emocional. 10Relação resumida e reorganizada de Walton (2008, p. 16-17). Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture (I) Não é certo que o mal exista. (I) Não é certo que o mal exista. Eva acredita que, se comer do fruto, morrerá. Satã joga duplamente com essa asserção. Por um lado, demonstra que é falsa de dois modos: nem o fruto em si causa a morte (A’), nem quem o proibiu punirá fatalmente seu consumo (A”). Por outro, aceita-a, propondo um novo conceito de ‘morte’ (O): em primeiro lugar, lembra a Eva que ela de fato não sabe o que significa ‘morrer’ (F); mais tarde, proporá que ‘morrer’ significa ‘ascender ontologicamente’ (O). Desse modo, ora parece descartar a asserção em que originalmente crê Eva, ora a reformula; ambas as atitudes, porém, podem ser harmonizadas: não é verdade que morrerá se ‘morrer’ significa ‘deixar de existir’; é verdade que morrerá se ‘morrer’ significa ‘ascender ontologicamente’. Figura 2. Classificação do diálogo entre Eva e Satã (Paradise Lost IX. 532-781). Satã deseja arruinar a humanidade; para tal, deve fazer com que Eva coma do fruto que lhe fora proibido. Esse objetivo prático, envolvendo tomada de decisões com vistas à ação, faz com que o diálogo seja uma ‘deliberação’. Fazer, porém, com que Eva coma do fruto não é o bastante. Como a queda do homem só se dará se for por escolha própria, Eva não pode ser coagida a comer; deve estar ‘convencida’ de que é de fato a melhor coisa a se fazer. Assim, a Serpente e ela deveriam entrar em acordo de que esta é a ação mais benéfica e racional que ela poderia tomar. A asserção “Eva deve comer do fruto” - explícita no comando “[...] reach, then, and freely taste” (“[...] tome os frutos, portanto, e coma livremente”, IX. 731, p. 395) - compõe a tese central avançada pela Serpente. Eva não avança tese própria, mas está em dúvida acerca de seu valor de verdade, posto que conhece motivos contrários ao consumo do fruto; na terminologia de Walton, seu papel é estar weakly opposed (2008, p. 12) à tese, questionando-a. Embora não questione abertamente os argumentos de Satã, estes visam a alcançar seu sistema de crenças e, portanto, Satã se adianta ao que supõe que ela poderia dizer. Além de questionar a eficácia da punição, também questiona sua justiça (C). 11No caso das personagens miltônicas, uma dificuldade inicial na aplicação do principle of charity é sua inocência. Eva não tem experiência do pecado, nem da morte. A argumentação de Satã é, em muitos pontos, próxima ao senso comum do leitor de Milton — por exemplo, quando afirma que comer do fruto da árvore do bem e do mal é uma transgressão pequena — , mas é difícil imaginar como Eva, que não tem noção do que seja transgredir, poderia julgar a gravidade de qualquer ato condenável ou interdito. Devemos, portanto, ter em mente que os argumentos de Satã são inteligíveis ao leitor e, por extensão, um leitor menos inquisitivo teria propensão a supor que são suficientemente inteligíveis a Eva. 12Trata-se de um diálogo, embora a parte estritamente argumentativa caiba apenas a Satã, uma vez que Eva não lhe oferece qualquer resposta após haver apontado a proibição de comer do fruto. 13Seria pertinente perceber como esse argumento dá mostras do alto valor da racionalidade em Paradise Lost. A Serpente se tornou ‘humana entre os animais’, ou seja: iguala-se aos homens em conhecimento, não em aparência. Do mesmo modo, portanto, Eva seria ‘deusa entre os homens’, mantendo-se humana, mas com conhecimento igual ao dos deuses. O conhecimento parece ser tratado como a característica mais importante dos seres vivos — mesmo Diretrizes para participantes de situações argumentativas Os participantes de uma situação argumentativa deveriam, em princípio, assumir certos compromissos que garantam o mais perfeito andamento da discussão. Dentre os pontos pertencentes a esse código de conduta, destaco os seguintes10: Devemos, contudo, ter em mente que analisar argumentos reais (ou seja, não elaborados com propósitos pedagógicos) é uma atividade sutil, devido ao fato de ser altamente inferencial; nem sempre nossos interlocutores explicitam premissas e Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture 238 Fernandes conclusão de modo suficientemente próximo a um modelo formal. No mais das vezes, temos de inferir ou algumas premissas (muitas vezes de caráter genérico) ou a própria conclusão. Assim, as conclusões abaixo expressas, bem como os argumentos constantes do Anexo, são o resultado de minha aplicação do principle of charity - a boa vontade em relação ao arguidor, que faz com que tentemos montar seus argumentos do modo mais convincente e válido possível (WALTON, 2008, p. 142) - aos versos de Milton11. C): ou é o caso que Eva deva comer do fruto, ou não é o caso que Eva deva comer do fruto. Os argumentos de Satã podem ser organizados em três frentes, como segue. C): ou é o caso que Eva deva comer do fruto, ou não é o caso que Eva deva comer do fruto. Os argumentos de Satã podem ser organizados em três frentes, como segue. Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Ataque à proibição de comer do fruto (A) Não é verdade que você morrerá (se desobedecer à proibição e comer do fruto). (A) Não é verdade que você morrerá (se desobedecer à proibição e comer do fruto). (C) A proibição de comer do fruto não é justa. (D) Deus não poderia punir com a morte a transgressão de comer do fruto. Após essas ressalvas, comecemos pela classificação do diálogo 12 de acordo com os parâmetros acima estabelecidos, conforme a Figura 2, abaixo: (J) Deus não pune (ou ameaça punir) por comer do fruto. (Quem pune ou ameaça punir por comer do fruto não é Deus). (K) Se alguém não é justo não é Deus. Situação Interlocutores e teses Objetivos Meios deliberação Serpente (Satã): Eva deve comer do fruto. (C) (Satã: A humanidade deve ser arruinada.) Eva: (¬C) acordo (vitória) convencimento Figura 2. Classificação do diálogo entre Eva e Satã (Paradise Lost IX. 532-781). Situação Interlocutores e teses Objetivos Meios deliberação Serpente (Satã): Eva deve comer do fruto. (C) (Satã: A humanidade deve ser arruinada.) Eva: (¬C) acordo (vitória) convencimento Figura 2. Classificação do diálogo entre Eva e Satã (Paradise Lost IX. 532-781). (L) Quem não é justo não deve ser temido nem obedecido. (F) Não é certo que a morte seja ruim. Argumentos em favor de se comer do fruto (G) Conhecer o bem e o mal é benéfico, desejável e recomendável. j (B) (H) Comer do fruto é benéfico, desejável e recomendável. (O) Morrer é benéfico, desejável e recomendável. (E) Deus deve reconhecer a virtude de Eva caso ela coma do fruto. Na segunda parte de sua fala, Satã pergunta a Eva que razões teriam os deuses para promulgarem uma proibição injusta (para ele já está estabelecido que é injusta [C]). Afirma, como vimos, que desejam ser adorados, o que só poderá ocorrer se a humanidade permanecer ignorante e, portanto, inferior a eles (M). Assim, além de questionar a justiça da proibição através das vantagens da transgressão, questiona-a através dos motivos de quem a promulgou - um argumento ad hominem que, rigorosamente falando, teria força apenas para lançar dúvidas, não para provar que a proibição é injusta. Ocorre, porém, que Satã já se deu ao trabalho de provar que é esse o caso (C). (N) É justo que os homens ascendam ontologicamente. (Igualando-se aos deuses.) Além de questionar o valor de verdade das asserções que vetam o consumo do fruto, Satã proporá teses que o recomendam. O testemunho da Serpente — o fato mesmo de a Serpente ser ‘capaz’ de dar testemunho — é prova de que seu consumo é benéfico, pois aumenta o conhecimento e a inteligência de quem o come (A’, B, H). Também afirma que, para seguir o bem e evitar o mal, é necessária consciência do que constitui a bondade e a maldade (G). Finalmente, não só nega que Deus puniria Eva por sua (justa) transgressão, mas afirma que o Altíssimo estaria satisfeito com ela, dados os benefícios que resultariam desse ato (E, J, K). A seguir, fazendo uso de um ceticismo empírico de que já lançou mão alhures14, mostrará que Eva não tem experiência sensível de ver os deuses produzindo algo que nos leve a concluir que têm força para criar o universo (Q, R). Aceita, porém, que os deuses preexistem (asserção de que nem ele nem Eva têm tampouco experiência sensível comprobatória), e que usam desta vantagem: como não vimos como o universo foi criado, podem dizer que foram eles. Se, antes, Satã derrubara ou reorganizara as crenças de Eva, aqui parte de asserções das quais sua vítima supostamente compartilha: o conhecimento é benéfico, a bondade é desejável, atos virtuosos são louváveis. Também devemos notar que, mais uma vez, Satã oscilará entre posições. (T) Se alguém age por avareza, não é um deus Eva acredita que os deuses são justos, que são superiores aos homens, que criaram o universo e que proibiram que a humanidade comesse do fruto da árvore do bem e do mal. A terceira estratégia de Satã para convencê-la a comer do fruto será lançar dúvidas sobre os deuses, sua justiça (C, D, K, L), seu papel na criação do universo (Q, R) e, portanto, seu direito mesmo a promulgar leis. Este é o momento mais marcadamente cético de sua argumentação, no qual predomina o questionamento de asserções — que talvez não sejam finalmente refutadas, mas cujo valor de verdade é suspenso. sem se tornar deuses, os homens lhes seriam suficientemente semelhantes para não mais lhes serem reverentes. Lógica luciferina comer o fruto tanto fazendo de Deus imagem favorável (como ser justo, há de louvá-la [E]) como desfavorável (J, K, L). Finalmente, questionará se os deuses têm mesmo direito a proibir o consumo do fruto. Jogará dúvidas acerca de seu poder para criarem o universo, e sobre a existência mesma da proibição, já que a árvore interdita está fisicamente ao alcance dos homens (e antes mesmo já dera a entender que somente Adão e Eva seriam suficientemente altos para alcançar o fruto, ou seja: é como se houvesse sido feito para que dele provassem [IX. 590-591, p. 392]) (M, P, Q, R, S). (I) Não é certo que o mal exista. Baseado nos efeitos benéficos e (portanto) recomendáveis do fruto (B, H), dirá que é injusto que tais benefícios estejam vetados aos homens; reforçará isso demonstrando que aos animais o fruto esteve disponível (já que a Serpente dele comeu). Dirá que, se se trata mesmo de transgressão, é pequena, e um deus justo não poderia aplicar tão pesado castigo a tão leve crime (J, K); complementará dizendo que transgredir, nesse caso, é ato de laudável coragem (E). Também buscará demonstrar que os deuses podem ter razões menos que nobres para proibir o fruto: desejam ser adorados, o que não poderá mais ocorrer se a humanidade se lhes igualar em conhecimento (M)13. Como se vê, trata-se de disputa do tipo (C w Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture 239 14Em sua contenda com Abdiel, quando afirma que ninguém foi testemunha da própria criação; assim, ninguém poderia reconhecer a Deus como o autor de seu ser para além de qualquer dúvida (V. 853-859, p. 322). Acta Scientiarum. Language and Culture Ataque aos deuses (P) Não sabemos quem de fato são os deuses. (P) Não sabemos quem de fato são os deuses. (P) Não sabemos quem de fato são os deuses. (Q) (R) Não é certo que os deuses hajam criado o universo. (S) Ou não é certo que haja mal no fato de a humanidade adquirir conhecimento, ou não é certo que os deuses hajam criado o universo. Também aqui, e de modo um pouco mais evidente, o comprometimento teórico de Satã oscila: ora aceita se tratar de uma proibição (rebatendo-a como injusta), ora aceita-a como proibição leve (rebatendo a punição como injusta), ora nega que seja efetiva (apontando condições práticas não cumpridas para que o fosse), ora nega que os deuses tenham mesmo o direito de a promulgar (apontando que podem não ser responsáveis pela criação do universo). (M) Os deuses proibiram a humanidade de comer do fruto para serem adorados pelos homens. Considerações finais contraexemplo não é suficiente para estabelecer a falsidade da ameaça de morte: sua ‘universalidade’ estaria certamente comprometida (não é verdade que ‘todos os seres’ morrerão ao comer do fruto, pois existe ‘ao menos um’ que comeu e não morreu), não sua eficácia em ao menos alguns casos. Pode-se constatar que Satã lança mão de argumentos céticos (abalando certezas sem substituí- las), argumentos que derrubam asserções pertencentes ao sistema de crenças de Eva e argumentos que o confirmam, avançando teses que deveriam ser, de acordo com esse sistema mesmo, verdadeiras. Seu objetivo, conforme apontado anteriormente, é prático (deseja que ela coma o fruto), mas só será alcançado de modo eficaz se a ação desempenhada por Eva for racional. O solilóquio de Eva ao final da fala de Satã (IX. 744- 779, p. 395-396), no qual reproduz para si mesma os argumentos propostos por seu arguidor, pode ser tomado como indicativo de que deseja agir somente se estiver convencida. Para Satã, porém, não importa qual linha de raciocínio a convença, contanto que ao menos ‘uma’ angarie convencimento. Por essa razão, não está particularmente preocupado com a coerência interna de sua bateria de argumentos. Em terceiro lugar, suas mentiras e sua oscilação entre teses que não se coadunam é instrumental para alcançar convencimento de um arguidor que não disponha de tempo para desemaranhar sua rede argumentativa ou condições de verificar premissas falsas, mas demonstra outro lapso como arguidor racional: Satã defende teses nas quais não acredita; embora questione a criação do universo pelos deuses enquanto fala com Eva (e, antes, Abdiel), em outro momento aceitara Deus como seu criador de modo inconteste (IV. 42-45, p. 278). Seu ceticismo é insincero. Finalmente, para falar de trechos circunvizinhos a esses argumentos citados, Satã interfere nas crenças de Eva de modo não argumentativo. A descrição que faz de Eva ao abordá-la (IX. 532-548, p. 391) já pressupõe e convida à aceitação de alguns dos pontos de que sua argumentação tratará: seus elogios a Eva pressupõem, por exemplo, sua semidivindade e soberania; afirma que sua beleza deveria participar da companhia dos anjos e que ter apenas Adão para apreciá-la é injusto. Tudo isso, se aceito, convida à ambição de se igualar aos deuses (N). Considerações finais Também, no momento em que afirma que Deus louvaria sua virtude ao transgredir a proibição (E), lança mão de elogios e a convida a imaginar um prêmio que provavelmente a torna volitivamente mais propensa a crer nas vantagens da proposta da Serpente. Apela, assim, emocionalmente ao orgulho e à vaidade de Eva (demonstradas em outros trechos do épico15) para que aceite uma opinião acerca de si mesma que é conforme a alguns de seus argumentos. Satã é um arguidor adequado? Está cumprindo todos os quesitos de uma deliberação racional? Não. De que modo, então, deixa de se portar como um arguidor estritamente racional? Em primeiro lugar, esconde sua carta de intenções; como vimos, um debate racional exige que os participantes sejam sinceros quanto a seus objetivos e seu arcabouço teórico; também devem concordar acerca do tipo de situação argumentativa de que tomam parte. Ao possuir a Serpente e usá-la para falar, disfarça sua identidade, escondendo assim de Eva que seu real interesse na deliberação é a ruína da humanidade, ou seja, disfarça a natureza de seus argumentos, que, embora pareçam avançar uma tese prática arrazoada (é racional que Eva coma do fruto), trabalham em prol de uma tese volitiva secreta (a humanidade deve ser arruinada). Eva pensa que a Serpente está sinceramente preocupada com o bem-estar da humanidade; a real intenção de seu interlocutor, que seria mais do que suficiente para lançar pesadas dúvidas acerca da validade de seus argumentos, e talvez a fizesse exigir provas bem mais sólidas do que lançar dúvidas ou oscilar entre pontos de vista discordes, é justamente sua ruína. Em linhas gerais, Satã se mostra um arguidor inteligentíssimo. Em primeiro lugar, é bem-articulado, e avança muito rapidamente grande número de argumentos, o que tanto pode convencer pela aparência de verdade como desnortear pela falta de tempo para seu interlocutor analisar o que ouve. Em segundo lugar, ataca uma mesma asserção de diversos modos; se perde em coerência, ganha em amplitude: Eva deve comer o fruto caso creia que Deus é justo; também deve comê-lo caso creia o contrário; deve comê-lo, pois a proibição é irreal ou simplesmente ineficaz; também deve comê-lo caso pense que é real, pois é injusta. Conforme dito anteriormente, os objetivos secretos de Em segundo lugar, Satã não só mente, mas se excede ao generalizar a partir da mentira. Argumentos em favor de se comer do fruto Tentará Eva a Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture 240 Fernandes 15Quando Eva é criada, enamora-se de seu reflexo em um lago; também, quando Adão a admoesta a não se separar dele devido ao perigo que os ronda, ofende-se por ser considerada mais fraca, o que faz com que insista na separação (respectivamente: IV. 453-467, p. 289; IX. Argument, p. 378). Acta Scientiarum. Language and Culture Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Received on December 9, 2011. Accepted on April 13, 2012. License information: 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. Considerações finais Possuir a Serpente lhe permite simular os efeitos do fruto; apresenta, portanto, um dado empírico crucial, mas inegavelmente falso. Essa mentira é tornada ainda mais perniciosa (pois ainda mais eficiente) quando generaliza hiperbolicamente a partir dela. A Serpente afirma que ‘nenhum’ ser morreria ao comer do fruto e que o fruto é benéfico, pois ela mesma foi beneficiada e não morreu (A”). Um único Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 241 Lógica luciferina Satã (extirpar vitória a Deus e à humanidade por meio da ruína desta) e sua motivação volitiva não repelem semelhantes práticas. HAMBLIN, C. L. Fallacies. Londres: Methuen, 1970. MARGUTTI PINTO, P. R. Introdução à lógica simbólica. Belo Horizonte: UFMG, 2001. Desse modo, o estudo argumentativo empreendido cumpriu seu propósito de auxiliar na compreensão da personagem central de Paradise Lost, e, com isso, auxiliar a compreensão do texto. De fato, Milton constrói Satã como uma personagem inteligente e convincente, porém desonesta. Leituras de Paradise Lost mais favoráveis à revolta do anjo caído podem talvez minorar esse último fato, concentrando-se (com certa razão) em questionar junto com Satã a supremacia ‘natural’ da Divindade. MILTON, J.; TESKEY, G. Paradise Lost (Norton critical edition). Londres/Nova Iorque: Norton, 2005. MILTON, J. Paradise Lost. In: HUGHES, M. Y. (Ed.). Complete works and major prose. Indianapolis: Hackett, 1957. p. 173-469. WALTON, D. Informal logic: a pragmatic approach. 2nd ed. Cambridge/Nova Iorque: Cambridge University Press, 2008. WALTON, D. Informal logic: a pragmatic approach. 2nd ed. Cambridge/Nova Iorque: Cambridge University Press, 2008. Acta Scientiarum. Language and Culture Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 A1. Tradução em prosa da fala de Satã16 A1. Tradução em prosa da fala de Satã16 [Primeira parte] Rainha do Universo, não acredite nas rígidas ameaças de morte: (A) você não morrerá. (A’) Como poderia morrer? Pela ação do fruto? Ele lhe dá vida através do conhecimento. (A”) Pela ação de quem a ameaça? Olhe para mim: toquei e provei do fruto, e não só estou vivo, (B) mas também alcancei uma vida mais perfeita do que a que me era destinada, ao me aventurar além de minha sorte. (C) Acaso o que aos animais é permitido estaria interdito aos homens? (D) Ou Deus acaso se enfureceria por transgressão tão pequena? (E) Ao invés de louvar sua destemida virtude — você, a quem a proclamada pena de morte ([F] o que quer que seja a Morte) (E, cont.) não impediu de obter o que lhe levaria a uma vida mais feliz, o conhecimento do Bem e do Mal: (G e H) quão justas são as coisas boas? Quanto às más ([I] se é que o mal é real), (G e H, cont.) por que não conhecê-las, se assim fica mais fácil evitá-las? (J) Deus, portanto, não pode feri-la e ser justo; (K) se não é justo, não é Deus; (L) não deve ser temido, nem obedecido. Seu modo mesmo da morte elimina o medo. [Segunda parte] Por que então o fruto foi proibido? [M] Que outra razão haveria senão causar assombro, para que vocês se mantivessem rebaixados e ignorantes a adorá-lo? Ele sabe que, no dia em que comerem do fruto, seus olhos — que agora parecem claros, mas que são opacos — alcançarão visão em tudo ampla e clara, e serão como deuses, conhecendo o bem e o mal como eles. [N] Mas que vocês se igualem aos deuses, assim como me igualei internamente aos humanos, não é senão alcançar a justa proporção das coisas. [O] Desse modo, talvez morram ao se desvestir do humano para investir-se do divino — morte desejável, embora usada como ameaça, e que nada pior que isso poderia trazer. [P] E quem são os deuses para o homem não deva se lhes igualar, participando do alimento divino? [Q] Os deuses existiam antes de nós, e tomam vantagem disso para formar nossa crença de que tudo provém deles. Questiono isso, pois vejo esta bela terra, produzir todas as coisas aquecidas pelo sol, enquanto nada lhes vejo produzir. Fernandes Fernandes Referências BRADSHAW, J. A concordance to the poetical works of John Milton. Londres: Swan Sonnenschein and Co., 1894. 242 A1. Tradução em prosa da fala de Satã16 [R] Se criaram todas as coisas, quem encerrou o conhecimento do bem e do mal nesta árvore, para que quem quer que dela coma o obtenha sem sua permissão? [S] E que mal haveria no fato de o homem vir a conhecer? Em que pode o seu conhecimento feri- los, ou esta árvore lhes oferecer que seja contrário à vontade dos deuses, se tudo a eles pertence? [T] Ou pode acaso a avareza habitar peitos celestes? Estas razões e muitas mais declaram sua necessidade a estes belos frutos. Humana deusa, tome-os, portanto, e coma livremente. Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 A’ PA’1: Comer do fruto aprimora a vida através do conhecimento. PA’1: Comer do fruto aprimora a vida através do conhecimento. PA’2: O que aprimora a vida não é mortal. PA’2: O que aprimora a vida não é mortal. q p CA’1: Logo, não é verdade que você morrerá se comer do fruto. (O fruto não é mortal.) CA’1: Logo, não é verdade que você morrerá se comer do fruto. (O fruto não é mortal A2. Montagem dos argumentos Legenda: A: argumento A. PA1: Primeira premissa do argumento A. CA: Conclusão do argumento A. Os argumentos serão diferenciados por meio de letras maiúsculas, em progressão alfabética. Ocasionalmente, a mesma asserção poderá figurar em mais de um argumento. A CA: Não é verdade que você morrerá (se desobedecer a proibição e comer do fruto). Acta Scientiarum. Language and Culture Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 Acta Scientiarum. Language and Culture A” A PA”1: Comi do fruto (desobedeci a proibição) e não morri. (Não fui morto por quem lançou a proibição.) CA”2: Logo, não é verdade que você morrerá se desobedecer a proibição. (Quem lançou a proibição não a matará.) Acta Scientiarum. Language and Culture Maringá, v. 35, n. 3 B PB1: Minha vida foi aprimorada ao comer do fruto. (Ao transgredir. Ao ser ousado.) PB2: O que aprimora a vida é benéfico, desejável e recomendável. CB: Logo, comer do fruto é benéfico, desejável e recomendável. 16Minha tradução. Trata-se de uma tradução literal, sem vistas à expressão literária. Acta Scientiarum. Language and Culture 243 Lógica luciferina C PC1: Não é justo que aos humanos seja proibido o que aos animais é permiti PC2: Comer do fruto é proibido aos homens. PC3: Comer do fruto é permitido aos animais. (Pois a Serpente é um animal CC: Logo, a proibição de comer do fruto não é justa. D PD1: Quem é justo não pune severamente uma transgressão pequena. PD2: Deus é necessariamente justo. PD3 A ameaça de morte é severa. PD4: A transgressão de comer do fruto é pequena. CD: Logo, Deus não poderia punir com a morte a transgressão de comer do E PE1 (= PD2): Deus é necessariamente justo. PE2: Quem é justo reconhece e louva as virtudes. PE3: A coragem é uma virtude. PE4: Eva, ao comer do fruto, seria corajosa (e virtuosa). CE: Logo, Deus deve reconhecer a virtude de Eva caso ela coma do fruto. F PF1: Para saber se algo é ruim, devemos conhecê-lo. PF2: Você não conhece a morte. CF: Logo, não é certo que a morte seja ruim. G PG1: Para praticar o bem e evitar o mal, deve-se conhecê-los. PG2: Praticar o bem e evitar o mal é benéfico, desejável e recomendável. CG: Logo, conhecer o bem e o mal é benéfico, desejável e recomendável. H PH1(= PB1): Comer do fruto dá conhecimento do bem e do mal. PH2 (= CG): Conhecer o bem e o mal é benéfico, desejável e recomendável CH (= CB): Logo, comer do fruto é benéfico, desejável e recomendável. I PI1: Para saber se algo existe, devemos conhecê-lo. PI2: Você não conhece o mal. CI: Logo, não é certo que o mal exista. J PJ1 (= PE1): Deus é necessariamente justo. PJ2 (semelhante a CC): A punição por comer do fruto não é justa. A” CJ: Logo, Deus não pune (ou ameaça punir) por comer do fruto. (Quem pu fruto não é Deus.) K PK1 (= PE1 = PJ1): Deus é necessariamente justo. C l j D C PC1: Não é justo que aos humanos seja proibido o que aos animais é permitido. PC2: Comer do fruto é proibido aos homens. PC3: Comer do fruto é permitido aos animais. (Pois a Serpente é um animal e comeu do fr CC: Logo, a proibição de comer do fruto não é justa. D PD1: Quem é justo não pune severamente uma transgressão pequena. PD2: Deus é necessariamente justo. PD3 A ameaça de morte é severa. PD4: A transgressão de comer do fruto é pequena. CD: Logo, Deus não poderia punir com a morte a transgressão de comer do fruto. D E PE1 (= PD2): Deus é necessariamente justo. PE2: Quem é justo reconhece e louva as virtudes. PE3: A coragem é uma virtude. PE4: Eva, ao comer do fruto, seria corajosa (e virtuosa). CE: Logo, Deus deve reconhecer a virtude de Eva caso ela coma do fruto. E CE: Logo, Deus deve reconhecer a virtude de Eva caso ela coma do fruto. F PF1: Para saber se algo é ruim, devemos conhecê-lo. PF2: Você não conhece a morte. CF: Logo, não é certo que a morte seja ruim. F PF1: Para saber se algo é ruim, devemos conhecê-lo. PF2: Você não conhece a morte. CF: Logo, não é certo que a morte seja ruim. G PG1: Para praticar o bem e evitar o mal, deve-se conhecê-los. PG2: Praticar o bem e evitar o mal é benéfico, desejável e recomendável. CG: Logo, conhecer o bem e o mal é benéfico, desejável e recomendável. G PG1: Para praticar o bem e evitar o mal, deve-se conhecê-los. PG2: Praticar o bem e evitar o mal é benéfico, desejável e recomendável. CG: Logo, conhecer o bem e o mal é benéfico, desejável e recomendável. H PH1(= PB1): Comer do fruto dá conhecimento do bem e do mal. PH2 (= CG): Conhecer o bem e o mal é benéfico, desejável e recomendável. CH (= CB): Logo, comer do fruto é benéfico, desejável e recomendável. H I PI1: Para saber se algo existe, devemos conhecê-lo. PI2: Você não conhece o mal. CI: Logo, não é certo que o mal exista. I PI1: Para saber se algo existe, devemos conhecê-lo. A” PI2: Você não conhece o mal. CI: Logo, não é certo que o mal exista. J PJ1 (= PE1): Deus é necessariamente justo. PJ2 (semelhante a CC): A punição por comer do fruto não é justa. CJ: Logo, Deus não pune (ou ameaça punir) por comer do fruto. (Quem pune ou ameaça punir por comer do fruto não é Deus.) J PJ1 (= PE1): Deus é necessariamente justo. PJ2 (semelhante a CC): A punição por comer do fruto não é justa. CJ: Logo, Deus não pune (ou ameaça punir) por comer do fruto. (Quem pune ou ameaça punir por comer do fruto não é Deus.) K PK1 (= PE1 = PJ1): Deus é necessariamente justo. CK: Logo, se alguém não é justo, não é Deus. K PK1 (= PE1 = PJ1): Deus é necessariamente justo. CK: Logo, se alguém não é justo, não é Deus. J j CK: Logo, se alguém não é justo, não é Deus. L Acta Scientiarum. Language and Culture L PL1: (Somente) Deus deve ser temido e obedecido. PL2: Quem não é justo não é Deus. CL: Logo, quem não é justo não deve ser temido nem obedecido. 244 Acta Scientiarum. Language and Culture Maringá, v. 35, n. 3, p. 233-244, July-Sept., 2013 R PR1: Se os deuses houvessem criado o universo, o conhecimento do bem e do mal não estaria encerrado em uma árvore no meio do Éden (i.e. ao alcance da humanidade). PR2: O conhecimento do bem e do mal está encerrado em uma árvore no meio do Éden. CR (= CQ): Logo, não é certo que os deuses hajam criado o universo. PR1: Se os deuses houvessem criado o universo, o conhecimento do bem e do mal não estaria encerrado em uma árvore no meio do Éden (i.e. ao alcance da humanidade). É PR2: O conhecimento do bem e do mal está encerrado em uma árvore no meio do Éden. CR (= CQ): Logo, não é certo que os deuses hajam criado o universo. S PS1 (= CG): Conhecer o bem e o mal é benéfico, desejável e recomendável. PS2: Se os deuses houvessem criado o universo, o conhecimento do bem e do mal encerrado em uma árvore no meio do Éden (i.e. ao alcance da humanidade) não poderia feri-los. CS ( CR): Logo, ou não é certo que haja mal no fato de a humanidade adquirir conhecimento, ou não é certo que os deuses hajam criado o universo. PS1 (= CG): Conhecer o bem e o mal é benéfico, desejável e recomendável. PS2: Se os deuses houvessem criado o universo, o conhecimento do bem e do mal encerrado em uma árvore no meio do Éden (i.e. ao alcance da humanidade) não poderia feri-los. PS2: Se os deuses houvessem criado o universo, o conhecimento do bem e do mal encerrado em uma árvore no meio do Éden (i.e. ao alcance da humanidade) não poderia feri-los. CS ( CR): Logo, ou não é certo que haja mal no fato de a humanidade adquirir conhecimento, ou não é certo que os deuses hajam criado o universo. N PN1: [Elogios feitos a Eva por Satã.] [ g p ] CN: É justo que os homens ascendam ontologicamente. (Igualando-se aos deuses.) [ g p ] CN: É justo que os homens ascendam ontologicamente. (Igualando-se aos deuses.) O PO1 ( PA’1,  PM3): O conhecimento faz ascender ontologicamente. (Iguala os animais aos homens e estes aos deuses.) PO2: Morrer significa “ascender ontologicamente”. (No caso dos animais, igualar-se aos homens; no da humanidade, aos deuses.) PO3: Ascender ontologicamente é benéfico, desejável e recomendável. CO: Logo, morrer é benéfico, desejável e recomendável. O PO1 ( PA’1,  PM3): O conhecimento faz ascender ontologicamente. (Iguala os animais aos homens e estes aos deuses.) PO2: Morrer significa “ascender ontologicamente”. (No caso dos animais, igualar-se aos homens; no da humanidade, aos deuses.) PO3: Ascender ontologicamente é benéfico, desejável e recomendável. CO: Logo morrer é benéfico desejável e recomendável P PP1: Os deuses afirmam haver criado o universo. PP2: Os deuses afirmam que a morte é temível. PP3 (= CO): Morrer é benéfico, desejável e recomendável. PP2 (= CQ): Não é certo que os deuses hajam criado o universo. CP: Logo, não sabemos quem de fato são os deuses. Q PQ1 (= PP1): Os deuses afirmam haver criado o universo. PQ2: A terra é produtiva. PQ3: Os deuses não são produtivos. (Não os vemos serem produtivos.) PQ4: Quem tem poder de criar o universo deveria ser produtivo. CQ: Não é certo que os deuses hajam criado o universo. R PR1: Se os deuses houvessem criado o universo, o conhecimento do bem e do mal não estaria encerrado em uma árvore no meio do Éden (i.e. ao alcance da humanidade). PR2: O conhecimento do bem e do mal está encerrado em uma árvore no meio do Éden. CR (= CQ): Logo, não é certo que os deuses hajam criado o universo. Q PQ1 (= PP1): Os deuses afirmam haver criado o universo. PQ2: A terra é produtiva. PQ3: Os deuses não são produtivos. (Não os vemos serem produtivos. PQ4: Quem tem poder de criar o universo deveria ser produtivo. CQ: Não é certo que os deuses hajam criado o universo. Q Fernandes ( ) p CM: Os deuses proibiram a humanidade de comer do fruto para serem adorados pelos homens. CM: Os deuses proibiram a humanidade de comer do fruto para serem adorados pelos homens. T T PT1: A avareza não é um sentimento divino. PT1: A avareza não é um sentimento divino. CT: Logo, se alguém age por avareza, não é um deus.
https://openalex.org/W2964423470	https://europepmc.org/articles/pmc6669981?pdf=render	English	null	Risk versus reward: host dependent parasite mortality rates and phenotypes in the facultative generalist Triphysaria versicolor	BMC plant biology	2,019	cc-by	9,344	© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Honaas et al. BMC Plant Biology (2019) 19:334 https://doi.org/10.1186/s12870-019-1856-1 Honaas et al. BMC Plant Biology (2019) 19:334 https://doi.org/10.1186/s12870-019-1856-1 Open Access Risk versus reward: host dependent parasite mortality rates and phenotypes in the facultative generalist Triphysaria versicolor Loren A. Honaas1,5* , Sam Jones1, Nina Farrell2, William Kamerow1, Huiting Zhang1, Kathryn Vescio1, Naomi S. Altman3, John I. Yoder4 and Claude W. dePamphilis1,2 Abstract Background: Parasitic plants engage in a complex molecular dialog with potential host plants to identify a host and overcome host defenses to initiate development of the parasitic feeding organ, the haustorium, invade host tissues, and withdraw water and nutrients. While one of two critical signaling events in the parasitic plant life cycle (germination via stimulant chemicals) has been relatively well-studied, the signaling event that triggers haustorium formation remains elusive. Elucidation of this poorly understood molecular dialogue will shed light on plant-plant communication, parasitic plant physiology, and the evolution of parasitism in plants. Results: Here we present an experimental framework that develops easily quantifiable contrasts for the facultative generalist parasitic plant, Triphysaria, as it feeds across a broad range of diverse flowering plants. The contrasts, including variable parasite growth form and mortality when grown with different hosts, suggest a dynamic and host-dependent molecular dialogue between the parasite and host. Finally, by comparing transcriptome datasets from attached versus unattached parasites we gain insight into some of the physiological processes that are altered during parasitic behavior including shifts in photosynthesis-related and stress response genes. Conclusions: This work sheds light on Triphysaria’s parasitic life habit and is an important step towards understanding the mechanisms of haustorium initiation factor perception, a unique form of plant-plant communication. 10 billion in crop damage annually [9, 10]. The Oroban- chaceae also provide unique opportunities to study para- sitism as it is the only plant family with the full range of parasitic lifestyles [11], plus a fully autotrophic sister lineage, Lindenbergia [12]. In addition to their usefulness for understanding the evolution of parasitism (and thus novel traits, [13, 14]), these plants display extremes of physiology and development that can help us understand many facets of plant biology. For example, strigolactones, long known as germination stimulants [15] for parasitic members of Orobanchaceae, were discovered in 2008 to be important plant hormones [16, 17], the likely receptors for which have been recently identified [18]. * Correspondence: loren.honaas@ars.usda.gov 1Intercollege Graduate Program in Plant Biology, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA 5Present address: Physiology and Pathology of Tree Fruits Research, USDA - Agricultural Research Service, Wenatchee, WA 98801, USA Full list of author information is available at the end of the article Background Triphysaria versicolor is a model parasitic plant in the family Orobanchaceae [1, 2], a family that represents one of a likely 12 independent origins of parasitism in flower- ing plants [3, 4]. T. versicolor is a facultative parasite, and a generalist that can parasitize a wide range of monocot and eudicot hosts, both in nature [5], and in the laboratory [6]. Other members of this family are a primary constraint to African agriculture [7], infesting 40% of all cereal crops in sub-Saharan Africa [8], and causing an estimated $US * Correspondence: loren.honaas@ars.usda.gov 1Intercollege Graduate Program in Plant Biology, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA 5Present address: Physiology and Pathology of Tree Fruits Research, USDA - Agricultural Research Service, Wenatchee, WA 98801, USA Full list of author information is available at the end of the article Strigolactones are also important signaling molecules perceived by arbuscular mycorrhizal (AM) fungi during symbiosis [19], suggesting that parasitic plants have evolved to eavesdrop on the molecular dialogue between Honaas et al. BMC Plant Biology (2019) 19:334 Page 2 of 12 Page 2 of 12 potential hosts and symbiotic fungi [20]. Interruption of this dialogue has been identified as one of the potential control points for weedy parasitic Orobanchaceae [21– 23]. However, the impact of altering strigolactone levels in the plant and in the rhizosphere as part of an effort to control parasitic weeds is still being explored. This is com- plicated by recent work reporting protective effects of AM fungi against Striga hermonthica in Sorghum [24]. An- other potential point of control is post attachment physi- ology of the parasite [10]. Post attachment resistance traits are usually polygenic and breeding programs have targeted these modes of resistance, though only partial and short- term resistance has been achieved [10]. A third point of control is the mechanism by which parasitic plants initiate haustorium formation [1], including the perception of haustorium inducing factors (HIFs). Raw host root exu- dates contain active HIFs including various quinones, hy- droquinones, phenolic acids and flavonoids [25]. It is likely that the considerable redundancy in host derived HIFs contributes to the broad host range of parasitic Oro- banchaceae [25]. It also presents the possibility that a complex HIF profile conveys host quality information, providing a point at which the parasite can evaluate its host in preparation for attachment [25]. The mechanism of this process is largely unknown, save the following ob- servations: 1) structurally diverse active HIFs all have a narrow window of redox potentials [25], 2) the quinone reductase TvQR1 is important for haustorium initiation in Triphysaria and acts very early in HIF perception [25, 26], and 3) that TvPirin is necessary for haustorium formation [27]. Interestingly, TvQR1 has a much greater allelic diver- sity than TvPirin, with the highest diversity in a protein domain that determines substrate specificity [28]. This di- versity may help explain Triphysaria’s ability to respond to a wide variety of host root exudates and hence feed across a broad host range. plants (i.e. T. eriantha), compared to Arabidopsis thali- ana, shows that Triphysaria has the ability to evaluate host quality [30]. Because Triphysaria does not require a host-derived germination simulant, host evaluation is uncoupled from germination, making the facultative generalist a useful model for characterization of HIF per- ception processes in parasitic plants. Importantly, the host range of Triphysaria overlaps with that of the weedy Orobanchaceae and provides a framework for dis- covery of host recognition and evaluation machinery that is shared family-wide. Previous work has shown that another facultative parasitic Orobanchaceae, Castilleja densiflora (syn. Orthocarpus densiflora) displays host dependent floral phenotypes [31] as well as host dependent survivorship [32]. Furthermore, phenotypic transitions to more vigorous growth, thought to occur after successful attachment, have been noted [32, our unpublished field observations]. Therefore, we hypothe- sized that Triphysaria would display host dependent phenotypes during interactions with various hosts that we could magnify by growing the parasite on distantly related plants that span the parasite’s host range. potential hosts and symbiotic fungi [20]. Interruption of this dialogue has been identified as one of the potential control points for weedy parasitic Orobanchaceae [21– 23]. However, the impact of altering strigolactone levels in the plant and in the rhizosphere as part of an effort to control parasitic weeds is still being explored. This is com- plicated by recent work reporting protective effects of AM fungi against Striga hermonthica in Sorghum [24]. An- other potential point of control is post attachment physi- ology of the parasite [10]. Post attachment resistance traits are usually polygenic and breeding programs have targeted these modes of resistance, though only partial and short- term resistance has been achieved [10]. A third point of control is the mechanism by which parasitic plants initiate haustorium formation [1], including the perception of haustorium inducing factors (HIFs). Raw host root exu- dates contain active HIFs including various quinones, hy- droquinones, phenolic acids and flavonoids [25]. It is likely that the considerable redundancy in host derived HIFs contributes to the broad host range of parasitic Oro- banchaceae [25]. It also presents the possibility that a complex HIF profile conveys host quality information, providing a point at which the parasite can evaluate its host in preparation for attachment [25]. The mechanism of this process is largely unknown, save the following ob- servations: 1) structurally diverse active HIFs all have a narrow window of redox potentials [25], 2) the quinone reductase TvQR1 is important for haustorium initiation in Triphysaria and acts very early in HIF perception [25, 26], and 3) that TvPirin is necessary for haustorium formation [27]. Interestingly, TvQR1 has a much greater allelic diver- sity than TvPirin, with the highest diversity in a protein domain that determines substrate specificity [28]. This di- versity may help explain Triphysaria’s ability to respond to a wide variety of host root exudates and hence feed across a broad host range. We selected a group of experimentally tractable host plant genera, based in part upon the survey by Thurman [5], that includes three eudicots (Arabidopsis, Medicago, and Solanum) and three monocots (Zea, Oryza, and Jun- cus). Here we describe experiments that reveal clines of easily quantifiable parasite phenotypes displayed by Tri- physaria while it fed across its host range. These pheno- types suggest that the generalist parasite may have the ability to evaluate host quality, and our framework pro- vides a means to evaluate parasite success nondestruc- tively throughout the parasite’s life cycle. Surprisingly, we found that phenotypes that indicate enhanced parasite vigor were strongly correlated with low parasite survivor- ship. We also show that direct parasite-host contact, not just host root exudate, is necessary for the development of a distinct growth phenotype. Finally, we developed image- based analytics that recapitulate destructive measurements that will allow us to capture phenotypic transitions during host-parasite interactions with non-destructive time course measurements. g In obligate parasites like Striga the commitment to haustorium formation (i.e. haustoriogenesis) is made at germination, because even though separate signaling events must occur to initiate haustoriogenesis, seed re- sources are quickly exhausted and provide only a few days of resources to effect successful attachment to a host root, without which the seedling dies [29]. There- fore, it is critical to coordinate haustorium formation with radicle growth and with regard to the proximity and orientation to the potential host root via the percep- tion of HIFs. In contrast, the commitment to haustorio- genesis in facultative hemiparasites, like Triphysaria, occurs via HIF perception by roots of ostensibly free-liv- ing plants. The facultative generalist parasite must also evaluate potential hosts during the free-living phase of growth to identify high quality versus low quality hosts. The general lack of self-haustorium formation, plus the reduced rate of haustorium formation on congeneric Results Co-culture across Triphysaria s host range The host range co-culture experiment was monitored daily and survivorship of Triphysaria was recorded weekly by counting surviving individuals. By the 5th week of green- house co-culture, parasites in the Solanum (p > 0.001), Zea (p = 0.001), Medicago (p = 0.003), and Arabidposis (p = 0.034) pots showed significantly fewer surviving individuals than the control pots (Fig. 1a.) Because the trend appeared to be toward very low parasite survivorship in the experi- mental treatments, the greenhouse experiment was ended Honaas et al. BMC Plant Biology (2019) 19:334 Page 3 of 12 Honaas et al. BMC Plant Biology Fig. 1 The characteristics of Triphysaria grown across its host range are significantly different from host-free plants and are often highly correlated. ANOVA (Dunnett-Hsu correction) statistical significance compared to the control p < 0.05 and p < 0.01. S=Solanum, Z = Zea, M = Medicago, A = Arabidopsis, J = Juncus, O=Oryza, C = host-free control. Pearson’s R2 for A vs. E = 0.86, D vs. E = 0.69 Fig. 1 The characteristics of Triphysaria grown across its host range are significantly different from host-free plants and are often highly correlated. ANOVA (Dunnett-Hsu correction) statistical significance compared to the control p < 0.05 and *p < 0.01. S=Solanum, Z = Zea, M = Medicago, A = Arabidopsis, J = Juncus, O=Oryza, C = host-free control. Pearson’s R2 for A vs. E = 0.86, D vs. E = 0.69 there were significant differences for Triphysaria grown with Zea (p = < 0.001) and Solanum (p = 0.0083). We hy- pothesized that the paleness of the plants was due to re- duced chlorophyll content, therefore we attempted to quantify the “pale” phenotype by estimating the red:green ratio of plants (as described in [33] because this method was useful to estimate changes in chlorophyll content in senescing wheat). We analyzed photographs of all surviv- ing individuals (See Fig. 2 for representative images from each treatment). The red:green ratio was significantly dif- ferent than the host-free control for Triphysaria grown with Solanum (p = 0.0247) and Zea (p = 0.0162) (Fig. 1e). The gradation of paleness was strongly correlated (R2 = 0.86) with survivorship rates (Fig. 1a) and moderately so with the mass length ratio (Fig. 1e, R2 = 0.69) suggesting that these phenotypes may be related. Results Considering all evi- dence together, the trend for Triphysaria grown with known hosts was fewer surviving individuals that were hardier and ostensibly less autotrophic. and several measurements, some destructive, were made to discover host dependent parasite phenotypes. Furthermore, because some of these patterns were very surprising, we employed very conservative statistics to avoid false posi- tives. Simple parasite growth parameters were significantly different than the control with hosts that induced the low- est survivorship. This included higher dry mass (Fig. 1b: for Zea p < 0.001 & Solanum p = 0.026) indicating that even though the parasites were less likely to survive with Solanum and Zea hosts, survivors accrued more tissue than free-living individuals. Compared to the gracile control plants, hosts which in- duced the highest parasite mortality also induced a novel phenotype – the survivors were “pale and plump” (Fig. 2: e.g. Solanum and Zea compared to the control), appar- ently due to shortened internodes and altered leaf morph- ology. We attempted to quantify the “plump” phenotype by integrating aspects of the growth parameter data. We integrated the dry mass (Fig. 1b) and plant height mea- surements (Fig. 1c) to produce a ratio to quantify the “plump” phenotype (Fig. 1d). Compared to the control, Because analyzing the plant images allowed us to quantify the “greenness” of plants, thereby confirming Page 4 of 12 Page 4 of 12 Honaas et al. BMC Plant Biology (2019) 19:334 Honaas et al. BMC Plant Biology instead collecting the flow through and then using it to sub-irrigate smaller pots containing Triphysaria only (Fig. 3a). We hypothesized that the “pale and plump” phenotype was a result of successful parasite attachment. Therefore, we predicted that this phenotype would be absent from the sub-irrigated pots which lacked direct host contact, yet these plants would be exposed to water soluble host exudates that have been shown to induce haustoria in Triphysaria [34]. Indeed the only parasites in the experiment that showed the phenotype were the Triphysaria that were grown in direct contact with the tomato host (Fig. 3b SlTv vs. others: see 3c & 3d for rep- resentative plant images). Fig. 2 Triphysaria displays host dependent phenotypes. Representative images of Triphysaria showing the average number of surviving plants in each treatment, plus controls. The host genus is listed above each set of parasites. Results The control plants were grown in identical conditions, in an identical circular arrangement, but without hosts There was a very weak pattern (paired t-test p = 0.04) that suggested parasite survivorship was lower in SlTv Sub (27 ± 9% surviving Triphysaria when watered with flow through from tomato only pots) than NC Sub (50 ± 6% surviving Triphysaria when watered with flow through from soil only pots), yet when we corrected for multiple comparisons the result was not significant. Thus, the sur- vivorship of parasites in this experiment was not signifi- cantly different, possibly for one or more of three reasons: 1) growing conditions were cooler, hence more favorable causing more co-cultured parasites to establish, 2) host- free plants, which, although they were more likely to survive without hosts, still showed a trend of decreasing survivorship and thus had more time to die (8 vs. 5 weeks), and 3) lack of statistical power – the control group was roughly 1/5 of the size of the multi-host experiment, which we designed with a very large control group (n = 45) based upon a power analysis from preliminary experi- ments (data not shown). Importantly, parasites in the sub- irrigated pots did not display the plump phenotype, supporting our hypothesis that host contact was required for this distinct phenotype. Fig. 2 Triphysaria displays host dependent phenotypes. Representative images of Triphysaria showing the average number of surviving plants in each treatment, plus controls. The host genus is listed above each set of parasites. The control plants were grown in identical conditions, in an identical circular arrangement, but without hosts visual observations and showing significant experimental contrasts, we attempted to recapitulate the “plumpness” (e.g. mass/height ratio) of plants as well. By analyzing each photograph to outline each plant, we generated perimeter to area ratios. This approach recapitulated our plant mass measurements (R2 = 0.87; see Fig. 1b vs. 1f) and showed a significant difference of parasites grown with Zea versus the host-free control. The correlation with the mass/height ratio was good, but lower (R2 = 0.72) indicating further refinement of the method is needed to accurately capture the experimental contrasts, namely a way to estimate plant height in a high through- put manner. Importantly, these image-based analytics showed us significant phenotypic differences between at least partly heterotrophic parasites and the autotrophic parasite controls. Differentially expressed genes in autotrophic vs. heterotrophic Triphysaria The Parasitic Plant Genome Project (PPGP; [35]; http:// ppgp.huck.psu.edu) hosts a publicly available compen- dium of life stage specific transcriptomes for species in- cluding Phelipanche (syn. Orobanche) aegyptiaca, Striga hermonthica and Triphysaria versicolor (see Yang et al., 2015). The observations we have made with Triphysaria feeding across its host range suggest that the parasite’s physiology is substantially altered in a host-dependent fashion. The PPGP transcriptome database includes data for Triphysaria grown with and without a host (M. trun- catula) for flowers/reproductive structures, shoots, and roots. We compared these previously analyzed digital gene expression datasets [13] to find gene activity that differed significantly between the autotrophic vs. hetero- trophic modes of the facultative parasite (Table 1). Co-culture with Solanum and subirrigation of Triphysaria Co-culture with Solanum and subirrigation of Triphysaria Observations in the first co-culture experiment led to hypotheses about the cause of the host-dependent phe- notypes. We sought to separate stimuli that induced the growth phenotypes, so we designed an experiment to isolate signaling cues that involved top watering larger co-culture pots as in the multi host experiment, but Honaas et al. BMC Plant Biology (2019) 19:334 Page 5 of 12 Honaas et al. BMC Plant Biology Fig. 3 The “plump” phenotype is dependent on direct host contact. a experimental design, Sl = S. lycopersicum, Tv = T. versicolor, NC = negative control; b box plot of area:perimeter ratios for all parasites in the experiment; c & d example Triphysaria images from pot SlTv (parasite + host) and Tv (parasite only) showing the “plump” phenotype that parasites display when grown with S. lycopersicum hosts. ANOVA (Tukey-Kramer correction) p < 0.001 Fig. 3 The “plump” phenotype is dependent on direct host contact. a experimental design, Sl = S. lycopersicum, Tv = T. versicolor, NC = negative control; b box plot of area:perimeter ratios for all parasites in the experiment; c & d example Triphysaria images from pot SlTv (parasite + host) and Tv (parasite only) showing the “plump” phenotype that parasites display when grown with S. lycopersicum hosts. ANOVA (Tukey-Kramer correction) *p < 0.001 Biological Process terms “biosynthetic process” and “carbo- hydrate metabolic process” are notably higher, respectively in root and shoot, in the feeding parasite compared to fully autotrophic Triphysaria. Consistent with elevated mortality rates in our experiment that suggest increased parasite stress, both in the roots and shoots of feeding parasites, “re- sponse to stress” category genes were strongly upregulated. These gene expression signals are correlated with altered growth patterns and provide candidate genes and processes to examine in future experiments. Not surprisingly, genes related to photosynthesis with the GO Biological Process term “photosynthesis” and Cellular Component term “thylakoid” are under-represented in Tri- physaria when it feeds on Medicago compared to the auto- trophic (host-free) mode of growth. Consistent with our previous work examining all parasite life stages [13], the Molecular Function GO term “peptidase activity” was over- represented in the feeding parasite’s root tissue and the Biological Process GO term “translation” was underrepre- sented among differentially expressed (DE) genes. The GO Page 6 of 12 Honaas et al. Co-culture with Solanum and subirrigation of Triphysaria BMC Plant Biology (2019) 19:334 Discussion selectivity [36–38], an observation widely made of para- Table 1 GO enrichment of differentially expressed genes in the feeding parasite support the observed host dependent phenotypes. Bold numbers indicate P < 0.05, adapted from [13] GOSlim Term DE genes p-value Vegetative Shoots upreg downreg ATPase activity 28 4 6.26E-06 peptidase activity 19 1 2.22E-05 carbohydrate metabolic process 13 3 1.78E-02 response to stress 13 2 5.98E-03 nucleus 12 2 1.09E-02 translation 3 15 2.76E-03 intracellular 3 16 1.50E-03 thylakoid 2 33 1.24E-09 protein complex 2 42 6.07E-13 photosynthesis 1 28 6.88E-09 structural constituent of ribosome 1 15 1.43E-04 ribosome 1 15 1.43E-04 Reproductive Shoots peptidase activity 20 4 9.56E-04 ion binding 18 51 1.21E-06 oxidoreductase activity 6 41 3.16E-09 transmembrane transport 3 14 5.03E-03 transport 3 19 2.22E-04 transmembrane transporter activity 3 16 1.50E-03 translation 2 12 5.18E-03 response to stress 1 8 1.74E-02 structural constituent of ribosome 1 8 1.74E-02 ribosome 1 8 1.74E-02 Roots ion binding 46 16 3.37E-05 peptidase activity 37 2 2.34E-10 response to stress 23 1 1.21E-06 cellular protein modification process 22 2 1.70E-05 kinase activity 20 2 6.66E-05 biosynthetic process 19 8 4.13E-02 DNA metabolic process 17 6 2.76E-02 DNA binding 16 6 4.35E-02 hydrolase activity, acting on glycosyl bonds 11 2 1.95E-02 lipid metabolic process 9 1 1.93E-02 translation 1 11 2.36E-03 intracellular 1 10 4.65E-03 Table 1 GO enrichment of differentially expressed genes in the feeding parasite support the observed host dependent phenotypes. old numbers indicate P < 0.05, adapted from [13] selectivity [36–38], an observation widely made of para- sitic angiosperms [39, 40]. Therefore, in order to gain insight into the host evaluation process, we set out to es- tablish a framework to observe phenotypic clines and transitions associated with host exposure across the Discussion Interpreting the responses of a generalist parasitic plant to a range of hosts Indeed, our observa- tions are consistent with previous work in a closely related facultative hemi-parasite [31, 32] as Triphysaria also displayed host dependent survivorship as well as host-dependent growth characteristics. Additionally, the distinct paleness of the “plump” individuals is concord- ant with significant under-representation of genes related to photosynthesis in the feeding parasite, sug- gesting increased heterotrophy compared to fully auto- trophic Triphysaria. Together these data show that the hemi-parasite Triphysaria displays clear host-dependent phenotypes that are suggestive of variable parasite suc- cess, or perhaps even host selectivity, though more work on this question is needed. In fact, Atsatt noted that fac- ultative members of Orobanchaceae (syn. Scrophularia- ceae) were ideal candidates to characterize host-specific parasite responses, in part because of the frequently ob- served enhanced vigor and more rapid growth after a presumed attachment to a host plant [31]. The low survivorship of discernably more successful individuals seems to indicate that, like recent work in pea suggests [46], these plants are engaging in risky be- havior possibly by allocating resources away from auto- trophic modes of growth. This risky behavior may be buffered or canalized by host plants for successfully at- tached parasites – like the increased survivorship of in- bred albino Orthocarpus purpurascens (syn. Castileja exserta) when grown with a host versus without [47]. It is therefore possible that the parasites die when attempt- ing to transition to heterotrophic modes of growth. In this way Triphysaria may be engaging in risk in a similar way as a forager on a negative energy budget [48] – when resources are so limited that survival is unlikely, risk prone behavior in the form of a gamble for a big payoff (in this case a successful union with a host plant root) may be the only viable strategy. These data shed light on a long-held hypothesis for which relevant data has been very limited – in fact Heide- Jørgensen makes the argument that the distinction be- tween facultative and obligate parasitism is irrelevant be- cause definitive evidence for facultative parasitism does not yet exist and is very hard to obtain [41]. He argues that a fully autotrophic mode of growth in a host-free sys- tem may be an artifact of highly favorable growth condi- tions in the lab that do not reflect growth conditions for facultative parasitic plants in nature. Discussion Interpreting the responses of a generalist parasitic plant to a range of hosts Previous work has suggested that other facultative gen- eralists in Orobanchaceae may show host preference or Honaas et al. BMC Plant Biology (2019) 19:334 Page 7 of 12 by Atsatt, who found the closely related facultative para- site, Castilleja densiflora (syn. Orthocarpus densiflora) has initially low survivorship when grown with hosts versus without, but after 2 months the parasites with hosts were more likely to survive [32]. Therefore, the initial low survivorship may reflect a gamble that pays off for successful parasites late in their life cycle when water stress increases late in the season in the northern California part of the parasite’s native range [32]. A lar- ger (necessarily due to high parasite mortality) and lon- ger controlled study, perhaps in the field, would help determine if the same long term trends are seen with Triphysaria when feeding on various hosts. parasite’s confirmed host range. Our observations of Tri- physaria shoots display a spectrum of phenotypic char- acteristics along the host range of the parasite. Typically, parasitic plant success is defined as a suc- cessful connection to a suitable host [41]. Because we (unpublished field and lab observations) and others [31] have noted transitions in parasite growth patterns that are thought to occur after successful attachment of para- sites to host roots (via haustoria), we reasoned that simi- lar obvious transitions in our experiment could be used as a proxy for successful attachment of Triphysaria to a suitable host. Triphysaria plants show a range of a “pale and plump” phenotype that is more pronounced on certain hosts than others. This parasite phenotype resulted from shortened internodes and stubbier, fleshier, and more pale leaves. Follow-up analyses of the parasite’s anatomy may provide some additional insight in the processes that drive these growth patterns. Of particular interest would be changes in leaf anatomy, as it is known that the related obligate parasites Striga gesnerioides and Alectra orobanchoides display diminished leaf morph- ology compared to free living relatives [42–45]. While the overall height and dry mass of the heterotrophic in- dividuals showed no clear trend, when used to calculate a mass:length ratio, it revealed a clear gradation that might be useful as a proxy for success of the parasite. This is because parasites that displayed the most dra- matic phenotypes (grown on Zea and Solanum, see Fig. 2) accrued more biomass compared to the more gracile individuals grown on other hosts. Experiment layout and watering The host range co-culture experiment was conducted in the College of Agricultural Sciences Greenhouse #111 at Penn State University from March 3 to May 19, 2014. The experiment was set up in a complete randomized block design (randomizer.org) with 27 pots in each of 5 blocks. Each block contained 6 hosts × 3 replicates plus 9 control [no host] pots each with 7 parasites. A custom drip irriga- tion system was made using watering timers (Orbit Digital 2-Outlet timer Model #: 27133), ¾” (~ 19 mm) irrigation tubing and various couplers and fittings widely available at hardware stores (Additional file 2: Figure S2). Weighted ir- rigation drippers were fitted into the ¾” irrigation tubing and standard garden hose ball-check valves were cali- brated to flow 100 mL/minute of water to each drip line outlet. Soil media was Sunshine mix #1 and sand (Quick- crete Medium - Lowes) mixed 1:1 (by volume) and mea- sured volumetrically into pots with a triple layer of newspaper in the bottom of each pot to prevent media loss. Solanum, Maize and Oryza were planted in large pots (2.5 gal, ~ 9.5 L) and received 200 mL of water twice per day, while Triphysaria (controls), Arabidopsis, Medi- cago and Juncus were planted in small pots (1 gal, ~ 3.8 L) and received 100 mL of water twice per day. The watering regime was calibrated to through-water pots with excess Seed germination g Triphysaria versicolor, Medicago truncatula (A17), and Zea mays (B73) seeds were obtained and germinated as described by [49]. Solanum lycopersicum (Heinz 57) seed were produced in the Penn State Biology Greenhouse. S. lycopersicum seeds were surface sterilized using a 50% bleach (5.25% hypochlorite) + 0.01% Triton X-100 (Sigma) solution for 30 min, then washed 10x with sterile distilled water and germinated on Triphysaria co-culture medium (1/4x Hoagland’s basal salt and nutrient mix, 7.5 g/L su- crose, 6 g/L plant tissue culture grade agarose, pH of 6.1). Oryza sativa subspecies Japonica cv. Nipponbare seeds were incubated in sterile distilled water at 28–37 °C until germination then transferred to Triphysaria co-culture medium. Arabidopsis thaliana (Col-0) seeds were surface sterilized using 70% ethanol + 0.01% Triton X-100 for 8 min, then washed with 100% ethanol, air-dried for ~ 15 min, and germinated on Triphysaria co-culture medium. Juncus effusus seed was obtained from a commercial vendor (http://www.everwilde.com/). J. effusus seed was washed 2–3 times with sterile distilled water, washed with 70% ethanol for 3–5 min, washed with undiluted commer- cial bleach (5.25% hypochlorite) for 40 min, washed with sterile molecular biology grade water 3–5 times, and ger- minated on Triphysaria co-culture medium. p Previous observations have shown that root exudates are sufficient to induce haustorium formation in Triphy- saria [30]. We therefore attempted to determine if the plump phenotype was due to exudates, induced tomato defenses, or direct host contact. We confirmed that the “plump” phenotype was dependent upon direct contact with a known host, supporting the hypothesis that the phenotype co-occurs with a switch to heterotrophy, not simply exposure to host exudates or allelopathic com- pounds. Although the signal was very weak that Sola- num exudate is sufficient to reduce survivorship, this hints that survivorship is linked to haustorium formation and can be induced without direct host contact. Using our image-based analytics, it should be possible to longi- tudinally monitor plant growth, and select individuals to excavate to search for haustoria when a significant con- trast appears in the non-destructive analysis. Discussion Interpreting the responses of a generalist parasitic plant to a range of hosts We contend that if autotrophy were a viable strategy, the plants would likely not risk costly haustorium formation on hosts that signifi- cantly increase mortality. Conversely, we have observed what seem to be autotrophic Triphysaria growing and flowering ~ 1 m away from any potential host plant in the field (unpublished). So while these data suggesting risky behavior by Triphysaria when grown with known hosts does support the hypothesis that Triphysaria is function- ally an obligate parasite [41], definitive support for this or the alternative hypothesis remains elusive. The host dependent phenotypes suggest increased parasite vigor, even though reduced overall survivorship for these heterotrophs was observed compared to more gracile host-free controls in our 5-week co-culture ex- periment. These observations are consistent with work We did attempt to excavate pots from the multi-host experiment to survey haustorium formation, but the very dense, wet, and sandy soil made this extremely difficult. While we frequently observed haustoria, we were unable Honaas et al. BMC Plant Biology (2019) 19:334 Honaas et al. BMC Plant Biology (2019) 19:334 Page 8 of 12 resulting from host contact was confirmed for each host (Additional file 1: Figure S1). to attribute connections to certain individuals, or even accurately count the tiny ~ 1 mm haustoria. Develop- ment of a co-culture system that would induce the phe- notypes we observed and that also allows researchers to monitor haustorium development with ease is needed. Conclusions Characterization of the elusive molecular dialogue be- tween parasitic plants and their host plants requires a tractable framework. To that end, we have created a framework that includes non-destructive methods for longitudinal studies and demonstrated that significant differences in easily quantifiable growth patterns are ob- tainable. Furthermore, these patterns of parasite growth and survival do shed light on long held questions about whether true facultative parasites exist in nature – our data suggest that Triphysaria is engaging in risky behav- ior to potentially parasitize certain hosts more than others. Our framework was optimized with hosts that have resources for molecular genomics work; this will fa- cilitate next steps to explore mechanisms of host choice or evaluation by parasitic plants, as well as the nature of the unique plant-plant molecular dialogue between para- sitic Orobanchaceae and their hosts. p y Seed germination Solanum lycopersicum (Heinz 57) and Triphysaria versico- lor seeds were obtained and germinated as described above. Co-culture across Triphysaria’s host range Host plants selection Co-culture across Triphysaria’s host range Host plants selection Putative hosts were selected from surveys by Thurman [5] and were further refined to include plants with pub- licly available genome or transcriptome sequence data resources anticipating molecular studies that leverage this experimental framework. Host plants selected for this project were Arabidopsis thaliana (Col-0), Medicago truncatula (A17), Solanum lycopersicum (Heinz 57), Zea mays (B73), Oryza sativa subspecies Japonica cv. Nip- ponbare, and Juncus effuses. Haustorium formation Honaas et al. BMC Plant Biology (2019) 19:334 Page 9 of 12 Page 9 of 12 water to maintain high levels of soil moisture. 5 mL granu- lar Osmocote Plus (15–9-12) was added to each irrigated pot and sticky cards were used to control insect pests dur- ing the experiment. were averaged, and this value was used to calibrate (86.05 ± 0.12 pixels/cm) the measurement function in ImageJ. The calibrated measurement tool was then used to measure the length from the cut plant (at soil surface) to the apex of each Triphysaria plant. To describe the “plump” phenotype observed in the experiment, we nor- malized the dry mass (in mg) of each Triphysaria by the height (in mm). Experiment layout and watering A sub-irrigation co-culture experiment was conducted in the College of Agricultural Sciences Greenhouse #85 at Penn State University in from September 16, 2014 to December 14, 2014. Treatments (10 each of Solanum, Solanum + Triphysaria, Triphysaria only, soil only con- trol, for a total of 40 pots) were randomly arranged (ran- domizer.org) and placed in two columns of 20. A drip irrigation system like that described above was used to water each large pot (2.5 gal, 4 L soil media) except that only one timer was used to regulate water to the whole experiment and calibrated to deliver 250 mL of water twice daily to each pot. These irrigated pots were placed at the high end of slightly inclined trays. One small pot (300 ml) with 7 Triphysaria only were placed one each at the low end of the inclined trays (that also contained the large pot) so as to be sub-irrigated with the flow- through of the larger pot. A small drainage port in each tray allowed for excess flow-through water to be drained away avoiding water logging of these smaller pots. Planting timeline g After germination (time on plates: Juncus 17d, Medicago and Maize 8d, Arabidopsis 7d, Solanum and Oryza - 6d) host plants were transplanted into soil media (see above) 2 weeks prior to co-culture and grown in a growth chamber (21 °C, 16/8 light/dark cycle) for 1 week (flats with domes for 3 days, with “cracked” domes for 4 days). 1 week prior to transplant to experimental pots, host plants were hardened off in the greenhouse where the experiment took place (3 days with “cracked” domes, 4 days without domes). ~ 6000 Triphysaria were germi- nated and grown in tandem batches at 16 °C ~ 25–35 days prior (Triphysaria germination is not highly syn- chronous) to co-culture. 1 day after host plants were added to experimental pots, germinated Triphysaria were transplanted serially (i.e. a researcher transplanted a suitable parasitic plant (defined as ≥1 cm root and first true leaves open) in one pot, then moved to the next pot, until the usable parasitic plants were exhausted). Seven Triphysaria plants were added to each host pot ~ 25 cm from the host stem, in a circle, at intervals of roughly 50° (~ 2.5 cm spacing) and Triphysaria only pots were planted in the same configuration sans host in small pots (see above). After initial planting the watering regime was supplemented by extensive hand misting with DI water (as needed during the first week ~ 4–5 times daily) to help all plants establish in the green- house. Survivorship at week 2 was very high and not significantly different for any treatment, indicating suc- cessful establishment. Data collection h The experiment was monitored daily. A survey of sur- vivorship was taken at 1 week intervals. When Triphy- saria die, they rapidly oxidize and wilt, so our definition of “survivor” was “upright Triphysaria with some green leaf tissue”. This definition could include dying individ- uals, though once senescence had begun, the plant was always counted as dead at the next week interval. On May 16, 2014, 5 weeks after the beginning of co-culture, all parasitic plants were harvested by cutting Triphysaria at the soil surface. All plants were photographed (Nikon D80) and then numbered and bagged for drying. Plants were dried for 48 h at 65 °C, at which time each Triphy- saria was weighed, replaced into the bag, and sealed in an airtight storage container. Plant height was calculated using ImageJ (http://imagej.nih.gov/). Each photograph was analyzed manually by measuring the length, in pixels, of 1 cm on the reference scale bar. These values Data analysis l Statistical analysis was done using SAS PROC MIXED [50] as a randomized complete block design with block as a random effect. Dunnett’s test [51] for differences be- tween the treatments (hosts) and the control (no host) was done for each response to control for multiple test- ing. The unequal allocation of replicates was done to optimize the power of Dunnett’s test. Each of the re- sponse variables was analyzed separately. Data analysis y Statistical analysis was done using SAS PROC MIXED [50] taking into account the pairing of the large and small pots. Tukey’s test for all pairwise comparisons was applied for each response to control for multiple testing. Image acquisition Images were taken with a D80 Nikon camera on a tripod at a fixed distance from the imaging stage, with fixed focus. Auto exposure was used to compensate for chan- ging light conditions throughout the day in the green- house. The plants were processed iteratively through each block. Because each block was randomized the ef- fect of dynamic lighting conditions would affect treat- ments randomly. The imaging stage had a dark felt background and metric/SAE rulers were fixed to the stage on both X and Y axes. Acknowledgements We thank Scott DiLoretto for use of the Horticulture greenhouse for these experiments and Anthony Omeis for additional support in the Biology Greenhouses at Penn State University. We also thank the Tomato Genetic Resource Center at The University of California, Davis, for Heinz 57 seeds, Charles Anderson for seeds of Arabidopsis thaliana Col-0, and Emily Helliwell and Yinong Yang for Oryza sativa seeds. Color normalization was performed using both the white reference (ruler) and plant ROIs by converting raw RGB values to XYZ, where the white reference was ap- plied to normalize ROI values for chromatic adaptation using Bradford matrices [53–55]. Conversion back to RGB color space provides the corrected values needed for Planting timeline Solanum and Triphysaria were germinated and grown prior to co-culture as described above. Co-culture and control pots were set up as described above. During the first week, watering was calibrated up (from 200 to 250 mL) to deliver sufficient water to sub-irrigated pots. Dur- ing this optimization phase, dead Triphysaria were re- placed as needed. Supplemental lighting from overhead Page 10 of 12 Page 10 of 12 Honaas et al. BMC Plant Biology (2019) 19:334 estimation of chlorophyll content through Green/Red ra- tio [33]. For the ImageJ macro, see Additional file 3. estimation of chlorophyll content through Green/Red ra- tio [33]. For the ImageJ macro, see Additional file 3. sodium vapor lamps was supplied from 8 am to 8 pm if light intensity dropped below 200μE. After the watering regime was established, 5 mL granular Osmocote Plus (15–9-12) was added to each top irrigated pot. Data analysis Statistical analysis was done using SAS PROC MIXED [50] using the mean values for the surviving plants in each pot. To account for the different numbers of survi- vors, weighted analysis was done using the number of surviving plants as weights. Block was included as a ran- dom effect. Dunnett’s test [51] for multiple comparisons with a control was done to determine differences be- tween the pots with hosts and the control. Unequal allo- cation of replicates was used to optimize the power of Dunnett’s test. Abbreviations HIF: Haustorium inducing factor; PPGP: Parasitic Plant Genome Project; ROI: Region of interest Data collection The experiment was monitored daily and data was col- lected as described above with the following exception: the duration of the experiment was ~ 8 weeks (59 days) instead of 5 weeks. Image segmentation and processing to estimate perimeter:area ratio Additional file 1: Figure S1. All hosts were verified by direct observation of haustorium formation by Triphysaria. A) Arabidopsis, B) Juncus, C) Medicago, D) Oryza, E) Solanum, F) Zea. (TIFF 4874 kb) All image segmentation was performed using ImageJ (https://imagej.nih.gov/ij/). Prior to segmentation the RGB images were either broken into separate channels or trans- formed into other color spaces for easier analysis: HSV, HSI, or CMYK. Once a particular channel was chosen the image was thresholded to capture the plant ROIs. The ROI could then be cleaned using an additional threshold- ing to capture the background within and outside the plant; this created a more clearly defined edge. From the refined ROI, the Particle Analyzer function was used to measure the area and the perimeter of the plant. The raw RGB data from the ROI was also obtained for the purpose of normalization against a white reference. The raw RGB data from the white reference was sampled and normal- ized using the grey world method [52]. Additional file 2: Figure S2. Images of experimental apparati and planting scheme. A) Seven Triphysaria were planted around each host equidistant from each other and the host plant. For control pots, the arrangement was identical, except without host plants. B) the watering control system. (TIFF 6785 kb) Additional file 2: Figure S2. Images of experimental apparati and planting scheme. A) Seven Triphysaria were planted around each host equidistant from each other and the host plant. For control pots, the arrangement was identical, except without host plants. B) the watering control system. (TIFF 6785 kb) Additional file 3: Archive containing the ImageJ macro. (IJM 2 kb) Non-destructive, image-based analysis Image acquisition Transcriptome analysis of Triphysaria versicolor (func- tional annotation and identification of differentially expressed (DE) genes in the parasite during free-living versus parasitic modes of growth) was reported previously [13]. The DE genes were subject to a Fisher’s exact test using GO slim terms for upregulated and down-regulated DE genes as the input table (fisher.test function in R), which identified enriched GO slim terms associated with upregulated or downregulated features for shoots, flowers, and roots; alpha was set to 0.05. References The population biology of closely related species of Orthocarpus. PhD thesis. Los Angeles: University of California; 1966. PhD thesis. Los Angeles: University of California; 1966. 7. Vurro M, Bonciani B, Vannacci G. Emerging infectious diseases of crop plants in developing countries: impact on agriculture and socio-economic consequences. Food Security. 2010;2(2):113–32. 7. Vurro M, Bonciani B, Vannacci G. Emerging infectious diseases of crop plants in developing countries: impact on agriculture and socio-economic consequences. Food Security. 2010;2(2):113–32. 33. Adamsen FJ, Pinter PJ, Barnes EM, LaMorte RL, Wall GW, Leavitt SW, Kimball BA. Measuring wheat senescence with a digital camera. Crop Sci. 1999;39(3):719–24. 34. Jamison DS, Yoder JI. Heritable variation in quinone-induced haustorium development in the parasitic plant Triphysaria. Plant Physiol. 2001;125(4):1870–9. 8. Rodenburg J, Riches CR, Kayeke JM. Addressing current and future problems of parasitic weeds in rice. Crop Prot. 2010;29(3):210–21. 35. Westwood JH, dePamphilis CW, Das M, Fernández-Aparicio M, Honaas LA, Timko MP, Wafula EK, Wickett NJ, Yoder JI. The parasitic plant genome project: new tools for understanding the biology of Orobanche and Striga. Weed Sci. 2012;60(2):295–306. 9. Ejeta G. The Striga scourge in Africa: a growing pandemic. In: Integrating new Technologies for Striga Control: towards ending the witch-hunt. Singapore: World Scientific Publishing Co; 2007. 10. Scholes JD, Press MC. Striga infestation of cereal crops - an unsolved problem in resource limited agriculture. Curr Opin Plant Biol. 2008;11(2):180–6. 36. Atsatt PR, Strong DR. The population biology of annual grassland Hemiparasites. I The Host Environment. Evolution. 1970;24(2):278–91. 11. Westwood JH, Yoder JI, Timko MP, dePamphilis CW. The evolution of parasitism in plants. Trends Plant Sci. 2010;15(4):227–35. 37. Werth CR, Riopel JL. A study of the host range of Aureolaria pedicularia (L.) Raf.(Scrophulariaceae). Am Midl Nat. 1979:300–6. p y g p Raf.(Scrophulariaceae). Am Midl Nat. 1979:300–6. 38. Gibson CC, Watkinson AR. Host selectivity and the mediation of competition by the root hemiparasite Rhinanthus minor. Oecologia. 1991;86(1):81–7. 12. Olmstead RG, DePamphilis CW, Wolfe AD, Young ND, Elisons WJ, Reeves PA. Disintegration of the Scrophulariaceae. Am J Bot. 2001;88(2):348–61. 38. Gibson CC, Watkinson AR. Host selectivity and the mediation of competition by the root hemiparasite Rhinanthus minor. Oecologia. 1991;86(1):81–7. 13. Yang Z, Wafula EK, Honaas LA, Zhang H, Das M, Fernandez-Aparicio M, Huang K, Bandaranayake PC, Wu B, Der JP, et al. Comparative transcriptome analyses reveal core parasitism genes and suggest gene duplication and repurposing as sources of structural novelty. Mol Biol Evol. 2015;32(3):767–90. 39. References 27. Bandaranayake PCG, Tomilov A, Tomilova NB, Ngo QA, Wickett N, dePamphilis CW, Yoder JI. The TvPirin gene is necessary for Haustorium development in the parasitic plant Triphysaria versicolor. Plant Physiol. 2012; 158(2):1046–53. 1. Bandaranayake PC, Yoder JI. Haustorium initiation and early development. In: Parasitic Orobanchaceae. Berlin: Springer; 2013. p. 61–74. 2. Goldwasser Y, Westwood J, Yoder J. The use of Arabidopsis to study interactions between parasitic angiosperms and their plant hosts. Arabidopsis Book. 2002;1:e0035. 2. Goldwasser Y, Westwood J, Yoder J. The use of Arabidopsis to study interactions between parasitic angiosperms and their plant hosts. Arabidopsis Book. 2002;1:e0035. 28. Ngo QA, Albrecht H, Tsuchimatsu T, Grossniklaus U. The differentially regulated genes TvQR1 and TvPirin of the parasitic plant Triphysaria exhibit distinctive natural allelic diversity. BMC Plant Biol. 2013;13:28. 3. Barkman TJ, McNeal JR, Lim SH, Coat G, Croom HB, Young ND, dePamphilis CW. Mitochondrial DNA suggests at least 11 origins of parasitism in angiosperms and reveals genomic chimerism in parasitic plants. BMC Evol Biol. 2007;7:248. 3. Barkman TJ, McNeal JR, Lim SH, Coat G, Croom HB, Young ND, dePamphilis CW. Mitochondrial DNA suggests at least 11 origins of parasitism in angiosperms and reveals genomic chimerism in parasitic plants. BMC Evol Biol. 2007;7:248. 29. Matusova R, Rani K, Verstappen FW, Franssen MC, Beale MH, Bouwmeester HJ. The strigolactone germination stimulants of the plant-parasitic Striga and Orobanche spp. are derived from the carotenoid pathway. Plant Physiol. 2005;139(2):920–34. 4. Bellot S, Cusimano N, Luo SX, Sun GL, Zarre S, Groger A, Temsch E, Renner SS. Assembled plastid and mitochondrial genomes, as well as nuclear genes, place the parasite family Cynomoriaceae in the Saxifragales. Genome Biol Evol. 2016;8(7):2214–30. 4. Bellot S, Cusimano N, Luo SX, Sun GL, Zarre S, Groger A, Temsch E, Renner SS. Assembled plastid and mitochondrial genomes, as well as nuclear genes, place the parasite family Cynomoriaceae in the Saxifragales. Genome Biol Evol. 2016;8(7):2214–30. 30. Yoder JI. A species-specific recognition system directs haustorium development in the parasitic plant Triphysaria (Scrophulariaceae). Planta. 1997;202(4):407–13. 5. Thurman LD. Genecological studies in Orthocarpus subgenus Triphysaria. Berkeley: University of California; 1966. 5. Thurman LD. Genecological studies in Orthocarpus subgenus Triphysaria. Berkeley: University of California; 1966. 31. Atsatt PR, Guldberg LD. Host influence on floral variability in Orthocarpus densiflorus (Scrophulariaceae). Plant Syst Evol. 1978;129(3):167–76. 6. Estabrook E, Yoder J. Plant-plant communications: rhizosphere signaling between parasitic angiosperms and their hosts. Plant Physiol. 1998;116(1):1–7. 32. Atsatt PR. Consent for publication Not applicable. Consent for publication Not applicable. 21. Yoder JI, Scholes JD. Host plant resistance to parasitic weeds; recent progress and bottlenecks. Curr Opin Plant Biol. 2010;13(4):478–84. The authors declare that they have no competing interests. 22. Irving LJ, Cameron DD. You are what you eat: interactions between root parasitic plants and their hosts. In: Advances in Botanical Research, Vol 50. Vol. 50; 2009. p. 87–138. Funding Thi 16. Umehara M, Hanada A, Yoshida S, Akiyama K, Arite T, Takeda-Kamiya N, Magome H, Kamiya Y, Shirasu K, Yoneyama K, et al. Inhibition of shoot branching by new terpenoid plant hormones. Nature. 2008;455(7210):195–200. Funding This research was supported by awards DBI-0701748 and IOS-1238057 to C.W.D. and J.H.Y., and by The United States Department of Agriculture's Agricultural Research Service. 17. Al-Babili S, Bouwmeester HJ. Strigolactones, a novel carotenoid-derived plant hormone. Annu Rev Plant Biol. 2015;66:161–86. Authors’ contributions C i d d i f Conception and design of study LAH, CWD, NSA, JIY; greenhouse work, experimental setup, plant cultivation LAH, SJ, NF, WK, KV, HZ, JIY; data analysis and presentation LAH, SJ, NF, WK, KV, HZ, NSA, CWD; wrote manuscript LAH, CWD. All authors read and approved the final manuscript. Page 11 of 12 Page 11 of 12 Page 11 of 12 Honaas et al. BMC Plant Biology (2019) 19:334 Honaas et al. BMC Plant Biology (2019) 19:334 Availability of data and materials 18. Conn CE, Bythell-Douglas R, Neumann D, Yoshida S, Whittington B, Westwood JH, Shirasu K, Bond CS, Dyer KA, Nelson DC. Convergent evolution of strigolactone perception enabled host detection in parasitic plants. Science. 2015;349(6247):540–3. Availability of data and materials Transcriptome data is available at http://ppgp.huck.psu.edu. Availability of data and materials Transcriptome data is available at http://ppgp.huck.psu.edu. Ethics approval and consent to participate Not applicable. Ethics approval and consent to participate Not applicable. 19. Akiyama K, Matsuzaki K, Hayashi H. Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi. Nature. 2005;435(7043):824–7. 20. Bouwmeester HJ, Roux C, Lopez-Raez JA, Becard G. Rhizosphere communication of plants, parasitic plants and AM fungi. Trends Plant Sci. 2007;12(5):224–30. Competing interests p g The authors declare that they have no competing interests. Author details 1 1Intercollege Graduate Program in Plant Biology, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA. 2Department of Biology, The Pennsylvania State University, University Park, PA 16802, USA. 3Department of Statistics and Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA. 4Department of Plant Sciences, University of California, Davis, CA 95616, USA. 5Present address: Physiology and Pathology of Tree Fruits Research, USDA - Agricultural Research Service, Wenatchee, WA 98801, USA. 23. Spallek T, Mutuku M, Shirasu K. The genus Striga: a witch profile. Mol Plant Pathol. 2013;14(9):861–9. 24. Lendzemo VW, Kuyper TW, Matusova R, Bouwmeester HJ, Van Ast A. Colonization by arbuscular mycorrhizal Fungi of Sorghum leads to reduced germination and subsequent attachment and emergence of Striga hermonthica. Plant Signal Behav. 2007;2(1):58–62. 25. Yoder JI. Host-plant recognition by parasitic Scrophulariaceae. Curr Opin Plant Biol. 2001;4(4):359–65. 26. Bandaranayake PC, Filappova T, Tomilov A, Tomilova NB, Jamison-McClung D, Ngo Q, Inoue K, Yoder JI. A single-electron reducing quinone oxidoreductase is necessary to induce haustorium development in the root parasitic plant Triphysaria. Plant Cell. 2010;22(4):1404–19. Received: 1 March 2018 Accepted: 30 May 2019 Received: 1 March 2018 Accepted: 30 May 2019 References Functional genomics of a generalist parasitic plant: laser microdissection of host-parasite interface reveals host- specific patterns of parasite gene expression. BMC Plant Biol. 2013;13(9):9. 50. Institute S: SAS/STAT 9.1 User's Guide the Reg Procedure:(Book Excerpt): SAS Institute; 2008. 50. Institute S: SAS/STAT 9.1 User's Guide the Reg Procedure:(Book Excerpt): SAS Institute; 2008. 51. Dunnett CW. New tables for multiple comparisons with a control. Biometrics. 1964;20(3):482–91. 52. Finlayson GD, Schiele B, Crowley JL. Comprehensive colour image normalization. In: European conference on computer vision. Berlin: Springer; 1998. p. 475–90. 52. Finlayson GD, Schiele B, Crowley JL. Comprehensive colour image normalization. In: European conference on computer vision. Berlin: Springer; 1998. p. 475–90. 53. Hunt R. Chromatic adaptation transforms and a colour inconstancy index. The Reproduction of Colour. 6th ed. 2004; p. 587–95. 54. Tooms MS. Colour reproduction in electronic imaging systems: photography, television, cinematography. Chichester: Wiley; 2016. 55. Luo M, Hunt R. A chromatic adaptation transform and a colour inconstancy index. Color Res Appl. 1998;23(3):154–8. References Shen H, Ye W, Hong L, Huang H, Wang Z, Deng X, Yang Q, Xu Z. Progress in parasitic plant biology: host selection and nutrient transfer. Plant Biol (Stuttg). 2006;8(2):175–85. 40. Runyon JB, Mescher MC, De Moraes CM. Volatile chemical cues guide host location and host selection by parasitic plants. Science (New York, NY). 2006;313(5795):1964–7. 14. Yoshida S, Cui S, Ichihashi Y, Shirasu K. The Haustorium, a specialized invasive organ in parasitic plants. Annu Rev Plant Biol. 2016;67:643–67. 15. Cook C, Whichard LP, Wall M, Egley GH, Coggon P, Luhan PA, McPhail A. Germination stimulants. II. Structure of strigol, a potent seed germination stimulant for witchweed (Striga lutea). J Am Chem Soc. 1972;94(17):6198–9. 41. Henning S. Heide-Jørgensen: introduction: the parasitic syndrome in higher plants. In: Joel DM, Gressel J, Musselman LJ, editors. Parasitic Page 12 of 12 Honaas et al. BMC Plant Biology (2019) 19:334 Honaas et al. BMC Plant Biology (2019) 19:334 Orobanchaceae: parasitic mechanisms and control strategies. Berlin: Springer; 2013. p. 1–14. 42. Press MC, Smith S, Stewart GR. Carbon acquisition and assimilation in parasitic plants. Funct Ecol. 1991;5(2):278–83. 43. Tuohy J, Smith EA, Stewart GR: The parasitic habit: trends in morphological and ultrastructural reductionism. In Proceedings of Biology and Control of Orobanche: 13–17 January 1986; Wageningen, Netherlands. Edited by S. J. ter Borg; pp. 86–95. 44. Shah N, Smirnoff N, Stewart GR. Photosynthesis and stomatal characteristics of Striga hermonthica in relation to its parasitic habit. Physiol Plantarum. 1987;69(4):699–703. 45. Press MC, Tuohy JM, Stewart GR. Gas exchange characteristics of the sorghum-striga host-parasite association. Plant Physiol. 1987;84(3):814–9. 46. Dener E, Kacelnik A, Shemesh H. Pea plants show risk sensitivity. Curr Biol. 2016;26(13):1763–7. 46. Dener E, Kacelnik A, Shemesh H. Pea plants show risk sensitivity. Curr Biol. 2016;26(13):1763–7. 47. Atsatt PR. Hemiparasitic flowering plants: phenotypic canalization by hosts. Nature. 1970;225(5238):1161–3. 47. Atsatt PR. Hemiparasitic flowering plants: phenotypic canalization by hosts. Nature. 1970;225(5238):1161–3. 48. Kacelnik A, Teson M. Risky theories—the effects of variance on foraging decisions. Integr Comp Biol. 1996;36(4):402–34. 49. Honaas LA, Wafula EK, Yang Z, Der JP, Wickett NJ, Altman NS, Taylor CG, Yoder JI, Timko MP, Westwood JH, et al. Functional genomics of a generalist parasitic plant: laser microdissection of host-parasite interface reveals host- specific patterns of parasite gene expression. BMC Plant Biol. 2013;13(9):9. 49. Honaas LA, Wafula EK, Yang Z, Der JP, Wickett NJ, Altman NS, Taylor CG, Yoder JI, Timko MP, Westwood JH, et al. Honaas et al. BMC Plant Biology (2019) 19:334 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
https://openalex.org/W4211109235	https://revistas.unisimon.edu.co/index.php/innovacioning/article/download/5546/5327, https://dialnet.unirioja.es/descarga/articulo/8178253.pdf	es		Revisión sistemática de literatura: MOOC K-12 y STEAM	Investigación e innovación en ingenierías	2,021	cc-by	10,765	Revisión sistemática de literatura: MOOC K-12 y STEAM Systematic literatura review: MOOC K-12 and STEAM Ginna Viviana Leytón Yela Víctor Andrés Bucheli Guerrero Universidad del Valle, Colombia Hugo Armando Ordoñez Erazo Universidad del Cauca, Colombia Resumen Recibido: 22/09/2021 Aceptado: 22/10/2021 Publicado: 13/12/2021 Correspondencia de autores: hugoordonez@unicauca.edu.co Objetivo: Identificar los trabajos de investigación centrados en los MOOC K-12 o STEAM, enfocados en el potencial de los entornos de educación, remodelando el marco educativo actual en la educación secundaria. Metodología: Se definieron ecuaciones para realizar las búsquedas de articulos publicados en conferencias y revistas mediante bases de datos bibliográficas, que permitieron sustraer los articulos para construir el corpus de referencias. Posteriormente, se analizan y sintetizan las investigaciones más significativas para el desarrollo de la revisión sistemática. Resultados: Esta revisión permitió identificar las tendencias, desafíos y oportunidades sobre MOOC K-12 o STEAM, identificando las implementaciones de cursos de ciencias de computación o pensamiento computacional en educación secundaria. Este tipo de iniciativas han obtenido resultados positivos, puesto que se han incrementado el número de estudiantes que seleccionan la programación informática como eje fundamental en el examen nacional para educación superior. Conclusiones: Las investigaciones se han centrado en su mayoría en la incorporación de la programación con cursos complementarios ofertados de forma opcional en educación secundaria, con un enfoque combinado, es decir, se llevan a cabo clases tradicionales con el apoyo de los MOOC. La gran mayoría de los MOOC para educación secundaria se han implementado específicamente para satisfacer las necesidades de los docentes y estudiantes de educación secundaria, con el fin de mejorar y apoyar el plan de estudios, y motivar a los estudiantes como futuros miembros activos en la creación de tecnología. Palabras clave: MOOC, cursos online masivos y abiertos, K-12, STEM, STEAM, educación secundaria, pensamiento computacional. Abstract Copyrith 2020 by Investigación e Innovación en Ingenierías Objective: To identify research works focused on MOOCs K-12 or STEAM, focused on the potential of education environments, reshaping the current educational framework in secondary education. Methodology: Equations were defined to search for articles published in conferences and journals using bibliographic databases, which allowed subtracting the articles to build the corpus of references. Subsequently, the most significant investigations for the development of the systematic review are analyzed and synthesized. Results: This review will identify trends, challenges, and opportunities on MOOC K-12 or STEAM, identifying the implementations of courses in computer science or computational thinking in education. This type of initiative has obtained favorable results since the number of students who select computer programming as a fundamental axis in the national exam for higher education has increased. Conclusions: Most of the research has focused on incorporating programming with complementary courses offered optionally in secondary education, with a combined approach, that is, traditional classes are carried out with the support of MOOCs. Most MOOCs for secondary education have explicitly been implemented to meet the needs of secondary education teachers and students to enhance and support the curriculum and motivate students as future active members in creating technology. Keywords: MOOC, massive open online courses, K-12, STEM, STEA. Como citar (IEEE): G. Leytón-Yela., V. Bucheli-Guerrero., y H. Ordoñez-Erazo. “Revisión sistemática de literatura: MOOC K-12 y STEAM”. Investigación e Innovación en Ingenierías, vol. 9, n°3, 57-81, 2021. DOI: https://doi.org/10.17081/invinno.9.3.5546 58 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo Introduction For some time now, there has been a great debate on incorporating computer science or programming at the primary and secondary school levels for students to be active members of society, not only as consumers of technology but also as creators of it in the future. Indeed, countries with more excellent technological experience have begun incorporating the K-12 curriculum in school education, starting in the early grades, and continuing through grade 12 [1]. Students acquire a knowledge base in problem-solving and computational thinking. Likewise, STEAM training presents strategies such as creating clubs, communities, programming, robotics tournaments, and even the teaching of critical thinking and programming languages implemented in their curricula. In the case of Europe, this is done through face-to-face, blended or virtual classes with the support of virtual platforms, code platforms, or massive open online courses [2]. As mentioned above, several computational tools support teaching, among them the open education system that has evolved in various forms. This open trend has developed with MOOCs, which aim at sharing and open access to knowledge. MOOCs are accessible to anyone, regardless of age, educational experience, or location [3]. There has been a growing interest in promoting and teaching programming to the global audience through MOOCs, such as Platzi, edX, Coursera, Udemy, and Udacity. This ever-increasing trend began to focus on lower educational levels, not only higher education. Since 2013, incorporating these types of courses as standalone initiatives for youth and K-12 educators has begun. A small but growing group of researchers [4, 5, 6, 7] have identified that MOOCs in K-12 education can help solve gaps in problem solving of all kinds by using valuable content and different learning strategies. In addition to the importance of equipping the new generations with skills in programming and computational thinking [8]. There is a great deal of academic research related to implementing K-12 or STEAM through platforms such as MOOCs. Still, it is necessary to establish which platforms successfully implemented globally, what beneficial results it has brought in the educational field, and what artificial intelligence (AI) techniques support the development of courses through MOOC-like platforms. The realization of this systematic literature review on the implementation of K-12, STEAM, or computer science or computational thinking courses in secondary education seeks to contribute relevant findings that have transformed education. The paper is organized as follows: the methodological process to select the relevant scientific articles on the subject and those that answer the open research questions defined in the Methodology are presented; then, the analysis of the results obtained in the systematic review classified in the three aspects mentioned above; finally, we showed the discussion of the results of the literature review. Methodology To develop this study, we implemented the systematic literature review methodology to identify evidencebased research, subtract relevant information for future studies, and create knowledge from publications directly related to programming MOOCs in secondary education. We included: • The bibliographic databases. • The identification criteria with the search equations. • The selection criteria allowed obtaining the corpus of references. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 59 Revisión sistemática de literatura: MOOC K-12 y STEAM The stages of the applied methodology are; inquiry, identification, selection, analysis, and synthesis of the selected articles [9]. Table 1. Questions and motivations. Research questions Motivation RQ.1. ¿What is the status of relevant studies on K-12 or STEAM MOOCs about programming MOOCs in secondary education, as a published since 2015? starting point in the recognition of basic concepts such as This question is intended to elicit specific information information about the origins. RQ.2. ¿What K-12 or STEAM MOOC platforms have been successfully implemented in secondary education? RQ.3. ¿How have K-12 MOOC or STEAM platforms transformed education? RQ.4. ¿What AI techniques have been implemented in K-12 or STEAM MOOC platforms? This question is intended to identify the main types of platforms that have achieved good results in secondary education. This question is intended to provide a description of the positive and negative transformation of MOOCs in secondary education. This question is intended to delve into the IA techniques implemented in MOOCs for high school, as a support in the teaching-learning process. Source: Own elaboration. Research questions and motivation for the study We determined a set of research questions and motivation for the study through this process, as shown in table 1. We identified scientific documents such as journals and conferences in Science Direct, Scopus, and Web of Science for the systematic literature review. In the searches, we found 110 papers, of which three were duplicated. Finally, we selected 100 scientific documents to form the final corpus. The following selection criteria made the selection. • Articles published from 2015 onwards. • Scientific publications in English and Spanish. • If there are articles in different bibliographic repositories, compare if they are the same and leave the most recent one. • If there are short and full versions of the same study, the latter is included. • Articles directly related to the subject matter (MOOC K-12 or STEAM). • Articles that present studies related to secondary education. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 60 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo Fig. 1 Stages of systematic review. Source: Own elaboration. Inquiry Stage 1, corresponding to the Inquiry, includes: • Verification of the existence of systematic literature review publications on MOOCs in the subareas of Engineering or Computer Science. • A general search is performed in the selected databases to verify the size of the reference corpus, using the keywords and the initial search string located in Table 2. • Identify the keywords needed in the search equation and the subject areas to consider in the search, selection, and analysis. Identification Stage 2 for identification includes: • With the definition of the research questions and the motivations of the study, we determined and selected the search equation, the relevant bibliographic databases, and the inclusion and exclusion criteria. For the definition of the search equations, we considered the keywords identified in the research topic as mentioned above. • A search is carried out to compile the reference corpus. Table 2 shows the search equations, the results obtained, and the results saved. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 61 Revisión sistemática de literatura: MOOC K-12 y STEAM Table 2. Search equations, data source and number of items retrieved for each search. No. Search string Source Recovered Scopus, Science Direct 51 TITLE-ABS-KEY ( ( ( ”Massive Open Online Course” OR mooc OR 1 moocs ) AND ”literature review” ) ) AND ( EXCLUDE ( PUBYEAR , 2014 ) OR EXCLUDE ( PUBYEAR, 2013 ) OR EXCLUDE ( PUBYEAR , 2012 ) ) ( “programming” AND ”computer programming” ”computational thinking” 2 AND (”Massive Open OR ) AND ( ”K-12” OR stem OR steam) Online Course” OR MOOC OR MOOCs ) AND (LIMIT-TO ( SUBJAREA, ”COMP” ) OR LIMIT-TO ( Scopus, Science Direct, Web of Science 71 SUBJAREA , ”ENGI” )) AND ( EXCLUDE ( EXACTKEYWORD , ”Higher Education” ) ) ( ( programming OR ”computer programming” OR ”computational thinking” ) AND ( ”massive open online 3 courses” OR ”MOOC” ) AND ( ”K-12” OR ”STEM” OR ”STEAM” Scopus, Science Direct 32 ) AND ”artificial intelligence” ) AND ( LIMIT- TO ( SUBJAREA , ”COMP” ) OR LIMIT-TO ( SUBJAREA, ”ENGI” ) ) Source: Own elaboration. Selection Stage 3 for Selection includes: • According to the inclusion and selection criteria, we analyzed the titles, abstracts, and keywords of the studies obtained in the searches. • The quality and relevance of the studies are evaluated by applying a Likert scale to determine whether the articles are transcendental to the study. • We only performed papers with a minimum score of 3 are selected, according to the rankings determined in the study. Analysis and synthesis. Stage 4 for Analysis and synthesis includes: • Obtain and analyze the content of selected studies in databases. • Using reference management software such as Zotero and Mendeley for complete document extraction and detailed bibliographic organization. • Conduct literature analysis within the study and across studies in the final corpus. • Perform a descriptive synthesis to identify the categories of Analysis, for this case, years of publication, authors, types of MOOC platform. • Answering the research questions defined in the methodology. The most notable features of the corpus are extracted. For this purpose, we used tools such as SARS and VOSViewer to identify other keywords and authors relevant to the research, to include them in the search equations. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 62 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo Results This section aims to present the most significant results of the systematic literature review carried out between February 2021 and June 2021. When identifying the articles in the selection stage, the strategies implemented in secondary education for learning programming through MOOC K-12 or STEAM type platforms are analyzed. In the same way, the benefits or advantages of implementing this type of course in secondary education and the IA techniques implemented in this type, of course, are identified. In this way, we answered each of the research questions defined in the methodology. Stage 1: Inquiry, previous literature review. Initially, prior consultation of systematic literature reviews related to MOOCs, published between 2015 and 2019, is carried out. This initial exploration allows identifying existing reviews about MOOCs that could support the search process through the methodologies applied in such reviews, the topics addressed, the issues resolved, and keywords established, in addition to being a starting point for the preliminary search processes in this research. We selected seven reviews of the literature on MOOCs. In 2018, there are systematic literature review studies [10, 11, 12]. The first one allows identifying the accessibility requirements of MOOCs around MOOC platforms and content providers, considered accessibility needs for students with disabilities, elderly students, and foreigners. The second examines MOOC implementations and evaluations, typically done by rating successes and challenges in MOOC revenue earned, acceptance, and completion rates. And the third focuses on understanding the implications of taking a MOOC for students, examining subjective considerations inherent in learning such as motivation, emotional and intellectual engagement. In 2020, there will be studies related to MOOC [13] about the origins and the evolution of MOOC concepts from 2012 to 2019. In addition, the article shows the MOOC-type platforms that have given language courses, their strengths, and limitations. In the same way, special mention is made of the specialized courses and didactic applications present in MOOCs. Another work [14] addresses the recommendation systems in MOOCs, emphasizing the need for this type of system, the proposed systems, and their implementation through videos, books, and the application of artificial intelligence. Finally, this year’s study [15] focuses on the K-12 MOOC model, in which consistency is sought with pedagogical methods that best suit how these types of students learn. A blended MOOC model can enhance learning and ensure teaching efficiency in introductory courses, which could help students prepare for college or as capstone courses for underachieving students or advanced courses that offer new teaching subjects. In 2021, the study [16] presents the current support for self-regulated learning in MOOCs, using technologies based on psychological models between 2010 and 2020. This study was working on student abandonment that mainly occurs in MOOCs due to the lack of methodologies supported by ICT, designed to help students self-regulate their learning. After this initial search, we carry out specific investigations to access the articles that can answer the research questions defined in the methodology. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 63 Revisión sistemática de literatura: MOOC K-12 y STEAM Table 3. Selected literature, MOOC reviews. Year Reference Research challenges in accessible MOOCs: a systematic literature review 2008–2016 [10]. 2018 A thematic literature review of the implementation of MOOCs - 2008 to 2018 [11]. Survey on understanding the implications of MOOCs in engineering education [12]. Recommender Systems for MOOCs: A Systematic Literature Survey (January 1, 2012 - July 12, 2019) [13]. 2020 MOOCs: Origins, Concept and Didactic Applications: A Systematic Review of the Literature (2012–2019) [14]. Research trends in K-12 MOOCs: A review of the published literature [15]. 2021 Self-Regulated Learning in Massive Online Open Courses: A State-of-the- Art Review [16]. Source: Own elaboration. Stage 2: Identification. At this stage, we carried out the searches defined in the methodology in Table 2. A total of 111 studies were identified, removing duplicate studies. Table 4 shows the distribution of the studies identified by type and year of publication, including the number in numerical value and percentage. Table 4. Distribution by type and year of the studies identified in the stage 2. 2015 2016 2017 2018 2019 2020 2021 Non-indexed journal articles 2 1 2 1 0 1 0 Indexed articles 8 7 13 24 19 29 4 Total studies 10 8 15 25 19 30 4 (9%) (7%) (14%) (23%) (17%) (27%) (4%) 7 (6%) 104 (94%) 111 Source: Own elaboration. Stage 3: Selection. In this stage, we filtered the number of studies selected, where we identified 60 out of the initial 111 in the previous step. We discarded studies that did not present specific topics or keywords concerning the research. The distribution of the selected studies is in Table 5. Table 5. Distribution by type and year of the studies identified in the stage 3. 2015 2016 2017 2018 2019 2020 2021 Non-indexed journal articles 2 0 2 1 0 1 0 Indexed articles 6 5 11 12 9 10 1 Total studies 8 5 13 13 9 11 1 (13%) (8%) (22%) (22%) (15%) (18%) (2%) 6 (10%) 54 (90%) 60 Source: Own elaboration. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 64 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo Stage 4: Analysis and synthesis. The selected and analyzed researchers answer the research questions defined in Table 1 of the methodology section. The following table (Table 5) shows detailed information about each one of the items in chronological order by year of publication, title, type of publication, source (Scholar Google GS, Scopus SCO, Science Direct SCD, Web of Science WOS), research dimension as well as the area of interest defined in [14] and implemented in this research. For being of great importance and relevance to this: • Origins, definitions, and concepts related to MOOCs. • MOOC platforms were implemented successfully. • MOOCs or specialized courses that have transformed secondary education. • IA techniques implemented in MOOCs). Table 6. Detailed information on the selected studies. Year Title Document type Source Investigation dimension GS thinking in teaching/programming, Computational thinking and 2015 the new learning 2015 Developing a Computer Programming MOOC [8] Computer programming and robotics in basic education [17] area Implementation of computational Article ecologies [2] 2015 Interest 4 school learning. Article SCD Methodology analysis for the creation of MOOCs. 1 Implementation of computational Article GS thinking in teaching/programming, 2, 3 school learning. A Purposeful MOOC to 2015 Alleviate Insufficient CS Education in Finnish Schools Article SCO Article SCD Article SCO Analysis of MOOC implementation in schools. 1, 2, 3 [18] 2015 Learning outside the classroom through MOOCs [19] A Global Snapshot of 2015 Computer Science Education Analysis of MOOC implementation in schools. 1, 2, 3 Implementation of computational in K-12 Schools [20] thinking in teaching/programming, 2,3 school learning. Designing for deeper learning 2015 in a blended computer science course for middle school Article WOS Article SCO Article SCO Analysis of MOOC implementation in schools. 2, 3 students [21] Programming web-course 2015 analysis: How to introduce computer programming? [22] Analysis of MOOC implementation in schools. 1, 2 Code Yourself” and “A 2016 Programar”: A bilingual MOOC for teaching computer science Analysis of MOOC implementation in schools. 1, 2, 3 to teenagers [23] Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 65 Revisión sistemática de literatura: MOOC K-12 y STEAM PC-01: Introduction to computational thinking: 2016 Educational technology Implementation of computational Article GS in primary and secondary thinking in teaching/programming, 1, 2, 3 school learning. education [24] A Review of Models for Introducing Computational 2016 Thinking, Computer Science Implementation of computational Article SCO and Computing in K–12 thinking in teaching/programming, 2 school learning. Education [25] The impact of STEM 2016 experiences on student selfefficacy in computational Implementation of computational Article SCO thinking [26] Computer Science & Technology Curriculum in UAE 1 school learning. Towards an Innovative 2016 thinking in teaching/programming, Implementation of computational Article SCO thinking in teaching/programming, 1 school learning. Public Schools System [27] Research challenges 2017 in accessible MOOCs: a systematic literature review State of the art SCO Article GS Article GS State of the art of MOOC. 1 2008–2016 [9] Can MOOCs Support Secondary 2017 Education in Computer Science? [28] Computational thinking 2017 in Colombian schools: international collaboration on Analysis of MOOC implementation in schools. 1, 2, 3 Implementation of computational thinking in teaching/programming, 1 school learning. innovation in education[29] The teaching of computer programming in Primary 2017 Education: current situation, analysis and presentation Implementation of computational Thesis GS thinking in teaching/programming, 2 school learning. of classroom experiences in Spain [30] Adoption of Computer 2017 Programming Exercises for Implementation of computational Article SCO Automatic Assessment [31] Principles to K-12 Students: A Article SCO Article SCO MOOC on Scratch [32] Teaching CS to CS teachers: 2017 Addressing the need for advanced content in K-12 of mathematics [34] in schools. 1, 2, 3 thinking in teaching/programming, 3 school learning. Computational thinking as an emergent learning trajectory Analysis of MOOC implementation Implementation of computational professional development [33] 2017 3 school learning. Teaching Software Engineering 2017 thinking in teaching/programming, Article SCO Analysis of MOOC implementation in schools. 2, 3 Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 66 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo Educating Computer Science 2017 Educators Online - A Racket MOOC for Elementary Math Article SCO Article SCO Analysis of MOOC implementation in schools. 2, 3 Teachers of Finland [35] A Tool for Introducing 2017 Computer Science with Automatic Formative Implementation of computational thinking in teaching/programming, 3 school learning. Assessment [36] Computational Thinking as 2017 Springboard for Learning Object-Oriented Programming Article SCO Article WOS Analysis of MOOC implementation in schools. 1, 2, 3 in an Interactive MOOC [37] Different underlying motivations and abilities 2017 predict student versus teacher Implementation of computational persistence in an online thinking in teaching/programming, 1, 2, 3 school learning. course [38] MOOC architecture model 2017 for computer programming State of the art SCO State of the art on MOOC. 1 Article SCO Evaluation of motivation in MOOC. 1, 2 State of the art SCO State of the art on MOOC. 1 Article SCO Article GS Article SCO State of the art SCO Article SCO Article SCO courses [39] Survey on understanding the 2018 implications of MOOCs in engineering education [12] A thematic literature review of 2018 the implementation of MOOCS - 2008 to 2018 [11] Troubleshooters for tasks of 2018 introductory programming MOOCs [7] Analysis of MOOC implementation in schools. 1, 2, 3, 4 ¿Can programming really be 2018 for everyone? Analysis of the experience within a MOOC. Analysis of MOOC implementation in schools. 1, 2 [40] Incorporating Computational Thinking in the Classrooms 2018 of Puerto Rico: How a MOOC Served as an Outreach and Analysis of MOOC implementation in schools. 1, 2, 3 Recruitment Tool for Computer Science Education [41] Teaching Software Engineering 2018 in K-12 Education: A Systematic State of the art on MOOC. 1, 2, 3 Mapping Study [42] Supporting Computational 2018 Thinking Development in K-6 [43] Improving Assessment of 2018 Computational Thinking Through a Comprehensive Framework [44] Analysis of MOOC implementation in schools. 2, 3 Implementation of computational thinking in teaching/programming, 3 school learning. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 67 Revisión sistemática de literatura: MOOC K-12 y STEAM 2018 Code ABC MOOC for math teachers [45] Analysis of MOOC implementation Article SCO State of the art SCO State of the art on MOOC. Article SCO thinking in teaching/programming, in schools. 2, 3 Computational Thinking and 2018 Online Learning: A Systematic 3 Literature Review [46] A Free-Choice Social Learning 2018 2018 Network for Computational Implementation of computational Thinking [47] school learning. Computational Thinking Implementation of computational in pre-university Blended Article SCD Learning classrooms [48] 2018 MOOC: Computational thinking (applied) for teachers [49] for playfully teaching programming concepts to Article SCO Article SCO What motivates enrolment in programming MOOCs? [51] Analysis of MOOC implementation in schools. 1, 2, 3 Implementation of computational thinking in teaching/programming, 1, 2, 3 school learning. school teachers [50] 2019 2, 3 school learning. A blended learning course 2019 thinking in teaching/programming, 3, 4 Article WOS Article SCO Article SCO Evaluation of motivation in MOOC. 1 Evaluating Digital Worksheets 2019 with Interactive Programming Exercises for K-12 Education Analysis of MOOC implementation in schools. 1, 2, 3 [52] Integrating Professional Tools 2019 in Programming Education with MOOCs [53] Preparing K-12 Teachers to 2019 Inspire Future Coders: It Doesn’t Have to be Complex Article SCO 1, 2, 3 thinking in teaching/programming, 2, 3 school learning. [54] as Collaboration Platform in a in schools. Implementation of computational Exploring the Role of Facebook 2019 Analysis of MOOC implementation Article SCO Article SCO Article GS K-12 MOOC [55] Analysis of MOOC implementation in schools. 1, 2, 3 MOOCs in Secondary 2019 Education - Experiments and Observations from German Analysis of MOOC implementation in schools. 1, 2, 3 Classrooms [56] Computational thinking for preservice 2019 teachers in Thailand: A Implementation of computational confirmatory thinking in teaching/programming, 3 school learning. factor analysis [57] Early Programming Education and Career Orientation: The 2019 Effects of Gender, Self-Efficacy, Motivation and Stereotypes Implementation of computational Article SCO thinking in teaching/programming, 3 school learning. [58] Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 68 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo MOOCs: Origins, Concept 2020 and Didactic Applications: A Systematic Review of the State of the art GS State of the art on MOOC. 1, 2, 3 State of the art SCO State of the art on MOOC. 1, 2, 3 State of the art GS State of the art on MOOC. 1, 3 Article SCO Article GS Literature (2012–2019) [14] Research trends in K-12 2020 MOOCs: A review of the published literature [15] Recommender Systems 2020 for MOOCs: A Systematic Literature Survey (January 1, 2012 - July 12, 2019) [13] A computer programming 2020 hybrid MOOC for Greek secondary education [5] Analysis of MOOC implementation in schools. 1, 2, 3 Modelo y lecciones aprendidas 2020 del proceso de creación de MOOCs para enseñar a Analysis of MOOC implementation in schools. 1, 2, 3 programar [6] Building a Community of STEM 2020 Educators in Nigeria Using the Implementation of computational Article SCO TeachAKid2Code Program [59] thinking in teaching/programming, 1, 2, 3 school learning. Computational thinking and 2020 assignment resubmission predict persistence in a Article SCO Article SCO Article SCO Analysis of MOOC implementation in schools. 1, 2, 3 computer science MOOC [60] EarSketch: An authentic, 2020 STEAM-based approach to computing education [61] Analysis of MOOC implementation in schools. 2, 3 Education in programming and mathematical 2020 learning: Functionality of a Implementation of computational programming language in thinking in teaching/programming, 3 school learning. educational processes [62] Programming MOOCs–different 2020 learners and different Article SCO Article SCO State of the art GS Evaluation of motivation in MOOC. 1, 2 motivation [63] Analyzing learners’ 2020 engagement and behavior in MOOCs on programming with Programming MOOC with the use of external tools. 1, 3 the Codeboard IDE [64] Self-Regulated Learning in 2021 Massive Online Open Courses: State of the art on MOOC. 1 A State-of-the-Art Review [16] Source: Own elaboration. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 Revisión sistemática de literatura: MOOC K-12 y STEAM Fig. 2 Distribution over time in the areas of interest in MOOC K-12 or STEAM defined in this research. Source: Own elaboration. Figure 2 shows the trend in the areas of interest researched over time. The year 2018 shows an increase in research corresponding to K-12 or STEAM MOOCs, focusing on MOOC-type courses or specialized courses in secondary education computer science and specifying the successfully implemented platforms. Furthermore, it is among the few years that present evidence of AI implementation in MOOCs for secondary education. Discussion The results obtained in this systematic review have shown that the number of studies related between MOOCs and K-12, STEM, or STEAM is significant. Some of them have found relevant information on MOOCs and K-12 education, STEAM, and computational thinking aimed at teachers and students. This section provides answers to each of the questions defined in the methodology, based on the main findings. Status of relevant research related to MOOC K-12 or STEAM. This section establishes the answer to the first research question, where we identified K-12 or STEAM courses implemented in secondary education through MOOC-type platforms. We considered the conceptual information corresponding to MOOCs, history, types of platforms cited in some studies. Of the 60 studies selected, 38 provide conceptual information on MOOCs, i.e., 63% of the studies show a general introduction to the type of platforms, basic concepts, history, and complementary information. It is relevant to mention the most significant findings in this field. Typically, studies start with a brief introduction to open learning as a critical factor to address the multiple challenges continuously presented worldwide, where information technologies have transformed education, provoking new ways of learning and new ways of offering education [8]. In this context, MOOCs have emerged as free online courses, easily accessible, to teach a global audience [3], in the case of this research to the early teaching of programming. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 69 70 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo The definition of the MOOC concept is coined by [3] as follows: “A MOOC is an online course with the option of open registration, a publicly shared curriculum, and expected outcomes. MOOCs integrate social networking, accessible online resources, and are facilitated by professional leaders in the field of study” [65]. The MOOC concept was known since 2008 and addressed by [14] through the University of Manitoba in Canada, which introduced the origin of the MOOC concept, alluding to "Connectivism and Connective Knowledge." Subsequently, the idea became relevant in 2011 with another Stanford University course on Artificial Intelligence, creating several MOOCs, mainly focused on educational technologies [64]. This includes various universities, organizations, and entrepreneurship companies that offer MOOCs, including leading universities such as the Massachusetts Institute of Technology (MIT), Harvard University, Stanford University, University of Edinburgh, and platforms designed as Coursera, edX, FutureLearn, Udemy, among others. The first, associated with Stanford, Princeton, and other universities, the second, founded by MIT and Harvard, the third was based by The Open University and, Udemy, which has been targeted for adult professionals and businesses [3]. Regarding the most significant characteristics of MOOCs, such as their open access, offered through the Internet and freely available on a massive scale, they also have a series of resources, start and end dates, and evaluation methods [3, 6, 14]. Regarding the characteristics of MOOCs aimed at K-12 teachers and students, studies have appeared in the literature. Since 2013 [32, 42, 52, 54], there have been studies on interaction in K-12 MOOCs and their benefits, as to the great potential in the enrichment of learning opportunities and the adaptation of pedagogical models according to this population [15]. On the other hand, we mentioned different forms of implementation, (I1) Support to curriculum that includes computational thinking, (I2) Preparation for tertiary education exams, (I3) Complementary courses for articulation process, and (I4) Complementary approaches offered optionally in secondary education. Table 7 specifies the jobs related to each of the K-12 or STEAM MOOC implementation forms. The 48% of the studies correspond to the implementation of K-12, STEAM, computational thinking in primary and secondary through an education curriculum. There are curriculum regulations or subjects with logical development and programming for problem-solving in various countries and institutions. In the second place, with 20%, studies related to preparation for tertiary education, where MOOCs in K-12 education are cited, have had a positive result in the professional development of students and teachers and preparation for national exams for access to higher education. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 71 Revisión sistemática de literatura: MOOC K-12 y STEAM Fig. 3 Distribution of the different types of implementations between 2015 and 2021. Source: Own elaboration. Table 7. Selected studies with implementation of K-12 or STEAM MOOCs. Area of interest 2015 2016 2017 2018 2019 2020 [2, 17, 19] [25] [28, 30] [43, 45, 49] [67, 53] [60] NA NA [37] [7, 48] [54] [5] NA [24] [29] NA NA [58, 63] [31, 32, 33, 34, [40, 41, 44, [50, 51, 52, 55, 35, 36, 38] 46, 47] 56, 57] Curriculum support that includes computational thinking (I1) Tertiary education exam preparation (I2) Complementary courses for articulation processes (I3) Complementary courses offered as an [8, 18, 20, option in secondary 21, 22] [3, 6, 26] [59, 61, 62] education (I4) Source: Own elaboration. Finally, in small percentages of 8% and 7%, there are studies related to complementary courses for articulation processes and complementary courses offered optionally in secondary education, respectively. Of the 60 articles selected, 10 (17%) do not specify the type of implementation since they show the systematic literature review of MOOCs, MOOC K-12 or STEAM, methodologies for MOOC development, among others. K-12 or STEAM MOOC platforms successfully implemented in secondary education Many studies provide information on the success of K-12 or STEAM MOOCs; 39 (65%) of the 60 studies selected delved into the success or limitations that have arisen in the implementation in secondary education. Among the results found, it is paramount to include computational thinking in primary and secondary education, as is the case of the K-12 curriculum [28], which incorporates the fundamental concepts and programming languages in education. Or also implemented individually as PC-01 as in studies [24, 29, 48]; where students successfully achieved the academic objectives established in the project, expressing a Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 72 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo feeling of satisfaction with participation in the course with motivating activities and creative interests, establishing these implementations both in high schools in the Dominican Republic and Colombia. For the methodology of this type of courses, video lessons, interactive tests with Scratch, evaluate, be evaluated, and participate in forums were used; this was a project supported by Renata and the University of the Basque Country. Another successful platform is the Core.org MOOC, which is part of an American nongovernmental organization with the same name, which has more significant influence worldwide with the promotion of programming in school, has the initiative that all students should learn to program, applying K-12 [1, 30]. Within its curriculum they include, formulating problems to be solved with the computer, organizing and analyzing logically, representing data with models, automating solutions with algorithmic thinking, identifying, analyzing, and implementing possible solutions, generalizing and transferring the solution. This knowledge is transversal in the educational systems, and the premise is that all students should learn to program. Both the K-12 curriculum and the Q2L curriculum [2] have been implemented in this same country. They design games and thinking systems that require computational thinking, using the Gamestar Mechanic software to develop video games. Given the deficit of engineers in Europe, a program called" Opening up Education" was created to teach programming to children and young people and thus raise students' interest in engineering. From this project, others emerged, such as the Autonomous Community of Madrid that incorporated the subject" Technology, programming and robotics" in high school, established by a decree in 2015 [2]. It is essential to train the teachers who implement the courses; this training is carried out with the support of Telefonica through a MOOC. Likewise, the Universidad Oberta de Catalunya [40] promotes activities to develop computational thinking, with programming to society, through a MOOC of introduction to programming with a learning-by-doing methodology for participants of all ages, this methodology emphasizes practice over theory. More than 2,000 people have participated in classroom training in the clubs and online courses. By converting thought into objects through algorithms, data structure, and procedures, personal knowledge is shared with others, making computational thinking participatory. On the other hand, in the United Kingdom, the educational reform law was created, which incorporates three core subjects: mathematics, science, and English, and additionally, the subject Computing, in which Computer Science is recognized as a rigorous academic discipline for the careers of the future, it is compulsory in primary and secondary schools [2]. It covers algorithms, data structures, and computer programming. The teaching is divided into stages KS1 to KS4 (from 5 to 16 years old). There are some criticisms from teachers for not carrying out prior training to implement this type of project. Therefore, recommended for learning assessment technologies such as online courses, for example, the MOOC entitled Cambridge GCSE Computing Online, designed to support the teaching and learning of Computing in schools. Another pioneer country in implementing computer programming and robotics in basic education curricula is Estonia [30]. They included the introduction of programming in its schools, starting in primary education, through the ProgeTiger program financed by the government and created in 1997, enabling schools to have a broadband connection, providing teacher training in the use of TIC. They also allow virtual learning environments to develop didactic material. Since 2012, pilot projects have been initiated at different educational levels. The teachers select the programs to be used in the subjects; they can also incorporate in their curriculum subjects from other technological areas, the objective being that the skills provided to the students in TIC support the digital progress of Estonia and are a source of Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 Revisión sistemática de literatura: MOOC K-12 y STEAM prosperity for the country [17]. One of the problems that have arisen is the availability of programming teachers; for this reason, the most advanced students have been asked to teach MOOC-type courses titled to instruct thousands of young people in secondary education. Similarly, in Finland, the University of Helsinki prepared a CS1 MOOC for high school students as a first pilot plan. They approach computer-related topics to motivate participation in more courses and even careers [64]. As mentioned above, one of the major problems is the preparation of teachers in programming, for this reason; in Finnish schools, mathematics teachers were prepared through a programming MOOC to support Finnish education; through workshops, the ABC MOOC, and the Racket MOOC [18, 28, 35, 45]. This type of strategy has been very successful in regulating programming in the subject of mathematics in Finland. In the case of Greece [4], they decided to create a programming MOOC through the blended learning approach with example and problem-solving. Also, they made PROG15 [5, 54], focusing on how this kind can help prepare for the national tertiary education exams. The students enrolled belong to different Greek vocational schools, and several teachers also enrolled in the course. The course was a great success, as 2,382 students took part in the Greek national computer programming exams, i.e., 12.2% of the population of secondary school students. Similarly, Uruguay and Edinburgh created a MOOC [3, 6], included in the primary and secondary school curricula on the formal study of computer skills and competencies. In addition, the article presents guidelines for developing a MOOC. This massive open online course, with access through the internet for free to many people, consists of videos (explanations or tutorials), texts, images, forums, and evaluations. The University of Edinburgh offers a development model, specifically from Coursera, aimed at young people aged 12 to 17 without programming skills for five weeks, obtained first with national participation. Subsequently, they participated in 117 countries; for this reason, the programming MOOC was developed in collaboration by the teams of the universities mentioned, resulting in a design with bilingual delivery. In Germany, implemented the first MOOC with more than 7000 high school participants was successful in learning the Python programming language. The success and performance rate of the MOOC MINTEC has been positive, especially for participants with previous skills or talents, the aptitude for teamwork, and how the students perceive the peer review evaluation method as a suitable method of assessment. Likewise, there are different European and intercontinental strategies for learning to program in children and young people [30], as described in table 8. In Spain there are several initiatives such as “Programamos Community, Young Programmers Club in Valladolid”, Citilab in Barcelona and Complubot in Madrid. This review provides an answer to the second research question: Which MOOC K-12 or STEAM platforms were successfully implemented in secondary education. Other countries implemented a great variety of strategies such as MOOCs and how these benefit children and young people with the learning of programming. Thus, more curriculums include teaching programming in primary and secondary education, with formal studies of computational skills and competencies. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 73 74 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo Transformation of education with the implementation of MOOC K-12 or STEAM platforms This section answered the third research question raised to determine which case studies have positively transformed secondary education. It identifies the potential, positive experiences, and benefits of MOOCs for students and teachers. Most of the reflections on K-12 or STEAM MOOCs emphasize the implementation of computational thinking and programming and show the potential of MOOCs in learning experiences for teachers and students. Similarly, some studies present the difficulties in implementing such MOOCs in a classroom as a support strategy, preparation for tertiary education, or modality of articulation with higher education. Given that only 20% of related studies have directly incorporated computer science curriculum or subject matter in K-12 education by educational regulation in some countries, it is necessary to study the research done as supplemental courses offered as electives or as course work in secondary education. Table 8. Strategies or projects for teaching programming to children and young people. Name of the Type strategy Description Platform type For kids with Scratch, learning programming CoderDojo Community languages and robotics with 675 clubs in 54 MOOC. countries. It is an open and informal peer-to-peer programming community. It has an event organized Code’s Cool Community by the European Commission every year, called CodeWeek.it. The two organizations collaborate with Collaboration in the creation of a MOOC. the creation of a MOOC. Didactic CS Unplugged Didactic material material It is a collection that aims to teach computer science incorporated in without the need for a computer. MOOCs. With the Hour of Code project, they seek to increase interest and participation in computer science Code.org Non-profit in schools. They advise the U.S. government in organization the design of new educational policies and have MOOC. recognized companies such as Apple and Microsoft that have joined the Code.org movement. It is an initiative of Google and Microsoft, Google, CS First Syllabus and Intellect, which collaborate with the Computer Arts Society (CAS) belonging to the British Computer MOOC. Society. It is a free online platform that offers free Codeacademy Interactive Platform programming courses in different programming languages that offers badges or medals for completing the exercises. Interactive platform created by a MOOC company. It is a platform that offers programming courses by Code school Platform managing practical lessons in which they manage incentives through prize draws. It is now called MOOC. Pluralsight. Scratch Application It was developed by MIT and allows learning Collaboration in the programming in a graphical and very intuitive way. creation of MOOCs. Source: Own elaboration. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 Revisión sistemática de literatura: MOOC K-12 y STEAM In general, many of the studies cited [15, 19, 28] show that MOOCs have the potential to be part of the positive influence in secondary education, presenting a reduction in dropout and support students in the preparation of their university studies. Among these is the preparation for future learning assessments with the MOOC FACT [21] for secondary education, hosted on OpenEdX; in which the outcome of the test is evidenced as promising, as well as positive indicators in the computer science curriculum, which triggered the curiosity to learn more in students. Similarly, different strategies have been implemented in Greece to transform secondary education through programming MOOCs called PROG15 and PROG16 with assistance from social networks such as Facebook [5, 54]. In the first version of the course, a reception of 12.2% of participants was achieved, according to the total number of students belonging to the different Greek vocational schools, thus achieving that more students take the computer programming exams for the national tertiary education exams. On the other hand, it is interesting how using social networks, the number of students increased, more students enrolled and participated more frequently through publications and participation in Facebook. Is this a more attractive place for interaction and collaboration of the course? In this sense, the Facebook groups presented a benefit, transformation, and positive comments complementing MOOC PROG15 and PROG16. In Germany, created systems called MOOCs MintEC [56] to support secondary education classrooms. Demonstrating that it is possible to incorporate the MOOC in the school where teachers have successfully used these courses, both in school, home, and extracurricular environments, as a key and enriching element in education. In the study [28], many of the teachers are skeptical about the integration of digital media, which is why other research seeks to train not only students but also teachers in computational thinking as in the study [18], where teachers in Finland challenged with the regulation of programming in the subject of mathematics. Many of the teachers do not know about organizing and incorporating into their school classes around programming tasks. Still, a programming MOOC supports the Finnish education system and teacher training with up-to-date teaching materials. This programming MOOC has provided opportunities for schools that lack strategies for implementing programming in the curriculum in Finland. In the researches [34, 35, 45], the MOOC ABC was implemented for students and to support mathematics teachers in preparing the programming in their classes. The result is encouraging since this type of resource has allowed the feasible preparation of teachers in teaching programming to take it to the classroom subsequently. Another paper [43] discusses the CSER digital technologies course curriculum in MOOC for the K-6 context, aligning key concepts from formal curriculum statements, teacher evaluations, discussion of key challenges and concerns. A community was created to share ideas of lessons learned and discuss critical challenges identified by teachers within the first-course cohort. So, by using programming MOOCs for teachers as well, favorable results are obtained. Teachers feel better prepared when planning upcoming lessons and better understand what high school students require [52]. Another vital transformation in secondary education occurred in studies [3, 6, 49]. Students from the first MOOC cohort in programming with better academic performance support new students through forums, providing students with a solid foundation in computer science that inspires them to develop their programming skills further. ”The MOOCs developed aim to contribute to logical and computational skills in young people, skills in demand today” [6]. Not only are programming MOOCs are found in the literature, and MOOCs include other essential competencies such as Software Engineering [32, 42]. The results demonstrate the feasibility and positive Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 75 76 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo contribution of having Software Engineering education in K-12; it was also possible to take the course at home with a guardian or parent, being a transforming factor of education by involving parents in this experiment. The results were positive; obtaining high grades. One of the main tasks challenging to control and transform in secondary education is the self-regulation of learning since most young people do not have habits for time management in online education as in the case of MOOCs. Therefore, a literature review on the current state of self-regulated learning support in MOOCs is cited [16], using technologies based on psychological models for 2010 and 2020. It is also essential since it allows working on student dropout that mainly occurs in MOOCs due to the lack of self-regulation in learning. Therefore, K-12 MOOCs have transformed secondary education through a blended classroom model, where the traditional classroom is worked with the support of MOOC-type tools or platforms to motivate students, as is the case of studies [41, 62, 67]. Artificial intelligence in K-12 or STEAM MOOC platforms for secondary education This section answers the fourth research question to determine which case studies have implemented artificial intelligence in programming MOOCs. Only 3 (5%) of the 60 projects present the incorporation of artificial intelligence in programming MOOCs for secondary education. In the project [7], implemented decision trees are used in education, and features are visualized to be easily understandable to those unfamiliar with programming in machine learning. The preprocessed data is used to train a decision tree model to visualize the skill structures that affect the learner in program development. On the Open School platform [47], online learning resources and programming exercises can compile and execute through intelligent tutoring and assessment, including automatically generating feedback to help students improve their learning outcomes. Another study is Alibi [36]. It is a Chatbot designed to support massive open online courses, including automatic formative assessment capabilities with immediate feedback at the homework level. The platform introduces high school students to computer science concepts innovatively, using state-of-the-art natural language processing techniques. As can be observed, there is no evidence in the selected studies of the explicit incorporation of IL techniques in programming MOOCs for secondary education. Very few specify the method they have implemented in summative and formative evaluation or assistance through Chatbot. In future work, it would be essential to develop tools that support the development of various tasks in MOOC-type courses, and even more so for high school students, who need more assistance as they are first-time learners of programming. Conclusions Advances in the development of open and online resources have become a transformative trend in traditional education, as incorporated into university curricula and the potential of K-12 MOOCs in secondary education. This systematic literature review article on the implementation of K-12, STEAM, or programming in secondary education through MOOCs for teachers and students, of which 60 relevant studies published between 2015 and 2021 are related. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 Revisión sistemática de literatura: MOOC K-12 y STEAM This research reveals how they have revolutionized and transformed secondary education for both students and teachers. Most MOOCs for secondary education have been implemented specifically to meet the needs of secondary education teachers and students to enhance and support the curriculum by incorporating computational thinking and programming. Therefore, the existence of a mixed approach is notable with the implementation of the MOOC. On the other hand, studies such as [4, 5, 54] have shown how the implementation of programming MOOCs in Greece has increased the number of students who select computer programming as a fundamental axis in the national examination for higher education. In addition to using social networks to motivate students in the development of the course, however, there are still difficulties in increasing the completion rate of this type of course and the preparation of teachers who support them. Regarding teacher preparation [34, 35, 45], countries such as Finland have obtained good results since before implementing programming in the subject of mathematics in secondary education; teachers were prepared with the necessary knowledge to teach classes using MOOC ABC. This type of initiative obtained favorable results implemented in other countries. The review also identified few studies related to certain aspects of MOOCs, such as the automation or implementation of IA; only 3 of the selected studies addressed the implemented technique, which would allow automating some processes charged to the teacher, as they are massive courses. In the future, it is necessary to expand the research that implements this type of techniques. Bibliographic References 1. Asociación de Maquinaria de Computación, Code.org, Asociación de Profesores de las Ciencias de la Computación, y Centro de Innovación Cibernética y Deepa Muralidhar de la Iniciativa Nacional de Matemáticas y Ciencias, «K – 12 Marco de las Ciencias de la Computación». 2016. 2. J. V. Berrocoso, M. R. F. Sánchez, y M. del C. G. Arroyo, «El pensamiento computacional y las nuevas ecologías del aprendizaje», Rev. Educ. Distancia RED, n.o 46, Art. n.o 46, oct. 2015, Accedido: sep. 23, 2020. [En línea]. Disponible en: https://revistas.um.es/red/article/view/240311 3. I. F. de Kereki y A. Manataki, «“Code Yourself” and “A Programar”: A bilingual MOOC for teaching computer science to teenagers», en 2016 IEEE Frontiers in Education Conference (FIE), oct. 2016, pp. 1-9. doi: 10.1109/FIE.2016.7757569. 4. F. Lazarinis, C. V. Karachristos, E. C. Stavropoulos, y V. S. Verykios, «A blended learning course for playfully teaching programming concepts to school teachers», Educ. Inf. Technol., vol. 24, n.o 2, pp. 1237-1249, mar. 2019, doi: 10.1007/s10639-018-9823-2. 5. P. Koutsakas, C. Karagiannidis, P. Politis, y I. Karasavvidis, «A computer programming hybrid MOOC for Greek secondary education», Smart Learn. Environ., vol. 7, n.o 1, 2020, doi: 10.1186/s40561-0200114-1. 6. I. F. D. Kereki y A. Manataki, «Modelo y lecciones aprendidas del proceso de creación de MOOCs para enseñar a programar», en Sexta Conferencia de Directores de Tecnología de Información, TICAL 2016 Gestión de las TICs para la Investigación y la Colaboración, sep. 2016, pp. 171-184. Accedido: sep. 23, 2020. [En línea]. Disponible en: https://www.research.ed.ac.uk/portal/en/publications/modeloy-lecciones-aprendidas-del-proceso-de-creacion-de-moocs-para-ensenar-a-programar(72f2a621887d-402b-b129-9d651a02446c).html Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 77 78 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo 7. M. Lepp et al., «Troubleshooters for tasks of introductory programming MOOCs», Int. Rev. Res. Open Distance Learn., vol. 19, n.o 4, pp. 56-75, 2018, doi: 10.19173/irrodl.v19i4.3639. 8. «Developing a Computer Programming MOOC», Procedia Comput. Sci., vol. 65, pp. 182-191, ene. 2015, doi: 10.1016/j.procs.2015.09.107. 9. S. Sanchez-Gordon y S. Luján-Mora, «Research challenges in accessible MOOCs: a systematic literature review 2008–2016», Univers. Access Inf. Soc., vol. 17, n.o 4, pp. 775-789, nov. 2018, doi: 10.1007/ s10209-017-0531-2. 10. S. Sanchez-Gordon y S. Luján-Mora, «Research challenges in accessible MOOCs: a systematic literature review 2008–2016», Univers. Access Inf. Soc., vol. 17, n.o 4, pp. 775-789, nov. 2018, doi: 10.1007/ s10209-017-0531-2. 11. T. Prinsloo y A. Ainslie, «A THEMATIC LITERATURE REVIEW OF THE IMPLEMENTATION OF MOOCS - 2008 TO 2018», Proc. 2018 AIS SIGED Int. Conf. Inf. Syst. Educ. Res., ene. 2018, [En línea]. Disponible en: https://aisel.aisnet.org/siged2018/34 12. J. Shailaja y S. Prathikantham, «Survey on understanding the implications of MOOCs in engineering education», 2018. 13. A. Khalid, K. Lundqvist, y A. Yates, «Recommender Systems for MOOCs: A Systematic Literature Survey (January 1, 2012 – July 12, 2019)», Int. Rev. Res. Open Distrib. Learn., vol. 21, n.o 4, pp. 255-291, jun. 2020, doi: 10.19173/irrodl.v21i4.4643. 14. F. J. Palacios Hidalgo, C. A. Huertas Abril, y M. a E. Gómez Parra, «MOOCs: Origins, Concept and Didactic Applications: A Systematic Review of the Literature (2012–2019)», Technol. Knowl. Learn., vol. 25, n.o 4, pp. 853-879, dic. 2020, doi: 10.1007/s10758-019-09433-6. 15. P. Koutsakas, G. Chorozidis, A. Karamatsouki, y C. Karagiannidis, «Research trends in K-12 MOOCs: A review of the published literature», Int. Rev. Res. Open Distance Learn., vol. 21, n.o 3, pp. 285-303, 2020. 16. J. Cerón et al., «Self-Regulated Learning in Massive Online Open Courses: A State-of-the-Art Review», IEEE Access, vol. 9, pp. 511-528, 2021, doi: 10.1109/ACCESS.2020.3045913. 17. J. M. C. Delgado, «Programación informática y robótica en la enseñanza básica», Av. En Supervisión Educ., n.o 24, Art. n.o 24, dic. 2015, doi: 10.23824/ase.v0i24.17. 18. J. Kurhila y A. Vihavainen, «A purposeful MOOC to alleviate insufficient cs education in Finnish schools», ACM Trans. Comput. Educ., vol. 15, n.o 2, 2015, doi: 10.1145/2716314. 19. T. Brahimi y A. Sarirete, «Learning outside the classroom through MOOCs», Comput. Hum. Behav., vol. 51, pp. 604-609, oct. 2015, doi: 10.1016/j.chb.2015.03.013. 20. P. Hubwieser et al., «A global snapshot of computer science education in K-12 schools», 2015, pp. 6583. doi: 10.1145/2858796.2858799. 21. S. Grover, R. Pea, y S. Cooper, «Designing for deeper learning in a blended computer science course for middle school students», Comput. Sci. Educ., vol. 25, n.o 2, pp. 199-237, 2015, doi: 10.1080/08993408.2015.1033142. 22. R. Da Silva Ribeiro, L. De Oliveira Brandao, T. V. M. Faria, y A. A. F. Brandao, «Programming webcourse analysis: How to introduce computer programming?», 2015, vol. 2015-February, n.o February. doi: 10.1109/FIE.2014.7044140. 23. I. F. De Kereki y A. Manataki, «“Code yourself” and “a programar”: A bilingual MOOC for teaching computer science to teenagers», 2016, vol. 2016-November. doi: 10.1109/FIE.2016.7757569. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 Revisión sistemática de literatura: MOOC K-12 y STEAM 24. X. Basogain, M. A. Olabe, J. C. Olabe, R. Ramírez, M. Del Rosario, y J. Garcia, «PC-01: Introduction to computational thinking: Educational technology in primary and secondary education», en 2016 International Symposium on Computers in Education (SIIE), sep. 2016, pp. 1-5. doi: 10.1109/ SIIE.2016.7751816. 25. F. Heintz, L. Mannila, y T. Farnqvist, «A review of models for introducing computational thinking, computer science and computing in K-12 education», 2016, vol. 2016-November. doi: 10.1109/ FIE.2016.7757410. 26. J. L. Weese, R. Feldhausen, y N. H. Bean, «The impact of STEM experiences on student self-efficacy in computational thinking», 2016, vol. 2016-June. 27. J. Al-Karaki et al., «Towards an innovative computer science & technology curriculum in UAE public schools system», 2016, vol. 10-13-April-2016, pp. 883-891. doi: 10.1109/EDUCON.2016.7474656. 28. C. T. Grella, T. Staubitz, R. Teusner, y C. Meinel, «Can MOOCs Support Secondary Education in Computer Science?», en Interactive Collaborative Learning, Cham, 2017, pp. 478-493. doi: 10.1007/978-3-31950337-0_45. 29. X. Basogain, Olabe, J. C. Olabe, M. Rico, L. Rodríguez, y M. Amortegui, «Pensamiento computacional en las escuelas de Colombia: colaboración internacional de innovación en la educación», undefined, 2017. /paper/Pensamiento-computacional-en-las-escuelas-de-de-en-Basogain-Olabe/92f3ece359f ae52e3d220653a017fa438aabe39c (accedido sep. 23, 2020). 30. H. Arranz de la Source, «La enseñanza de la programación informática en Educación Primaria: situación actual, análisis y presentación de experiencias de aula en España», dic. 2017, Accedido: sep. 29, 2020. [En línea]. Disponible en: http://dspace.uib.es/xmlui/handle/11201/3892 31. Y. T. Yu, C. M. Tang, C. K. Poon, y J. W. Keung, «Adoption of computer programming exercises for automatic assessment ’ issues and caution», 2017, pp. 555-564. 32. F. Hermans y E. Aivaloglou, «Teaching software engineering principles to K-12 students: A MOOC on scratch», 2017, pp. 13-22. doi: 10.1109/ICSE-SEET.2017.13. 33. D. Leyzberg y C. Moretti, «Teaching CS to CS teachers: Addressing the need for advanced content in K-12 professional development», 2017, pp. 369-374. doi: 10.1145/3017680.3017798. 34. P. Niemel, T. Partanen, M. Harsu, L. Leppänen, y P. Ihantola, «Computational thinking as an emergent learning trajectory of mathematics», 2017, pp. 70-79. doi: 10.1145/3141880.3141885. 35. T. Partanen, P. Niemelä, L. Mannila, y T. Poranen, «Educating computer science educators online a racket MOOC for elementary math teachers of Finland», 2017, vol. 1, pp. 47-58. doi: 10.5220/0006257800470058. 36. L. Benotti, M. C. Martínez, y F. Schapachnik, «A Tool for Introducing Computer Science with Automatic Formative Assessment», IEEE Trans. Learn. Technol., vol. 11, n.o 2, pp. 179-192, 2018, doi: 10.1109/ TLT.2017.2682084. 37. J. Krugel y P. Hubwieser, «Computational thinking as springboard for learning object-oriented programming in an interactive MOOC», 2017, pp. 1709-1712. doi: 10.1109/EDUCON.2017.7943079. 38. R. M. Higashi, C. D. Schunn, y J. B. Flot, «Different underlying motivations and abilities predict student versus teacher persistence in an online course», Etrd-Educ. Technol. Res. Dev., vol. 65, n.o 6, pp. 14711493, dic. 2017, doi: 10.1007/s11423-017-9528-z. 39. B. Yulianto, H. Prabowo, R. Kosala, y M. Hapsara, «MOOC architecture model for computer programming courses», 2017, pp. 35-40. doi: 10.1109/ICIMTech.2016.7930298. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 79 80 Ginna Viviana Leytón Yela, Víctor Andrés Bucheli Guerrero, Hugo Armando Ordoñez Erazo 40. D. Bañeres, C. Casado, A. Ornellas, E. Planas, J. Prieto, y M. Serra, «¿Realmente la programación puede ser para todos? Análisis de la experiencia dentro de un MOOC», Actas Las Jorn. Sobre Enseñ. Univ. Informática, vol. 3, n.o 0, Art. n.o 0, jun. 2018. 41. P. O. Franco, J. Carroll-Miranda, M. L. Delgado, E. G. López, y G. R. Gómez, «Incorporating Computational Thinking in the classrooms of Puerto Rico: How a MOOC served as an outreach and recruitment tool for Computer Science Education», 2018, vol. 2018-January, pp. 296-301. doi: 10.1145/3159450.3159544. 42. F. C. Pinheiro, C. G. von Wangenheim, y R. M. Filho, «Teaching Software Engineering in K-12 education: A systematic mapping study», Inform. Educ., vol. 17, n.o 2, pp. 167-206, 2018, doi: 10.15388/infedu.2018.10. 43. K. Falkner, R. Vivian, y N. Falkner, «Supporting Computational Thinking Development in K-6», 2018, pp. 126-133. doi: 10.1109/LaTICE.2018.00031. 44. D. Basso, I. Fronza, A. Colombi, y C. Pahl, «Improving assessment of computational thinking through a comprehensive framework», presentado en ACM International Conference Proceeding Series, 2018. doi: 10.1145/3279720.3279735. 45. P. Niemelä, T. Partanen, L. Mannila, T. Poranen, y H.-M. Järvinen, «Code ABC MOOC for math teachers», Commun. Comput. Inf. Sci., vol. 865, pp. 66-96, 2018, doi: 10.1007/978-3-319-94640-5_4. 46. C. Kirwan, E. Costello, y E. Donlon, «Computational thinking and online learning: A systematic literature review», 2018, vol. 2018-November, pp. 650-657. 47. H. Jamil, «A free-Choice social learning network for computational thinking», 2018, pp. 69-71. doi: 10.1109/ICALT.2018.00023. 48. X. Basogain, M. Á. Olabe, J. C. Olabe, y M. J. Rico, «Computational Thinking in pre-university Blended Learning classrooms», Comput. Hum. Behav., vol. 80, pp. 412-419, mar. 2018, doi: 10.1016/j. chb.2017.04.058. 49. I. F. De Kereki, «MOOC: Computational thinking (applied) for teachers», 2018, vol. 2018-July. doi: 10.18687/LACCEI2018.1.1.26. 50. F. Lazarinis, C. V. Karachristos, E. C. Stavropoulos, y V. S. Verykios, «A blended learning course for playfully teaching programming concepts to school teachers», Educ. Inf. Technol., vol. 24, n.o 2, pp. 1237-1249, 2019, doi: 10.1007/s10639-018-9823-2. 51. P. Luik et al., «What motivates enrolment in programming MOOCs?», Br. J. Educ. Technol., vol. 50, n.o 1, pp. 153-165, ene. 2019, doi: 10.1111/bjet.12600. 52. S. Serth, R. Teusner, J. Renz, y M. Uflacker, «Evaluating Digital Worksheets with Interactive Programming Exercises for K-12 Education», 2019, vol. 2019-October. doi: 10.1109/FIE43999.2019.9028680. 53. L. Carter y C. Crockett, «Preparing K-12 teachers to inspire future coders: It doesn’t have to be complex», 2019, vol. 2018-October. doi: 10.1109/FIE.2018.8658762. 54. P. Koutsakas, E. Syritzidou, A. Karamatsouki, y C. Karagiannidis, «Exploring the role of facebook as collaboration platform in a K-12 MOOC», Commun. Comput. Inf. Sci., vol. 993, pp. 31-48, 2019, doi: 10.1007/978-3-030-20954-4_3. 55. T. Staubitz, R. Teusner, y C. Meinel, «MOOCs in secondary education - Experiments and observations from German classrooms», 2019, vol. April-2019, pp. 173-182. doi: 10.1109/EDUCON.2019.8725138. 56. Aumgri y S. Petsangsri, «Computational thinking for preservice teachers in Thailand: A confirmatory factor analysis», Espacios, vol. 40, p. 12, sep. 2019. 57. E. Aivaloglou y F. Hermans, «Early programming education and career orientation: The effects of gender, self-efficacy, motivation and stereotypes», 2019, pp. 679-685. doi: 10.1145/3287324.3287358. Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 Revisión sistemática de literatura: MOOC K-12 y STEAM 58. I. C. Emembolu, C. Emembolu, K. Umechukwu, A. Sulaiman, y O. Aderinwale, «Building a Community of STEM Educators in Nigeria Using the TeachAKid2Code Program», Adv. Intell. Syst. Comput., vol. 1135 AISC, pp. 536-545, 2020, doi: 10.1007/978-3-030-40271-6_53. 59. C. Chen, G. Sonnert, P. M. Sadler, y D. J. Malan, «Computational thinking and assignment resubmission predict persistence in a computer science MOOC», J. Comput. Assist. Learn., vol. 36, n.o 5, pp. 581-594, 2020, doi: 10.1111/jcal.12427. 60. R. Moore, D. Edwards, J. Freeman, B. Magerko, T. McKlin, y A. Xambo, «EarSketch: An authentic, STEAMbased approach to computing education», 2016, vol. 2016-June. 61. R. García-Perales y A. Palomares-Ruiz, «Education in programming and mathematical learning: Functionality of a programming language in educational processes», Sustain. Switz., vol. 12, n.o 23, pp. 1-15, 2020, doi: 10.3390/su122310129. 62. P. Luik et al., «Programming MOOCs–different learners and different motivation», Int. J. Lifelong Educ., vol. 39, n.o 3, pp. 305-318, 2020, doi: 10.1080/02601370.2020.1780329. 63. J. M. Gallego-Romero, C. Alario-Hoyos, I. Estévez-Ayres, y C. Delgado Kloos, «Analyzing learners’ engagement and behavior in MOOCs on programming with the Codeboard IDE», Educ. Technol. Res. Dev., vol. 68, n.o 5, pp. 2505-2528, 2020, doi: 10.1007/s11423-020-09773-6. 64. J. Kurhila y A. Vihavainen, «A Purposeful MOOC to Alleviate Insufficient CS Education in Finnish Schools», ACM Trans. Comput. Educ., vol. 15, n.o 2, p. 10:1-10:18, abr. 2015, doi: 10.1145/2716314. 65. A. McAuley, B. Stewart, G. Siemens, y D. Cormier, «The MOOC model for digital practice.» Revista Investigación e Innovación en Ingenierías, vol. 9, n°3, pp. 57-81, 2021 DOI: https://doi.org/10.17081/invinno.9.3.5546 81
https://openalex.org/W4297238113	https://discovery.ucl.ac.uk/10156556/1/nutrients-14-03948-v2.pdf	English	null	Quantity and Source of Protein during Complementary Feeding and Infant Growth: Evidence from a Population Facing Double Burden of Malnutrition	Nutrients	2,022	cc-by	13,915	Citation: Kittisakmontri, K.; Lanigan, J.; Wells, J.C.K.; Manowong, S.; Kaewarree, S.; Fewtrell, M. Quantity and Source of Protein during Complementary Feeding and Infant Growth: Evidence from a Population Facing Double Burden of Malnutrition. Nutrients 2022, 14, 3948. https:// doi.org/10.3390/nu14193948 Academic Editor: Yajun Xu Received: 6 August 2022 Accepted: 19 September 2022 Published: 23 September 2022 Keywords: protein intake; early-life nutrition; complementary feeding; animal source foods; double burden of malnutrition; infant growth; insulin; insulin-like growth factor-1 nutrients nutrients nutrients nutrients nutrients Quantity and Source of Protein during Complementary Feeding and Infant Growth: Evidence from a Population Facing Double Burden of Malnutrition ri 1,2,* , Julie Lanigan 1, Jonathan C. K. Wells 1 , Suphara Manowong 3, Sujitra Kaewarree 3 Kulnipa Kittisakmontri 1,2,* and Mary Fewtrell 1 Kulnipa Kittisakmontri 1,2,* and Mary Fewtrell 1 Kulnipa Kittisakmon and Mary Fewtrell 1 1 Childhood Nutrition Research Centre, Department of Population, Policy and Practice, Research and Teaching Department, University College London Great Ormond Street Institute of Child Health, London WC1N 1EH, UK 2 Division of Nutrition, Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand 3 Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand * Correspondence: kulnipa.k@cmu.ac.th; Tel.: +66-53-936-477 3 Department of Pediatrics, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand * Correspondence: kulnipa.k@cmu.ac.th; Tel.: +66-53-936-477 Abstract: Background: While high protein intake during infancy may increase obesity risk, low qualities and quantities of protein contribute to undernutrition. This study aimed to investigate the impact of the amount and source of protein on infant growth during complementary feeding (CF) in a country where under- and overnutrition co-exist as the so-called the double burden of malnutrition. Methods: A multicenter, prospective cohort was conducted. Healthy term infants were enrolled with dietary and anthropometric assessments at 6, 9 and 12 months (M). Blood samples were collected at 12M for IGF-1, IGFBP-3 and insulin analyses. Results: A total of 145 infants were enrolled (49.7% female). Animal source foods (ASFs) were the main protein source and showed a positive, dose–response relationship with weight-for-age, weight-for-length and BMI z-scores after adjusting for potential confounders. However, dairy protein had a greater impact on those parameters than non-dairy ASFs, while plant-based protein had no effect. These ﬁndings were supported by higher levels of IGF-1, IGFBP-3 and insulin following a higher intake of dairy protein. None of the protein sources were associated with linear growth. Conclusions: This study showed the distinctive impact of different protein sources during CF on infant growth. A high intake of dairy protein, mainly from infant formula, had a greater impact on weight gain and growth-related hormones. 1. Introduction Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. The double burden of malnutrition (DBM)—the coexistence of under- and overnutrition—represents an emerging public health problem globally, especially for those in lower- and middle-income countries (LMICs) where eating habits are being transformed towards Westernized diets and lifestyles [1]. The World Health Organization (WHO) has emphasized the need for “double-duty actions” to prevent both forms of malnutrition [2]. While undernutrition and overweight were initially considered to affect different groups, they are increasingly recognized to occur within individuals through the life course3. This might potentially affect younger age groups, resulting in the combination of poor linear growth and overweight; however, evidence is lacking. This framework also recognizes that the two forms of malnutrition may have common risk factors in the form of unhealthy diets and environments [3,4]. Optimizing early-life nutrition by improving maternal nutrition, encouraging exclusive breastfeeding, and promoting appropriate complementary feeding (CF) are among the important actions identiﬁed to overcome the DBM [5]. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). https://www.mdpi.com/journal/nutrients Nutrients 2022, 14, 3948. https://doi.org/10.3390/nu14193948 2 of 18 Nutrients 2022, 14, 3948 2 of 18 The CF period is one of great change, when infants are introduced to foods other than milk [6] and protein intake increases; the percent of energy from protein (%PE) typically rises from around 5% to 15% when complementary foods become the major energy source for breastfed infants [7]. Protein is a key macronutrient promoting growth. However, to our knowledge, no studies have yet focused on the association between protein intake and growth in the context of the DBM. Previous studies have typically investigated the impact of protein intake in early life in populations facing either undernutrition or overnutrition [8], but not both. In high-income settings, research has highlighted the association between “too much” dietary protein in early life and the increased risk of overweight/obesity in later childhood, while previous studies in resource-limited countries have focused on the association of “too little” high-quality protein with undernutrition and, particularly, stunting. 1. Introduction There are no studies from LMICs investigating the effect of high protein intake in early life on the growth of infants and young children [8]. Furthermore, it is unclear whether all protein sources (i.e., dairy protein, non-dairy animal-based protein (ABP) and plant-based protein (PBP)) have the same effect on growth [9–12]. The most robust evidence, from a large, multi-center randomized controlled trial (RCT) in ﬁve European countries, reported that high protein intake from formula during infancy signiﬁcantly increased weight gain, but not linear growth, in children aged 2 and 6 years [13,14]. g g y Additionally, this RCT demonstrated that infants who received high-protein formula had signiﬁcantly higher plasma insulin-like growth factor-1 (IGF-1) and urine C-peptide, an insulin derivative, at 6 months of age compared to those fed with low-protein formula or that were breastfed. However, the impact of other protein sources during CF was not investigated. During infancy, nutrition is the most important factor promoting growth via the GH–IGF axis [15], while amino acids derived from dietary protein are associated with IGF-1 and insulin secretion [16–20]. The aim of this study was to investigate associations of the amount and source of protein intake during the CF period with the growth of infants in Thailand, where the DBM is prevalent. Furthermore, plasma insulin, serum IGF-1 and insulin-like growth factor binding protein 3 (IGFBP-3) were also measured to support the clinical outcomes. We aimed to tackle a key question in the context of the DBM: how a speciﬁc component of infant diet may relate to markers of both undernutrition and overweight in early life. 2. Subjects and Methods Dietary intakes were converted to energy and nutrients using the Institute of Nutrition Mahidol University Calculation (INMUCAL)-Nutrient program version 4.0 (2018) developed by the Institute of Nutrition, Mahidol University, Bangkok, Thailand [23]. This programme provided information on total energy consumption (kcal/day), crude intakes of all macronutrients (g/day) and 8 micronutrients, as well as the caloric distribution from each macronutrient. In addition, the program also separately reported protein and iron intake from ASFs and plant-based foods (Figure S1). Venous blood samples were obtained at 12 months of age. In total, approximately 2 mL of venous blood was obtained and kept at −20 ◦C until analyses were undertaken. Serum IGF-1 and IGFBP-3 were analyzed by a solid-phase, enzyme-labeled chemiluminescent immunometric assay using the IMMULITE® 2000 system (Siemens Healthcare Diagnostics Products Inc., Devault, PA, USA). The intra- and inter-assay variation in these tests was less than 8%. Plasma insulin was analyzed by an electrochemiluminescence technique using the COBAS® e411 analyzer (Roche Diagnostics Inc., Basel, Switzerland). The repeatability and intermediate precision of this technique were less than 5%. p q Statistical analyses were performed using SPSS (IBM Corp. Released 2019. IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY, USA: IBM Corp). The sample size calculation showed that at least 126 infants were needed to see differences of 0.5 z-score in WLZ at 12 months old between infants who regularly received red meat and those who did not [24]. For analyses, non-parametric data were natural log (Ln)-transformed prior to use in the regression models. Conditional growth status was calculated as z-score deviation from average size of the study population at 12 months of age, controlling for baseline size at 6 months. Simple linear regression was used to develop a formula predicting the average size of the study population at 12 months, while a positive and negative result indicated larger or smaller size than expected at follow-up, respectively, given their earlier size [25]. Demographic data, prevalence of malnutrition, CF practices and nutrient intake are described as means ± standard deviation (SD) and percentages depending on data characteristics. To investigate associations between protein intake and outcomes of interest, bivariate correlation and general linear models were performed. Pearson’s correlations were used to demonstrate relationships between the variables. 2. Subjects and Methods A multicenter, prospective, cohort study was conducted at three well-baby clinics in Chiang Mai, Thailand, between June 2018 and May 2019. Healthy term infants with birth weight ≥2500 g were recruited at age 4–6 months. Exclusion criteria were: infants with any underlying or chronic diseases; known cases of, or recovery from, protein-energy malnutrition; or those who regularly received medication except mineral and vitamin supplementation. Parents and legal guardians were provided with study information and gave written informed consent before enrollment. Ethics approval was obtained from the University College London Ethics committee, United Kingdom (Approval ID: 12551/001), and the Ethics Committee of the Faculty of Medicine, Chiang Mai University, Thailand (Approval ID: PED-2561-05287). pp Data including demographics of infants, family characteristics, growth measurements and dietary assessments were collected at 6, 9 and 12 months (M) during routine child health surveillance clinic visits. Body weight and the recumbent length of infants were measured by trained health professionals using an electronic scale (TScale Electronics Mfg. Co., Ltd., Kunshan, Taiwan, precision ± 5 g) and a standard wooden measuring board (precision ± 0.1 cm). The weight-for-age (WAZ), weight-for-length (WLZ), BMI (BMIZ) and length-for-age (LAZ) z-scores (standard deviation scores) were calculated using WHO Anthro version 3.2.2 [21]. Stunting, wasting and underweight were deﬁned as LAZ, WLZ or WAZ <−2 standard deviation score (SDS), respectively. Overweight and obesity were Nutrients 2022, 14, 3948 3 of 18 deﬁned as WLZ more than +2 SDS [22]. The primary outcome was conditional growth at 12 months (see below). deﬁned as WLZ more than +2 SDS [22]. The primary outcome was conditional growth at 12 months (see below). ( ) Dietary intake was estimated using a food frequency questionnaire (FFQ) for the semi-quantitative estimation of habitual intake alongside a 24 h recall interview (24-HR) at all time points, and a 3-day food record (3-DFR) was also collected at 9 and 12 months for quantitative estimation. Initially, dietary data from the 3-DFRs were used to estimate the average energy and nutrient intakes at 9 and 12 months of age, while the 24-HRs were used to estimate those intakes at 6 months of age. However, in cases where the-3DFR was missing, dietary intake from the 24-HR was used instead. The FFQ was used to conﬁrm the portion size if data from either the 3-DFR or 24-HR were unclear. 2. Subjects and Methods Regression analysis was used to investigate the association between the main predictor (protein intake) and the primary outcome (conditional growth at 12 months old) and secondary outcomes, including insulin, IGF-1 and IGFBP-3. In order to investigate the effect of different protein sources, protein intakes were also divided into 3 groups: (1) milk protein—breast milk, formula, cow’s milk and other dairy products; (2) non-dairy ABP—meats, eggs and meat products; (3) PBP—cereals and legumes. Covariates in the regression models were selected using a directed acyclic graph (DAG). The DAG is considered as a statistical approach to identify confounding variables and reduce the risk of selection bias when estimating causality in observational studies. The DAG applied in this study was created using DAGitty.net version 3.0, 2020 [26]. To demonstrate the magnitude of effect, both correlation and regression coefﬁcients were also reported with their 95% conﬁdence intervals (CIs). Statistical signiﬁcance was deﬁned by a p-value less than 0.05. 4 of 18 Nutrients 2022, 14, 3948 N—number; SD—standard deviation. 1 Minimum wage in Chiang Mai was THB 320 per day during the study period. 2 Average monthly income of Thai families reported by the National Statistical Ofﬁce of Thailand 2019 was THB 26,018. 3.3. Complementary Feeding Practices and Nutrient Intakes Notably, 44.1% of infants were exclusively breastfed until 6 months of age, while 36.6% of all infants continued to receive only breast milk along with complementary foods until 12 months of age (Table 2). The mean age of introduction of CF was 5.7 ± 0.6 months. The most common ﬁrst complementary food was rice with cooked egg yolk, while other non-dairy proteins such as meats and organ meats were introduced later. Mean protein intake during CF rapidly increased and reached its highest value at 12 months. In general, infants consumed more dietary protein than the Dietary Reference Intake for Thais 2020 (Thai DRI), as well as the intake recommended by the WHO. At 9 and 12 months of age, protein intakes were 2 to 3 times higher than the Thai and international recommendations (Figure 1). The average percentage of energy from protein (%PE) was 7.8, 12.6 and 15.6% at 6, 9 and 12 months of age, respectively. Table 2. Introduction of complementary feeding and milk feeding practices (n = 145). Variable Results Age of ﬁrst introduction of complementary foods (months), mean ± SD 5.7 ± 0.6 Age of introduction of each food group (months), mean ± SD - Rice 5.7 ± 0.6 - Fruits 5.8 ± 0.6 - Vegetables 5.9 ± 0.5 - Eggs 6.0 ± 0.5 - Meats 6.3 ± 0.9 - Dairy products (excluding infant/follow-on formula) 9.9 ± 2.2 Breastfeeding practices - Exclusive breastfeeding until 6 months of age, n (%) 64 (44.1%) - Receiving only breast milk alongside complementary foods until 12 months of age, n (%) 53 (36.6%) - Duration of exclusive breastfeeding (months), mean ± SD 4.4 ± 2.0 - Duration of predominant breastfeeding (months), mean ± SD 8.4 ± 4.4 Formula and dairy products - Receiving formula feeding, n (%) 87 (60.0) - Receiving unfortiﬁed cow’s milk before 12 months of age, n (%) 21 (14.5) - Duration of formula feeding (months), median (IQR) 3 (0, 9) N—number; SD—standard deviation; IQR—interquartile range. Table 2. Introduction of complementary feeding and milk feeding practices (n = 145). Figure 1 shows mean protein intake (g/kg/day) of infants during complementary feed- ing. 3.2. Prevalence of Malnutrition in the Study Population 3.2. Prevalence of Malnutrition in the Study Population At 12 months of age, the percentages of infants with wasting, underweight and stunt- ing were 3.5, 4.1 and 4.8%, respectively, while only one infant (0.7%) was overweight. No infants in this cohort were classiﬁed as obese, or both wasted and stunted. According to the parental reports, over one-third of mothers and nearly two-thirds of fathers had overweight or obesity. Therefore, the prevalence of DBM at household level where underweight infants lived with parents who had overweight/obesity was 6.2% of all families. 3.1. Demographic Data One hundred and ﬁfty healthy term infants were enrolled. There were four dropouts, and one infant was excluded due to developing a multiple food-protein allergy during the study period (Figure S2). Data from 145 infants (96.7%) were thus available for analysis. As shown in Table 1, there were almost equal numbers of male and female infants, while nearly two thirds of the infants were ﬁrst-born. Mean parental age was around 30 years, and more than 50% attained at least a college degree. The majority of infants were living in extended, middle-class families where most families received a higher monthly income than the minimum wage in Thailand. Table 1. Demographic data and family characteristics (n = 145). Demographic Data Results Infants Sex, female (n, %) 72 (49.7) Gestational age, weeks (means ± SD) 38.8 ± 1.0 Route of delivery (n, %) - Vaginal delivery 96 (66.2%) - Caesarean section 49 (33.8%) Child order, ﬁrst born (n, %) 93 (64.1%) Birth anthropometry (means ± SD) - Body weight, kg 3.2 ± 0.4 - Length, cm 49.3 ± 1.9 - Head circumference, cm 33.3 ± 1.4 Parents Parental age, years old (means ± SD) - Mothers 29.8 ± 5.7 - Fathers 32.0 ± 5.9 Parental BMI, kg/m2 (means ± SD) - Mothers 22.8 ± 4.0 - Fathers 24.7 ± 3.6 Maternal educational attainment (n, %) - Did not receive formal education 2 (1.4) - Below bachelor’s degree 74 (51.0) - Bachelor’s degree and above 69 (47.6) Family characteristics Results Main caregivers (n, %), choose more than 1 - Mothers 134 (92.4) - Fathers 6 (4.1) - Grandparents 17 (11.7) - Others 2 (1.4) Family type (n, %) - Nuclear family 50 (34.5) - Extended family 95 (65.5) Main ﬁnancial providers (n, %), choose more than 1 - Mother 92 (63.5) - Father 140 (96.6) - Grandparents 13 (9.0) - Others 2 (1.4) Family income per month 1, 2, THB (n, %) - less than 10,000 11 (7.6) - 10,000–29,999 65 (44.8) - 30,000–49,999 51 (35.2) - ≥50,000 18 (12.4) N—number; SD—standard deviation. 1 Minimum wage in Chiang Mai was THB 320 per day during the study period. 2 Average monthly income of Thai families reported by the National Statistical Ofﬁce of Thailand 2019 was THB 26,018. Table 1. Demographic data and family characteristics (n = 145). Nutrients 2022, 14, 3948 5 of 18 5 of 18 Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes g p (g/ g/ y) g p y feeding. Compared to the recommendations suggested by the Thai dietary recommended intake (Thai DRI), the Institute of Medicine (IOM), the World Health Organization (WHO)/Food and Agriculture Organization (FAO)/United Nations International Chil- dren’s Emergency Fund (UNICEF) and the European Food Safety Authority (ESFA), the protein intake of this study population rapidly increased from 6 to 12 months and was higher than all recommendations at 9 and 12 months of age. 3.4. Association between Dietary Protein and Growth Outcomes Infants were categorized into three groups based on the average %PE from 6 to 12 months of age; those in the highest and lowest quartiles had %PE ≥12.9% and ≤10.9%, respectively while the median group received protein between these values Infants in Infants were categorized into three groups based on the average %PE from 6 to 12 months of age; those in the highest and lowest quartiles had %PE ≥12.9% and ≤10.9%, respectively, while the median group received protein between these values. Infants in the highest quartile had signiﬁcantly higher WAZ, WLZ and BMIZ at 12 months, while there was no signiﬁcant difference in LAZ between groups (Table 3). Conditional weight- related z-scores (i.e., WAZ, WLZ and BMIZ) of infants in the high protein intake group were signiﬁcantly higher compared to the median and low protein intake groups (Figure 2: 95%CIs shown in Table S1), indicating that infants in the high protein intake group gained more weight than expected, given their baseline z-score at 6 months of age. However, there was no difference in the prevalence of all forms of malnutrition (i.e., underweight, wasting, stunting and overweight/obesity) between protein intake groups. the highest quartile had significantly higher WAZ, WLZ and BMIZ at 12 months, while there was no significant difference in LAZ between groups (Table 3). Conditional weight- related z-scores (i.e., WAZ, WLZ and BMIZ) of infants in the high protein intake group were significantly higher compared to the median and low protein intake groups (Figure 2: 95% CIs shown in Table S1), indicating that infants in the high protein intake group gained more weight than expected, given their baseline z-score at 6 months of age. How- ever, there was no difference in the prevalence of all forms of malnutrition (i.e., under- weight, wasting, stunting and overweight/obesity) between protein intake groups. Table 3. Comparison of growth among infants in different protein intake groups 1. 3.3. Complementary Feeding Practices and Nutrient Intakes Compared to the recommendations suggested by the Thai dietary recommended intake (Thai DRI), the Institute of Medicine (IOM), the World Health Organization (WHO)/Food and Agriculture Organization (FAO)/United Nations International Children’s Emergency Fund (UNICEF) and the European Food Safety Authority (ESFA), the protein intake of this study population rapidly increased from 6 to 12 months and was higher than all recommendations at 9 and 12 months of age. 6 of 18 6 of 18 6 of 18 6 of 18 Nutrients 2022, 14, 3948 Nutrients 2022, 14, x FOR Figure 1. Comparison of protein intake (protein weight ratio) between this study and the Thai and international recommendations. Figure 1. Comparison of protein intake (protein weight ratio) between this study and the Thai and international recommendations. Figure 1. Comparison of protein intake (protein weight ratio) between this study and the Thai and international recommendations. Figure 1. Comparison of protein intake (protein weight ratio) between this study and the Thai and international recommendations. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes L 3 12M WAZ 0.10 −0.45 −0.60 0.70 (0.24, 1.17 4) 0.55 (0.15, 0.96 4) 0.15 (−0.26, 0.56) WLZ 0.25 −0.30 −0.39 0.64 (0.14, 1.16 4) 0.55 (0.11, 0.99 4) 0.10 (−0.35, 0.54) BMIZ 0.29 −0.19 −0.31 0.60 (0.07, 1.13 5) 0.48 (0.02, 0.94 5) 0.12 (−0.34, 0.58) LAZ −0.19 −0.55 −0.64 0.45 (−0.07, 0.96) 0.35 (−0.09, 0.80) 0.10 (−0.35, 0.54) CI—conﬁdence interval. 1 Groups were classiﬁed by average percent protein energy (%PE) from all food sources at 6–12 months: high intake (H) infants received %PE in the highest quartile; median intake (M) infants received %PE in between the highest and lowest quartile; low intake (L) infants received %PE in the lowest quartile. 2 One-way ANOVA (eta-squared); 3 post hoc analysis (Bonferroni’s test); 4 p < 0.01; 5 p < 0.05. BMIZ 0.09 −0.24 −0.26 0.34 (−0.14, 0.83) 0.32 (−0.10, 0.74) 0.02 (−0.40, 0.44) LAZ −0.17 −0.48 −0.69 0.52 (−0.01, 1.05) 0.32 (−0.14, 0.77) 0.20 (−0.24, 0.65) 12M WAZ 0.10 −0.45 −0.60 0.70 (0.24, 1.17 4) 0.55 (0.15, 0.96 4) 0.15 (−0.26, 0.56) WLZ 0.25 −0.30 −0.39 0.64 (0.14, 1.16 4) 0.55 (0.11, 0.99 4) 0.10 (−0.35, 0.54) BMIZ 0.29 −0.19 −0.31 0.60 (0.07, 1.13 5) 0.48 (0.02, 0.94 5) 0.12 (−0.34, 0.58) LAZ −0.19 −0.55 −0.64 0.45 (−0.07, 0.96) 0.35 (−0.09, 0.80) 0.10 (−0.35, 0.54) CI—confidence interval. 1 Groups were classified by average percent protein energy (%PE) from all food sources at 6–12 months: high intake (H) infants received %PE in the highest quartile; median intake (M) infants received %PE in between the highest and lowest quartile; low intake (L) infants received %PE in the lowest quartile. 2 One-way ANOVA (eta-squared); 3 post hoc analysis (Bonfer- roni’s test); 4 p < 0.01; 5 p < 0.05. CI—conﬁdence interval. 1 Groups were classiﬁed by average percent protein energy (%PE) from all food sources at 6–12 months: high intake (H) infants received %PE in the highest quartile; median intake (M) infants received %PE in between the highest and lowest quartile; low intake (L) infants received %PE in the lowest quartile. 2 One-way ANOVA (eta-squared); 3 post hoc analysis (Bonferroni’s test); 4 p < 0.01; 5 p < 0.05. intake (M) infants received %PE in between the highest and lowest quartile; low intake (L) infants received %PE in the lowest quartile. 2 One-way ANOVA (eta-squared); 3 post hoc analysis (Bonfer- roni’s test); 4 p < 0.01; 5 p < 0.05. Figure 2. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes 2 One-way ANOVA (eta-squared); 3 post hoc analysis (Bonferroni’s test); 4 p < 0.01; 5 p < 0.05. WLZ 0.14 −0.22 −0.24 0.38 (−0.10, 0.86) 0.36 (−0.06, 0.77) 0.02 (−0.39, 0.43) BMIZ 0.09 −0.24 −0.26 0.34 (−0.14, 0.83) 0.32 (−0.10, 0.74) 0.02 (−0.40, 0.44) LAZ −0.17 −0.48 −0.69 0.52 (−0.01, 1.05) 0.32 (−0.14, 0.77) 0.20 (−0.24, 0.65) 12M WAZ 0.10 −0.45 −0.60 0.70 (0.24, 1.17 4) 0.55 (0.15, 0.96 4) 0.15 (−0.26, 0.56) WLZ 0.25 −0.30 −0.39 0.64 (0.14, 1.16 4) 0.55 (0.11, 0.99 4) 0.10 (−0.35, 0.54) BMIZ 0.29 −0.19 −0.31 0.60 (0.07, 1.13 5) 0.48 (0.02, 0.94 5) 0.12 (−0.34, 0.58) LAZ −0.19 −0.55 −0.64 0.45 (−0.07, 0.96) 0.35 (−0.09, 0.80) 0.10 (−0.35, 0.54) CI—confidence interval. 1 Groups were classified by average percent protein energy (%PE) from all food sources at 6–12 months: high intake (H) infants received %PE in the highest quartile; median intake (M) infants received %PE in between the highest and lowest quartile; low intake (L) infants received %PE in the lowest quartile. 2 One-way ANOVA (eta-squared); 3 post hoc analysis (Bonfer- roni’s test); 4 p < 0.01; 5 p < 0.05. Figure 2. Comparison of conditional growth outcomes among infants consuming different protein intakes. Figure 2 illustrates conditional growth status at 12 months. Infants who consumed protein in the highest quartile (black bar) had significantly higher conditional WAZ, WLZ and BMIZ compared to infants receiving protein in the median (dark grey bar) and lowest quartile (light grey bar). Although conditional LAZ was higher in the high protein intake Figure 2. Comparison of conditional growth outcomes among infants consuming different protein intakes. Figure 2 illustrates conditional growth status at 12 months. Infants who consumed protein in the highest quartile (black bar) had signiﬁcantly higher conditional WAZ, WLZ and BMIZ compared to infants receiving protein in the median (dark grey bar) and lowest quartile (light grey bar). Although conditional LAZ was higher in the high protein intake Table 3. Cont. −0.24 0 48 Table 3. Cont. Growth Parameters High (n = 36) Median (n = 73) Low (n = 36) Mean Difference 2 (95%CI) H vs. L 3 H vs. M 3 M vs. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes Growth Parameters High (n = 36) Median (n = 73) Low (n = 36) Mean Difference 2 (95% CI) H vs. L 3 H vs. M 3 M vs. L 3 6M WAZ −0.14 −0.40 −0.50 0.36 (−0.13, 0.85) 0.27 (−0.16, 0.69) 0.09 (−0.33, 0.52) Table 3. Comparison of growth among infants in different protein intake groups 1. Growth Parameters High (n = 36) Median (n = 73) Low (n = 36) Mean Difference 2 (95%CI) H vs. L 3 H vs. M 3 M vs. L 3 6M WAZ −0.14 −0.40 −0.50 0.36 (−0.13, 0.85) 0.27 (−0.16, 0.69) 0.09 (−0.33, 0.52) WLZ 0.02 −0.06 −0.05 0.07 (−0.46, 0.59) 0.08 (−0.37, 0.54) −0.02 (−0.47, 0.44) BMIZ −0.08 −0.14 −0.16 0.08 (−0.46, 0.59) 0.06 (−0.40, 0.52) 0.02 (−0.44, 0.48) LAZ −0.15 −0.55 −0.66 0.50 (−0.01, 1.01) 0.40 (−0.05, 0.84) 0.11 (−0.34, 0.55) 9M WAZ 0.03 −0.46 −0.59 0.62 (0.16, 1.08 5) 0.49 (0.09, 0.89 4) 0.13 (−0.27, 0.53) WLZ 0.14 −0.22 −0.24 0.38 (−0.10, 0.86) 0.36 (−0.06, 0.77) 0.02 (−0.39, 0.43) BMIZ 0.09 −0.24 −0.26 0.34 (−0.14, 0.83) 0.32 (−0.10, 0.74) 0.02 (−0.40, 0.44) LAZ −0.17 −0.48 −0.69 0.52 (−0.01, 1.05) 0.32 (−0.14, 0.77) 0.20 (−0.24, 0.65) the highest quartile had significantly higher WAZ, WLZ and BMIZ at 12 there was no significant difference in LAZ between groups (Table 3) Condi Table 3. Comparison of growth among infants in different protein intake groups 1. Nutrients 2022, 14, 3948 9M WAZ 7 of 18 53) Table 3. Cont. Growth Parameters High (n = 36) Median (n = 73) Low (n = 36) Mean Difference 2 (95%CI) H vs. L 3 H vs. M 3 M vs. L 3 12M WAZ 0.10 −0.45 −0.60 0.70 (0.24, 1.17 4) 0.55 (0.15, 0.96 4) 0.15 (−0.26, 0.56) WLZ 0.25 −0.30 −0.39 0.64 (0.14, 1.16 4) 0.55 (0.11, 0.99 4) 0.10 (−0.35, 0.54) BMIZ 0.29 −0.19 −0.31 0.60 (0.07, 1.13 5) 0.48 (0.02, 0.94 5) 0.12 (−0.34, 0.58) LAZ −0.19 −0.55 −0.64 0.45 (−0.07, 0.96) 0.35 (−0.09, 0.80) 0.10 (−0.35, 0.54) CI—conﬁdence interval. 1 Groups were classiﬁed by average percent protein energy (%PE) from all food sources at 6–12 months: high intake (H) infants received %PE in the highest quartile; median intake (M) infants received %PE in between the highest and lowest quartile; low intake (L) infants received %PE in the lowest quartile. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes 12 months was significantly associated with conditional growth outcomes, whilst p n intake from 6–9 months of age was not. Thus, only protein intake from 9–12 mon age was included in the subsequent analyses. intake from 6–9 months of age was not. Thus, only protein intake from 9–12 months of age was included in the subsequent analyses. 12 months was significantly associated with conditional growth outcomes, whilst p n intake from 6–9 months of age was not. Thus, only protein intake from 9–12 mon age was included in the subsequent analyses. Table 4. Pearson’s correlations between protein intakes during two different periods (6–9 and 9–12 months of age) and conditional growth. ble 4. Pearson’s correlations between protein intakes during two different periods (6–9 and 9 nths of age) and conditional growth Table 4. Pearson’s correlations between protein intakes during two different periods (6–9 and 9–12 months of age) and conditional growth. ble 4. Pearson’s correlations between protein intakes during two different periods (6–9 and onths of age) and conditional growth Table 4. Pearson’s correlations between protein intakes during two different periods (6–9 and 9–12 months of age) and conditional growth. C di i l Average %PE 6–9 M Average %PE 9–12 M ble 4. Pearson’s correlations between protein intakes during two different periods (6–9 and 9 onths of age) and conditional growth. Average %PE 6 9M Average %PE 9 12M Table 4. Pearson’s correlations between protein intakes during two different periods (6–9 and 9–12 months of age) and conditional growth. Conditional Average %PE 6–9 M Average %PE 9–12 M r p-Value r p-Value WAZ 0.17 0.04 0.26 0.002 WLZ 0.16 0.06 0.23 0.006 BMIZ 0.12 0.16 0.20 0.02 LAZ 0.09 0.26 0.07 0.39 %PE—percent protein energy; r—correlation coefﬁcient; WAZ—weight-for-age z-score; WLZ—weight-for-length z-score; BMIZ—body mass index z-score; LAZ—length-for-age z-score. able 4. Pearson’s correlations between protein intakes during two different periods (6–9 and 9 onths of age) and conditional growth. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes Comparison of conditional growth outcomes among infants consuming different protein intakes. Figure 2. Comparison of conditional growth outcomes among infants consuming different protein intakes. Figure 2. Comparison of conditional growth outcomes among infants consuming different protein intakes. Figure 2. Comparison of conditional growth outcomes among infants consuming different protein intakes. Figure 2. Comparison of conditional growth outcomes among infants consuming different protein intakes. Figure 2. Comparison of conditional growth outcomes among infants consuming different protein intakes. Figure 2 illustrates conditional growth status at 12 months. Infants who consumed protein in the highest quartile (black bar) had significantly higher conditional WAZ, WLZ and BMIZ compared to infants receiving protein in the median (dark grey bar) and lowest quartile (light grey bar). Although conditional LAZ was higher in the high protein intake group compared with other groups, there was no significant difference. Figure 2 illustrates conditional growth status at 12 months. Infants who consumed protein in the highest quartile (black bar) had signiﬁcantly higher conditional WAZ, WLZ and BMIZ compared to infants receiving protein in the median (dark grey bar) and lowest quartile (light grey bar). Although conditional LAZ was higher in the high protein intake group compared with other groups, there was no signiﬁcant difference. According to CF recommendations in Thailand [27] (Table S2), infants should be given three main meals (i.e., breakfast, lunch, and dinner) from 9 months; thus, protein intakes during the early (6–9 months old) and later stages (9–12 months old) of CF were expected to be quite different. Therefore, average %PE during the early and later CF peri- ods were separated for univariate analyses investigating the association of protein intake According to CF recommendations in Thailand [27] (Table S2), infants should be given three main meals (i.e., breakfast, lunch, and dinner) from 9 months; thus, protein intakes during the early (6–9 months old) and later stages (9–12 months old) of CF were expected to be quite different. Therefore, average %PE during the early and later CF periods were separated for univariate analyses investigating the association of protein intake with conditional growth outcomes. The results in Table 4 indicate that protein intake from 9–12 months was signiﬁcantly associated with conditional growth outcomes, whilst protein Nutrients 2022, 14, 3948 8 of 18 intake from 6–9 months of age was not. Thus, only protein intake from 9–12 months of age was included in the subsequent analyses. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes Predictor and Co-Variates Conditional WAZ Conditional WLZ β 95%CI β 95%CI %PE 9–12 M 0.11 0.03, 0.18 1 0.12 0.05, 0.20 1 Duration of predominant BF 0.02 −0.05, 0.08 0.02 −0.05, 0.09 Type of milk 9–12 M 0.10 −0.25, 0.45 0.19 −0.16, 0.55 Non-protein energy 6–9 M 0.002 0, 0.004 0.002 0, 0.004 Non-protein energy 9–12 M <0.001 −0.001, 0.002 −0.001 −0.002, 0.001 Maternal education 0.06 −0.07, 0.18 0.05 −0.08, 0.17 Frequency of illness −0.02 −0.16, 0.12 −0.03 −0.17, 0.12 Maternal BMI −0.02 −0.06, 0.03 −0.01 −0.05, 0.03 Maternal age 0.001 −0.03, 0.03 0.001 −0.03, 0.03 Figure 3. Directed acyclic graphs demonstrating co-variates of the association between protei take and linear growth/ponderal growth. Figure 3. Directed acyclic graphs demonstrating co-variates of the association between protein intake and linear growth/ponderal growth. gure 3. Directed acyclic graphs demonstrating co-variates of the association between protei e and linear growth/ponderal growth. Figure 3. Directed acyclic graphs demonstrating co-variates of the association between protein intake and linear growth/ponderal growth. Figure 3 illustrates DAGs predicting conditional growth status (for either linea nderal growth) by protein intake during the CF period (main predictor). Black arro dicate causal paths between the main predictors and outcomes. Dashed-grey arro present bias paths. Boxes with black frames show potential confounders. All covariates suggested by the DAGs were included in the multiple regression m . There was no association between conditional LAZ and %PE from 9 to 12 months her covariates (Table 5). However, %PE from 9 to 12 months was associated with c tional WAZ, WLZ, and BMIZ (95% CI varied between 0.02 and 0.20). Considering All covariates suggested by the DAGs were included in the multiple regression models. There was no association between conditional LAZ and %PE from 9 to 12 months, or other covariates (Table 5). However, %PE from 9 to 12 months was associated with conditional WAZ, WLZ, and BMIZ (95%CI varied between 0.02 and 0.20). Considering different protein sources, only %PE from milk/dairy and non-dairy protein from 9 to 12 months were signiﬁcantly associated with the weight-related parameters, while PBP was not (Table 5). Protein intake from milk had a stronger association with conditional weight- related parameters compared to other protein sources based on effect size (regression co-efﬁcient (β)). Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes Conditional Average %PE 6–9M Average %PE 9–12M r p-Value r p-Value WAZ 0.17 0.04 0.26 0.002 WLZ 0.16 0.06 0.23 0.006 BMIZ 0.12 0.16 0.20 0.02 LAZ 0.09 0.26 0.07 0.39 PE percent protein energy; r correlation coefficient; WAZ weight for age z score; WLZ According to the DAGs (Figure 3), the suggested covariates for the multiple linear regression model investigating the association of protein intake with linear growth were duration of predominant breastfeeding, type of milk feeding, non-protein energy intake at 6–12 months, maternal education, frequency of illness and family income. For ponderal growth including WAZ, WLZ and BMIZ, the DAG suggested duration of predominant breastfeeding, type of milk feeding, non-protein energy, maternal education, frequency of illness, maternal BMI and maternal age as covariates. ight-for-length z-score; BMIZ—body mass index z-score; LAZ—length-for-age z-score. According to the DAGs (Figure 3), the suggested covariates for the multiple lin gression model investigating the association of protein intake with linear growth w ration of predominant breastfeeding, type of milk feeding, non-protein energy int 6–12 months, maternal education, frequency of illness and family income. For ponde owth including WAZ WLZ and BMIZ the DAG suggested duration of predomin Figure 3 illustrates DAGs predicting conditional growth status (for either linear or ponderal growth) by protein intake during the CF period (main predictor). Black arrows indicate causal paths between the main predictors and outcomes. Dashed-grey arrows represent bias paths. Boxes with black frames show potential confounders. owth including WAZ, WLZ and BMIZ, the DAG suggested duration of predomin eastfeeding, type of milk feeding, non-protein energy, maternal education, frequency ness, maternal BMI and maternal age as covariates. Figure 3. Cont. Figure 3. Cont. 9 of 18 9 o 9 of 18 9 Nutrients 2022, 14, 3948 nts 2022, 14, x FOR PEE Figure 3. Directed acyclic graphs demonstrating co-variates of the association between protein take and linear growth/ponderal growth. Figure 3 illustrates DAGs predicting conditional growth status (for either linear ponderal growth) by protein intake during the CF period (main predictor). Black arro indicate causal paths between the main predictors and outcomes. Dashed-grey arro represent bias paths. Boxes with black frames show potential confounders. All covariates suggested by the DAGs were included in the multiple regression m els. There was no association between conditional LAZ and %PE from 9 to 12 months other covariates (Table 5). Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes However, %PE from 9 to 12 months was associated with c ditional WAZ, WLZ, and BMIZ (95% CI varied between 0.02 and 0.20). Considering d ferent protein sources, only %PE from milk/dairy and non-dairy protein from 9 to months were significantly associated with the weight-related parameters, while PBP w not (Table 5). Protein intake from milk had a stronger association with conditional weig related parameters compared to other protein sources based on effect size (regression efficient (𝛽)). To differentiate the effect of milk protein from breast milk and that from dairy/inf formula on conditional growth outcomes, %PE from breast milk was subtracted from %PE from formula, cow’s milk and other dairy products. The resulting variable was cal “%PE from dairy vs. breast milk”. As shown in Figure 4, this variable was directly asso ated with weight-related parameters, suggesting that greater %PE from formula milk a dairy rather than breast milk was significantly associated with higher conditional WA WLZ and BMIZ. The findings suggest that a 1% increase in daily %PE from formula and dairy from to 12 months of age was associated with a 0.18 (95%CI, 0.03, 0.32) and 0.16 (95%CI, 0 0 30) standard deviation score (SDS) increase in conditional WAZ and WLZ respective Figure 3. Directed acyclic graphs demonstrating co-variates of the association between protein intake and linear growth/ponderal growth. All covariates suggested by the DAGs were included in the multiple regression models. There was no association between conditional LAZ and %PE from 9 to 12 months, or other covariates (Table 5). However, %PE from 9 to 12 months was associated with conditional WAZ, WLZ, and BMIZ (95%CI varied between 0.02 and 0.20). Considering different protein sources, only %PE from milk/dairy and non-dairy protein from 9 to 12 months were signiﬁcantly associated with the weight-related parameters, while PBP was not (Table 5). Protein intake from milk had a stronger association with conditional weight- related parameters compared to other protein sources based on effect size (regression co-efﬁcient (β)). Table 5. 1 Multiple linear regression analyses investigating associations between protein intake from all sources at 9–12 months and conditional growth. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes ent protein sources, only %PE from milk/dairy and non-dairy protein from 9 to nths were significantly associated with the weight-related parameters, while PBP w (T bl 5) P t i i t k f ilk h d t i ti ith diti l i Table 5. 1 Multiple linear regression analyses investigating associations between protein intake from all sources at 9–12 months and conditional growth. not (Table 5). Protein intake from milk had a stronger association with conditional weig related parameters compared to other protein sources based on effect size (regression efficient (𝛽)). To differentiate the effect of milk protein from breast milk and that from dairy/inf formula on conditional growth outcomes, %PE from breast milk was subtracted from %PE from formula, cow’s milk and other dairy products. The resulting variable was cal “%PE from dairy vs. breast milk”. As shown in Figure 4, this variable was directly ass ated with weight-related parameters, suggesting that greater %PE from formula milk a dairy rather than breast milk was significantly associated with higher conditional WA WLZ and BMIZ. The findings suggest that a 1% increase in daily %PE from formula and dairy from Predictor and Co-Variates Conditional WAZ Conditional WLZ β 95%CI β 95%CI %PE 9–12 M 0.11 0.03, 0.18 1 0.12 0.05, 0.20 1 Duration of predominant BF 0.02 −0.05, 0.08 0.02 −0.05, 0.09 Type of milk 9–12 M 0.10 −0.25, 0.45 0.19 −0.16, 0.55 Non-protein energy 6–9 M 0.002 0, 0.004 0.002 0, 0.004 Non-protein energy 9–12 M <0.001 −0.001, 0.002 −0.001 −0.002, 0.001 Maternal education 0.06 −0.07, 0.18 0.05 −0.08, 0.17 Frequency of illness −0.02 −0.16, 0.12 −0.03 −0.17, 0.12 Maternal BMI −0.02 −0.06, 0.03 −0.01 −0.05, 0.03 Maternal age 0.001 −0.03, 0.03 0.001 −0.03, 0.03 Nutrients 2022, 14, 3948 10 of 18 10 of 18 Table 5. Cont. Table 5. Cont. Table 5. Cont. Predictor and Co-Variates Conditional BMIZ Conditional LAZ β 95%CI β 95%CI %PE 9–12 M 0.10 0.02, 0.18 2 0.01 −0.07, 0.09 Duration of predominant BF 0.03 −0.04, 0.10 −0.02 −0.08, 0.05 Type of milk 9–12 M 0.23 −0.13, 0.59 −0.19 −0.56, 0.18 Non-protein energy 6–9 M 0.002 −0.01. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes 0.004 <0.001 −0.002, 0.003 Non-protein energy 9–12 M <0.001 −0.002, 0.001 0.001 −0.001, 0.003 Maternal education 0.08 −0.05, 0.20 −0.05 −0.18, 0.09 Frequency of illness −0.01 −0.15, 0.14 0.001 −0.15, 0.15 Maternal BMI −0.01 −0.05, 0.03 N/A N/A Maternal age 0.01 −0.02, 0.04 N/A N/A Family income N/A N/A 0.12 −0.10. 0.34 2 Multiple linear regression analyses investigating associations between protein intakes from different food sources at age 9–12 months and conditional growth. y ltiple linear regression analyses investigating associations between protein intakes from different food sour months and conditional growth. nalyses investigating associations between protein intakes from different food sources at age 9–12 months and conditional growth. y 2 Multiple linear regression analyses investigating associations between protein months and conditional growth. g Predictor and Co-Variates Conditional WAZ Conditional WLZ β 95%CI β 95%CI %PE Milk/dairy 0.18 0.03, 0.32 2 0.16 0.01, 0.30 2 %PE Non-dairy ASFs 0.10 0.02, 0.18 2 0.12 0.04, 0.20 1 %PE Plant-based foods 0.15 −0.15, 0.45 0.16 −0.15, 0.46 Duration of predominant BF 0.02 −0.05, 0.09 0.02 −0.05, 0.09 Type of milk 9–12 M −0.04 −0.46, 0.09 0.13 −0.30, 0.56 Non-protein energy 6–9 M 0.002 0, 0.004 0.002 0, 0.004 Non-protein energy 9–12 M <0.001 −0.001, 0.002 −0.001 −0.002, 0.001 Maternal education 0.05 −0.07, 0.18 0.05 −0.08, 0.17 Frequency of illness −0.02 −0.16, 0.12 −0.02 −0.16, 0.12 Maternal BMI −0.01 −0.06, 0.03 −0.01 −0.05, 0.03 Maternal age 0.01 −0.03, 0.04 0.003 −0.03, 0.04 Predictor and Co-Variates Conditional BMIZ Conditional LAZ β 95%CI β 95%CI %PE Milk/dairy 0.13 −0.02, 0.28 0.07 −0.08, 0.21 %PE Non-dairy ASFs 0.10 0.01, 0.18 2 <0.001 −0.09, 0.09 %PE Plant-based foods 0.14 −0.16, 0.45 0.001 −0.31, 032 Duration of predominant BF 0.03 −0.04, 0.10 −0.01 −0.08, 0.06 Type of milk 9–12 M 0.17 −0.26, 0.60 −0.32 −0.76, 0.12 Non-protein energy 6–9 M 0.002 −0.01. 0.004 <0.001 −0.002, 0.003 Non-protein energy 9–12 M <0.001 −0.002, 0.001 0.001 −0.001, 0.003 Maternal education 0.07 −0.06, 0.20 −0.05 −0.19, 0.08 Frequency of illness −0.002 −0.15, 0.14 −0.002 −0.15, 0.14 Maternal BMI −0.01 −0.05, 0.03 N/A N/A Maternal age 0.01 −0.02, 0.05 N/A N/A Family income N/A N/A 0.13 −0.10. 0.35 β—regression coefﬁcient; CI—conﬁdence interval; %PE—percent protein energy; BF—breastfeeding; WAZ—weight-for-age z-score; WLZ—weight-for-length z-score; BMIZ—body mass index z-score; LAZ—length- for-age z-score; N/A—not analyzed; 1 p < 0.01; 2 p < 0.05. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes β—regression coefﬁcient; CI—conﬁdence interval; %PE—percent protein energy; BF—breastfeeding; WAZ—weight-for-age z-score; WLZ—weight-for-length z-score; BMIZ—body mass index z-score; LAZ—length- for-age z-score; N/A—not analyzed; 1 p < 0.01; 2 p < 0.05. To differentiate the effect of milk protein from breast milk and that from dairy/infant formula on conditional growth outcomes, %PE from breast milk was subtracted from the %PE from formula, cow’s milk and other dairy products. The resulting variable was called “%PE from dairy vs. breast milk”. As shown in Figure 4, this variable was directly associated with weight-related parameters, suggesting that greater %PE from formula milk and dairy rather than breast milk was signiﬁcantly associated with higher conditional WAZ, WLZ and BMIZ. 11 of 18 eastfeed- index z 11 of 18 eastfeed- i d Nutrients 2022, 14, 3948 o e; A e g o age o e; N/A o a a y e ; p 0 0 ; p 0 0 Figure 4 Scatter plots and linear regression statistics for conditional growth outcomes by %PE from Figure 4. Scatter plots and linear regression statistics for conditional growth outcomes by %PE from dairy source vs. breast milk at 9–12 months. Figure 4. Scatter plots and linear regression statistics for conditional growth outcomes by %PE from dairy source vs. breast milk at 9–12 months. Figure 4. Scatter plots and linear regression statistics for conditional growth outcomes by %PE from dairy source vs. breast milk at 9–12 months. Figure 4. Scatter plots and linear regression statistics for conditional growth outcomes by %PE from dairy source vs. breast milk at 9–12 months. Figure 4. Scatter plots and linear regression statistics for conditional growth outcomes by %PE from dairy source vs. breast milk at 9–12 months. Figure 4. Scatter plots and linear regression statistics for conditional growth outcomes by %PE from dairy source vs. breast milk at 9–12 months. Figure 4. Scatter plots and linear regression statistics for conditional growth outcomes by %PE from dairy source vs. breast milk at 9–12 months. Figure 4. Scatter plots and linear regression statistics for conditional growth outcomes by %PE from dairy source vs. breast milk at 9–12 months. Scatter plots demonstrate associations between the percentage of protein energy from dairy sources (i.e., formula and cow’s milk), subtracted from the percentage of pro- tein energy from breast milk (%PE dairy source vs. breast milk) and conditional growth at 12 months. 12 Months of Age EW In order to investigate the association between dietary protein and weight-related growth parameters, IGF-1, IGFBP-3 and insulin were investigated at 12 months of age. Milk protein was the only food source that showed a signiﬁcantly positive association with circulating IGF-1, IGFBP-3 and insulin (Table 6). However, a stronger association was found b “% f d b lk” d h G l l h dairy source vs. breast milk, and conditional WAZ, WLZ and BMIZ, but not conditional LAZ. In order to investigate the association between dietary protein and weight-related growth parameters, IGF-1, IGFBP-3 and insulin were investigated at 12 months of age. d i b t ilk d diti l WAZ WLZ d BMIZ b t t diti l In order to investigate the association between dietary protein and weight-related growth parameters, IGF-1, IGFBP-3 and insulin were investigated at 12 months of age. Milk protein was the only food source that showed a signiﬁcantly positive association with circulating IGF-1, IGFBP-3 and insulin (Table 6). However, a stronger association was found between “%PE from dairy vs. breast milk” and the IGF-1 level, suggesting the consumption of more %PE from formula and dairy than from breast milk was associated more strongly with IGF-1 level. dairy source vs. breast milk, and conditional WAZ, WLZ and BMIZ, but not conditional LAZ. 3.5. Association between Dietary Protein Intake and Blood Levels of IGF-1, IGFBP-3 and Insulin at 12 Months of Age Table 6. Pearson’s correlation between %PE at 9–12 months and blood levels of IGF-1, IGFBP-3 and insulin at 12 months of age. In order to investigate the association between dietary protein and weight-related growth parameters, IGF-1, IGFBP-3 and insulin were investigated at 12 months of age. Milk protein was the only food source that showed a significantly positive association Table 6. Pearson’s correlation between %PE at 9–12 months and blood levels of IGF-1, IGFBP-3 and insulin at 12 months of age. Protein Intake (%PE) from Correlation Coefﬁcients (r) IGF-1 (ng/mL) IGFBP-3 (ng/mL) Insulin (µU/mL) All food sources 0.11 0.13 0.03 • Milk/Dairy • %PE from dairy vs. breast milk 0.33 1 0.38 1 0.20 2 0.21 2 0.20 2 0.20 2 Non-dairy ASFs −0.16 −0.04 −0.14 Plant-based foods −0.11 −0.09 −0.06 ASFs—animal source foods; %PE—percent protein energy; 1 p < 0.001; 2 p < 0.05. 12 Months of Age EW In order to investigate the association between dietary protein and weight-related growth parameters, IGF-1, IGFBP-3 and insulin were investigated at 12 months of age. Milk protein was the only food source that showed a significantly positive association with circulating IGF-1, IGFBP-3 and insulin (Table 6). However, a stronger association was found between “%PE from dairy vs. breast milk” and the IGF-1 level, suggesting the consumption of more %PE from formula and dairy than from breast milk was associated more strongly with IGF-1 level. As shown in Figure 5, there were positive dose–response relationships of “%PE from dairy vs. breast milk” with all growth-related hormones after adjusting for sex. A 1% greater %PE from formula and dairy was associated with increasing blood concentrations of IGF-1, IGFBP-3 and insulin by 2.34 (95%CI 1.44, 3.23) ng/mL, 33.41 (95%CI 9.46, 57.37) ng/mL and 4 (95%CI 1, 7) %, respectively. Mean IGF-1, IGFBP-3 and insulin stratified by sex are given in Table S3. As shown in Figure 5, there were positive dose–response relationships of “%PE from dairy vs. breast milk” with all growth-related hormones after adjusting for sex. A 1% greater %PE from formula and dairy was associated with increasing blood concentrations of IGF-1, IGFBP-3 and insulin by 2.34 (95%CI 1.44, 3.23) ng/mL, 33.41 (95%CI 9.46, 57.37) ng/mL and 4 (95%CI 1, 7) %, respectively. Mean IGF-1, IGFBP-3 and insulin stratiﬁed by sex are given in Table S3. Table 6. Pearson s correlation between %PE at 9–12 months and blood levels of IGF-1, IGFBP-3 and insulin at 12 months of age. Protein Intake (%PE) from Correlation Coefficients (r) IGF-1 (ng/mL) IGFBP-3 (ng/mL) Insulin (µU/mL) All food sources 0.11 0.13 0.03 • Milk/Dairy 0 33 1 0 20 2 0 20 2 g Scatter plots illustrate the associations between the percentage of protein energy from dairy sources (i.e., formula and cow’s milk) subtracted from the percentage of protein energy from breast milk (%PE dairy source vs. breast milk) and blood concentrations of IGF-1, IGFBP-3 and insulin at 12 months. The scatter plots show dose–response, positive associations between %PE dairy source vs. breast milk, and all laboratory markers after controlling of sex. y • %PE from dairy vs. breast milk 0.33 1 0.38 1 0.20 2 0.21 2 0.20 2 0.20 2 Non-dairy ASFs −0.16 −0.04 −0.14 Plant-based foods −0.11 −0.09 −0.06 ASFs—animal source foods; %PE—percent protein energy; 1 p < 0.001; 2 p < 0.05. Figure 1 shows mean protein intake (g/kg/day) of inf 3.4. Association between Dietary Protein and Growth Outcomes The scatter plots show dose–response, positive associations between %PE The ﬁndings suggest that a 1% increase in daily %PE from formula and dairy from 9 to 12 months of age was associated with a 0.18 (95%CI, 0.03, 0.32) and 0.16 (95%CI, 0.01, 0.30) standard deviation score (SDS) increase in conditional WAZ and WLZ, respectively, after adjusting for other protein sources, duration of predominant BF, non-protein energy consumption, type of milk feeding, maternal age, maternal education, maternal BMI, and frequency of illness. q y A 1% increase in daily %PE from non-dairy ASFs from 9 to 12 months was also associated with a 0.10 (95%CI 0.02, 0.18), 0.12 (95%CI 0.04, 0.20) and 0.10 (95%CI 0.01, 0.18) SDS increase in conditional WAZ, WLZ and BMIZ, respectively, after adjusting for other protein sources, duration of predominant BF, non-protein energy consumption, type of milk feeding, maternal age, maternal education, maternal BMI and frequency of illness. g g q y Scatter plots demonstrate associations between the percentage of protein energy from dairy sources (i.e., formula and cow’s milk), subtracted from the percentage of protein energy from breast milk (%PE dairy source vs. breast milk) and conditional growth at 12 months. The scatter plots show dose–response, positive associations between %PE dairy source vs. breast milk, and conditional WAZ, WLZ and BMIZ, but not conditional LAZ. Nutrients 2022, 14, 3948 12 of 18 12 of 18 3.5. Association between Dietary Protein Intake and Blood Levels of IGF-1, IGFBP-3 and Insulin at 12 Months of Age EW 12 of 18 4. Discussion Scatter plots illustrate the associations between the percentage o dairy sources (i.e., formula and cow’s milk) subtracted from the per ergy from breast milk (%PE dairy source vs. breast milk) and blood c 1, IGFBP-3 and insulin at 12 months. The scatter plots show dose–re ciations between %PE dairy source vs. breast milk, and all laborato trolling of sex. 4. Discussion This cohort study demonstrated that infants living in Chiang sumed more dietary protein, mainly from ASFs, than Thai and WH during the CF period. More importantly, the main results indicate was significantly associated with weight-related parameters (i.e., W This cohort study demonstrated that infants living in Chiang Mai, Thailand, consumed more dietary protein, mainly from ASFs, than Thai and WHO recommendations during the CF period. More importantly, the main results indicated that protein intake was signiﬁcantly associated with weight-related parameters (i.e., WAZ, WLZ, and BMIZ) during the CF period after adjusting for potential confounders. Considering protein sources, the results showed a different impact of protein from diary and non-dairy ASFs. The predominant association with weight gain was from dairy protein—mainly formula and unfortiﬁed cow’s milk—whereas non-dairy ABP showed a lesser impact. Protein intake from formula and unfortiﬁed cow’s milk also showed positive associations with circulating IGF-1, IGFBP-3 and insulin at 12 months of age in a dose–response manner, independent of infant sex. There was no association of protein intake with linear growth markers, or of PBP with conditional growth outcomes in this cohort. g y g p during the CF period after adjusting for potential confounders. sources, the results showed a different impact of protein from diary The predominant association with weight gain was from dairy prot and unfortified cow’s milk—whereas non-dairy ABP showed a less take from formula and unfortified cow’s milk also showed positive culating IGF-1, IGFBP-3 and insulin at 12 months of age in a dose– dependent of infant sex. There was no association of protein intak markers, or of PBP with conditional growth outcomes in this cohort In contrast to a recent review highlighting that infants and you consumed less ABP compared with those from high-income sett g In contrast to a recent review highlighting that infants and young children in LMICs consumed less ABP compared with those from high-income settings [28], this cohort showed that infants living in northern Thailand consumed more dietary protein from ASFs than from plant-based foods during the CF period. 4. Discussion It could be assumed that, in some LMICs, especially upper-middle-income countries such as Thailand, CF is now shifting towards a “Western style” diet. Recently, a cross-sectional study [29] and data from the national survey [30] in Thailand also reported that over 80% of protein in complementary foods came from ASFs. These ﬁndings are relevant to the current global situation in which many LMIC countries are transitioning to Western diets with high amounts of ASFs, even though this change may occur at very different rates across different countries [31,32]. p g showed that infants living in northern Thailand consumed more ASFs than from plant-based foods during the CF period. It could be a LMICs, especially upper-middle-income countries such as Thailan towards a “Western style” diet. Recently, a cross-sectional study [2 national survey [30] in Thailand also reported that over 80% of prote foods came from ASFs. These findings are relevant to the current glo many LMIC countries are transitioning to Western diets with high am though this change may occur at very different rates across differen Considering the relation between protein intake and growth found that infants consuming protein in the highest quartile, with a m 13%, had significantly higher weight-related z-scores at 12 months those who had lower protein intakes. Interestingly, the median %P intake group was similar to a report based on European population al [33] found that most studies in European countries showing a s g g y y Considering the relation between protein intake and growth outcomes, this study found that infants consuming protein in the highest quartile, with a median %PE of nearly 13%, had signiﬁcantly higher weight-related z-scores at 12 months of age compared to those who had lower protein intakes. Interestingly, the median %PE of the high protein intake group was similar to a report based on European populations [33]. Michaelsen et al. [33] found that most studies in European countries showing a signiﬁcant association between high protein intake and BMI at 12 months reported a %PE around 13%. Therefore, some experts agreed to recommend an upper limit of protein intake around 15%, with the aim of reducing the risk of childhood obesity in their populations [4,34,35]. In contrast, current international recommendations only recommend safe levels: lower limits of protein intake considered necessary to adequately support the normal growth of infants/children [36–38]. 12 Months of Age EW Figure 5. Cont. Figure 5. Cont. 13 of 18 13 of 18 Nutrients 2022, 14, 3948 Figure 5. Scatter plots and regression statistics of blood levels of IGF-1, IG months of age by %PE from dairy vs. breast milk at 9–12 months (stratified Figure 5. Scatter plots and regression statistics of blood levels of IGF-1, IGFBP-3 and insulin at 12 months of age by %PE from dairy vs. breast milk at 9–12 months (stratiﬁed by sex). Figure 5. Scatter plots and regression statistics of blood levels of IGF-1, IG months of age by %PE from dairy vs. breast milk at 9–12 months (stratified Figure 5. Scatter plots and regression statistics of blood levels of IGF-1, IGFBP-3 and insulin at 12 months of age by %PE from dairy vs. breast milk at 9–12 months (stratiﬁed by sex). 4. Discussion Given the dramatically increasing prevalence of overweight/obesity in young children in many LMICs, an upper limit of protein intake should be considered for international recommendations, and more studies in this speciﬁc context should be encouraged. Nutrients 2022, 14, 3948 14 of 18 14 of 18 Furthermore, our ﬁndings also indicated dose–response associations between ABP and weight-related parameters regardless of the type of milk received or how much energy was provided from carbohydrate and fat, although the effects of dairy and non-dairy protein were different to some extent. When considering the concept of conditional growth [39], the outcomes can be interpreted as indicating that every 1% increase in %PE from either dairy or non-dairy ABP at 9–12 months of age is associated with a positive deviation in WAZ, WLZ and BMIZ from the expected values at 12 months, based on growth parameters at 6 months. Thus, these results suggest that higher protein intake from ASFs is associated with more rapid weight gain than the infant’s expected growth trajectory. Underpinning these clinical ﬁndings, our laboratory results showed that the higher consumption of dairy protein, mainly from formula and cow’s milk, signiﬁcantly increased levels of circulating IGF-1, IGFBP-3 and insulin, which are the main hormonal regulators of human growth and may relate to increased adiposity [40,41]. A possible mechanism explaining the greater effect of dairy protein over other protein sources is the high proportion of leucine, a potent factor stimulating IGF-1 secretion in dairy protein compared to other food sources (14% vs. 8% of amino acids in dairy and meats, respectively) [42]. In addition, some evidence indicates that leucine also plays an essential role in the activation of the mammalian target of rapamycin (mTOR), which is the major regulator of growth and metabolism homeostasis in humans [43]. [ ] To our knowledge, this is the ﬁrst evidence from an LMIC demonstrating an associa- tion between high protein intake and rapid weight gain, and the possible mechanism of this association through IGF-1, IGFBP-3 and insulin. More importantly, this cohort also showed the distinctive effect of different protein sources on infant growth, as previous evidence on this issue was inconclusive. The latest systematic review and meta-analysis examining the relationship between high protein intake and growth and risk of childhood overweight/obesity included no studies from LMICs [44]. 4. Discussion Therefore, such countries should ideally adopt recommen- dations related to dietary protein based on data from their population and avoid making assumptions by using dietary data from other countries. less, for countries such as Thailand facing the DBM and nutritional transition, using either approach could be problematic. Therefore, such countries should ideally adopt recommen- dations related to dietary protein based on data from their population and avoid making assumptions by using dietary data from other countries. More importantly, the distinctive effects of different protein sources should be taken into account when considering recommendations for dietary protein during the CF period. Current evidence suggests that dairy protein from formula and cow’s milk can promote rapid weight gain and could contribute to childhood obesity [54], while non-dairy ABP has a lesser impact on weight gain according to this cohort and other studies [55,56]. Therefore, to optimize protein intake during the CF period, nutritional policies focused on decreasing the intake of dairy protein, such as reducing the protein content in infant and follow-on formula and avoiding cow’s milk whilst encouraging mothers to continue breastfeeding throughout the ﬁrst year of life, should be integrated into CF practices. In addition, non- dairy ABP enriched with essential micronutrients such as iron, zinc, iodine, and vitamin A should be promoted to provide adequate micronutrients whilst avoiding a high intake of dairy protein. y p Finally, limitations of this study should be noted. First, the results from the present cohort cannot infer causality between dietary protein and rapid weight gain due to the observational study design. The association could be interpreted either way, and it is not possible to conclude whether dietary protein contributes to greater weight gain or whether parents of faster-growing infants provide more food, including protein. However, DAGs were applied to appropriately identify potential confounders and to avoid overadjustment and selection bias [57]. Second, the null effect of PBP on growth outcomes should be interpreted with caution because the PBP consumed by infants in this cohort was mainly cereals, whereas legumes and grains containing higher protein quantity and quality, which may be more frequently used in other LMICs, were rarely consumed. 4. Discussion This systematic review con- cluded that there is adequate evidence supporting a possibly causal effect of high protein, especially ABP, on BMI (dose–response effect), while limited evidence suggests that high protein intake may affect weight gain/weight-for-age score and the risk of childhood obesity. However, there were several inconclusive results, including the effect of high protein on linear growth and body composition [44]. The present study did not demon- strate a relationship between high protein intake and overweight/obesity due to the very small number of infants who were overweight/obese at 12 months old. However, there is evidence justifying the concern about the potential impact of high protein intake on overweight/obesity in this population. In 2019, the prevalence of overweight/obesity among Thai infants and young children aged less than 5 years rose to 12.7% [45] compared to the previous national surveys in 2009 and 2016 (8.5% and 8.2%, respectively) [46,47]. The daily protein intake reported in 2013 was similar to the present study; infants and young children aged 6 to less than 36 months had dietary protein intakes about 3 times higher than the Thai recommendations and nearly 80% of the protein was derived from ASFs, while total energy consumption was not different from the recommendation [30,48,49]. Notably, the literature from LMICs generally considers ABP as a preferred protein source due to its beneﬁcial effect in preventing undernutrition [44,50,51]. Theoretically, protein from ASFs should provide adequate amounts of essential amino acids to meet the requirements of infants and children in order to prevent stunting [52]. A recent systematic review of studies on infants and children aged 6–60 months in LMICs did not ﬁnd any signiﬁcant associations between the consumption of ASFs and growth outcomes includ- ing weight, length/height and head circumference, though the included studies showed high heterogeneity [53]. The literature thus illustrates how ‘optimal’ protein intakes and sources during CF may differ in high-income and low-income settings. Reducing protein in complementary foods in European countries and the United States may help prevent childhood over- weight/obesity, while promoting the consumption of ABPs in many low-income countries might mitigate the burden of wasting, stunting and micronutrient deﬁciencies. Nonethe- Nutrients 2022, 14, 3948 15 of 18 15 of 18 less, for countries such as Thailand facing the DBM and nutritional transition, using either approach could be problematic. 4. Discussion Third, the lack of a signiﬁcant association between protein intake and linear growth may be due to lack of statistical power, as the sample size was calculated based on the expected difference in WLZ at 12 months between infants consuming ASF regularly and those who did not. Fourth, by assessing change in size between 6 and 12 months, and assessing complementary feeding during this period, some of the variability in growth that we quantiﬁed may have occurred prior to the dietary exposure. However, this makes any associations of growth and complementary feeding that we detect conservative. Lastly, it should be noted that “extra” weight gain from increasing intake of ABP cannot be assumed to indicate higher body fatness without additional evidence from body composition analysis, and we do not yet know how our ﬁndings will translate into the risk of overweight/obesity at later ages. 5. Conclusions The present cohort provides evidence from a middle-income country that different protein sources may have contrasting inﬂuences on infant growth. While high protein intake from ASFs, especially formula and cow’s milk, during the CF period was associated with higher weight gain in a dose–response manner, the study did not ﬁnd an effect on linear growth. Importantly, higher levels of IGF-1, IGFBP-3 and insulin in infants consuming higher amounts of protein from formula and cow’s milk provided mechanistic support for the clinical ﬁndings. However, further studies in populations facing the DBM and nutritional transition are needed to conﬁrm the key ﬁndings from this cohort and to investigate the relationship between dietary protein and body composition. A longer follow-up period is also needed to see whether the study population consuming higher protein have a greater risk of overweight/obesity at later ages. Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/nu14193948/s1, Figure S1: An example of nutrient values reported from the INMUCAL program, Figure S2: A participant ﬂow chart, Table S1: Means, standard deviation (SD) and 95% conﬁdence intervals (CI) of conditional growth among protein intake groups, Table S2: Complementary feeding recommendations for Thai infants, Table S3: Comparison of Nutrients 2022, 14, 3948 16 of 18 average IGF-1, IGFBP-3 and insulin concentrations between female and male infants at 12 months of age. average IGF-1, IGFBP-3 and insulin concentrations between female and male infants at 12 months of age. Author Contributions: K.K., M.F. and J.L. designed the research (project conception, development of overall research plan, and study oversight); K.K. conducted research and data collection; J.C.K.W. guided conditional growth calculations; S.M. and S.K. analyzed serum IGF-1 and IGFBP-3; K.K. analyzed data and performed statistical analysis; K.K. wrote the paper; K.K. and M.F. had primary responsibility for the ﬁnal content; M.F., J.L. and J.C.K.W. provided critical comments for the revision. All authors have read and agreed to the published version of the manuscript. Funding: This study is funded by the Childhood Nutrition Research Centre, UCL Great Ormond Street Institute of Child Health. Research at UCL Great Ormand Street Institute of Child Health is supported by the NIHR Great Ormond Street Hospital Biomedical Research Centre. References 1. Winichagoon, P. Thailand nutrition in transition: Situation and challenges of maternal and child nutrition. Asia Pac. J. Clin. Nutr. 2013, 22, 6–15. [PubMed] 2. Dietz, W.H. Double-duty solutions for the double burden of malnutrition. Lancet 2017, 390, 2607–2608. 3. Wells, J.C.; Sawaya, A.L.; Wibaek, R.; Mwangome, M.; Poullas, M.S.; Yajnik, C.S.; Demaio, A. The double burden of malnutrition: Aetiological pathways and consequences for health. Lancet 2020, 395, 75–88. [CrossRef] 4. Popkin, B.M.; Corvalan, C.; Grummer-Strawn, L.M. Dynamics of the double burden of malnutrition and the changing nutrition reality. Lancet 2020, 395, 65–74. [CrossRef] 5. Hoffman, D.; Arts, M.; Begin, F. The “First 1,000 Days+” as Key Contributor to the Double Burden of Malnutrition. Ann. Nutr. Metab. 2019, 75, 99–102. [CrossRef] 6. Fewtrell, M.; Bronsky, J.; Campoy, C.; Domellöf, M.; Embleton, N.; Mis, N.F.; Hojsak, I.; Hulst, J.M.; Indrio, F.; Lapillonne, A.; et al. Complementary Feeding: A Position Paper by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) Committee on Nutrition. J. Pediatr. Gastroenterol. Nutr. 2017, 64, 119–132. [CrossRef] 7. Michaelsen, K.F.; Grummer-Strawn, L.; Begin, F. Emerging issues in complementary feeding: Global aspects. Matern. Child Nutr. 2017, 13, e12444. [CrossRef] ; Greer, F.R. Protein needs early in life and long-term health. Am. J. Clin. Nutr. 2014, 99, 718S–722S. [CrossR 8. Michaelsen, K.F.; Greer, F.R. Protein needs early in life and long-term health. Am. J. Clin. Nutr. 2014, 99 y g 9. Scaglioni, S.; Agostoni, C.; De Notaris, R.; Radaelli, G.; Radice, N.; Valenti, M.; Giovannini, M.; Riva, E. Early macronutrient intake and overweight at ﬁve years of age. Int. J. Obes. Relat. Metab. Disord. 2000, 24, 777–781. [CrossRef] g y g 10. Hoppe, C.; Rovenna Udam, T.; Lauritzen, L.; Mølgaard, C.; Juul, A.; Fleischer Michaelsen, K. Animal protein intake, serum insulin-like growth factor I, and growth in healthy 2.5-y-old Danish children. Am. J. Clin. Nutr. 2004, 80, 447–452. [CrossRef] 11. Morgan, J.; Taylor, A.; Fewtrell, M. Meat consumption is positively associated with psychomotor ou 24 months of age. J. Pediatr. Gastroenterol. Nutr. 2004, 39, 493–498. [CrossRef] [PubMed] g 12. Thorisdottir, B.; Gunnarsdottir, I.; Palsson, G.I.; Halldorsson, T.I.; Thorsdottir, I. Animal protein intake at 12 months is associated with growth factors at the age of six. Acta Paediatr. 2014, 103, 512–517. [CrossRef] [PubMed] g g 13. Koletzko, B.; von Kries, R.; Closa, R.; Escribano, J.; Scaglioni, S.; Giovannini, M.; Beyer, J.; Demmelmair, H.; Gruszfeld, D.; Dobrzanska, A.; et al. 5. Conclusions Institution Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the University College London Ethics committee, United Kingdom (Approval ID: 12551/001), and the Ethics Committee of the Faculty of Medicine, Chiang Mai University, Thailand (Approval ID: PED-2561-05287). Informed Consent Statement: Informed consent was obtained from all subjects involved in the study Acknowledgments: The authors are grateful to all parents and infants who participated in this cohort. We also thank the healthcare staff at the three study sites for their help and support, the head and secretary of the Department of Pediatrics, Faculty of Medicine, Chiang Mai University for facilitating the Ethics approval. Conﬂicts of Interest: MF receives an unrestricted donation for infant nutrition research from Philips. The remaining authors have no potential conﬂict of interest. 14. Weber, M.; Grote, V.; Closa-Monasterolo, R.; Escribano, J.; Langhendries, J.P.; Dain, E.; Giovannini, M.; Verduci, E.; Gruszfeld, D.; Socha, P.; et al. Lower protein content in infant formula reduces BMI and obesity risk at school age: Follow-up of a randomized trial. Am. J. Clin. Nutr. 2014, 99, 1041–1051. [CrossRef] [PubMed] References Lower protein in infant formula is associated with lower weight up to age 2 y: A randomized clinical trial. Am. J. Clin. Nutr. 2009, 89, 1836–1845. [PubMed] 14. Weber, M.; Grote, V.; Closa-Monasterolo, R.; Escribano, J.; Langhendries, J.P.; Dain, E.; Giovannini, M.; Verduci, E.; Gruszfeld, D.; Socha, P.; et al. Lower protein content in infant formula reduces BMI and obesity risk at school age: Follow-up of a randomized trial. Am. J. Clin. Nutr. 2014, 99, 1041–1051. [CrossRef] [PubMed] Nutrients 2022, 14, 3948 17 of 18 15. Rzehak, P.; Grote, V.; Lattka, E.; Weber, M.; Gruszfeld, D.; Socha, P.; Closa-Monasterolo, R.; Escribano, J.; Giovannini, M.; Verduci, E.; et al. Associations of IGF-1 gene variants and milk protein intake with IGF-I concentrations in infants at age 6 months-results from a randomized clinical trial. Growth Horm. IGF Res. 2013, 23, 149–158. [CrossRef] 16. Wan, X.; Wang, S.; Xu, J.; Zhuang, L.; Xing, K.; Zhang, M.; Zhu, X.; Wang, L.; Gao, P.; Xi, Q.; et al. Dietary IGF-1 secretion mediated by PPARgamma activation. PLoS ONE 2017, 12, e0173174. [CrossRef] X.; Wang, S.; Xu, J.; Zhuang, L.; Xing, K.; Zhang, M.; Zhu, X.; Wang, L.; Gao, P.; Xi, Q.; et al. Dietary protein-in secretion mediated by PPARgamma activation. PLoS ONE 2017, 12, e0173174. [CrossRef] y g [ ] 17. Wu, L.; Liao, P.; He, L.; Feng, Z.; Ren, W.; Yin, J.; Duan, J.; Li, T.; Yin, Y. Dietary L-arginine supplementation protects weanling pigs from deoxynivalenol-induced toxicity. Toxins 2015, 7, 1341–1354. [CrossRef] 18. Zhang, S.; Zeng, X.; Ren, M.; Mao, X.; Qiao, S. Novel metabolic and physiological functions of branched chain amino acids: A review. J. Anim. Sci. Biotechnol. 2017, 8, 10. [CrossRef] 18. Zhang, S.; Zeng, X.; Ren, M.; Mao, X.; Qiao, S. Novel metabolic and A review. J. Anim. Sci. Biotechnol. 2017, 8, 10. [CrossRef] 19. Nissen, S.L.; Abumrad, N.N. Nutritional role of the leucine metabolite β-hydroxy β-methylbutyrate (HMB). J. Nutr. Biochem. 1997, 8, 300–311. [CrossRef] rad, N.N. Nutritional role of the leucine metabolite β-hydroxy β-methylbutyrate (HMB). J. Nutr. Biochem ossRef] 20. Mogami, H.; Yura, S.; Itoh, H.; Kawamura, M.; Fujii, T.; Suzuki, A.; Aoe, S.; Ogawa, Y.; Sagawa, N.; Konishi, I.; et al. Isocaloric high-protein diet as well as branched-chain amino acids supplemented diet partially alleviates adverse consequences of maternal undernutrition on fetal growth. Growth Horm. IGF Res. 2009, 19, 478–485. [CrossRef] 21. World Health Organization. References WHO Anthro for Personal Computers Version 3.2.2: Software for Assessing Growth and Development of the World’s Children 2011 [Updated 2019, Cited 2022 April]. Available online: https://www.who.int/tools/child-growth- standards/software (accessed on 1 May 2021). ( y ) 22. World Health Organization. WHO Child Growth Standards: Length/Height-for-Age, Weight-for-Age, Weight-for-Length, Weight-for- Height and Body Mass Index-for-Age: Methods and Development; WHO: Geneva, Switzerland, 2006. g y f g p 23. Institute of Nutrition, Mahidol University. INMUCAL-Nutrients, Version 4.0; Mahidol University: Nakorn Pathom, Thailand, 2018. 23. Institute of Nutrition, Mahidol University. INMUCAL-Nutrients, Version 4.0; Mahidol University: Nakorn Pathom, Thailand, 2018. 24. Olaya, G.A.; Lawson, M.; Fewtrell, M.S. Efﬁcacy and safety of new complementary feeding guidelines with an emphasis on red 23. Institute of Nutrition, Mahidol University. INMUCAL-Nutrients, Version 4.0; Mahidol University: Nakorn Pathom, Thailand, 2018. 24. Olaya, G.A.; Lawson, M.; Fewtrell, M.S. Efﬁcacy and safety of new complementary feeding guidelines with an emphasis on red meat consumption: A randomized trial in Bogota Colombia Am J Clin Nutr 2013 98 983 993 [CrossRef] [PubMed] y y 24. Olaya, G.A.; Lawson, M.; Fewtrell, M.S. Efﬁcacy and safety of new complementary feeding guidelines meat consumption: A randomized trial in Bogota, Colombia. Am. J. Clin. Nutr. 2013, 98, 983–993. [Cro p g 25. Kittisakmontri, K.; Lanigan, J.; Wells, J.C.K.; Fewtrell, M. The Impact of Dietary Protein in Complementary Foods on Infant Growth and Body Composition in a Population Facing the Double Burden of Malnutrition: Protocol for a Multicenter, Prospective Cohort Study. JMIR Res. Protoc. 2020, 9, e18112. [CrossRef] [PubMed] y 26. Textor, J.; Van der Zander, B.; Gilthorpe, M.S.; Li´skiewicz, M.; Ellison, G.T. Robust causal inference usin The R package ‘dagitty’. Int. J. Epidemiol. 2016, 45, 1887–1894. [CrossRef] p g g y 27. Bureau of Health Promotion. Mother and Child Health Handbook; Veteran Press: Bangkok, Thailand, 201 28. Parikh, P.; Semba, R.; Manary, M.; Swaminathan, S.; Udomkesmalee, E.; Bos, R.; Poh, B.K.; Rojroongwasinkul, N.; Geurts, J.; Sekartini, R.; et al. Animal source foods, rich in essential amino acids, are important for linear growth and development of young children in low- and middle-income countries. Matern. Child Nutr. 2022, 18, e13264. [CrossRef] [PubMed] 29. Thaweekul, P.; Surapolchai, P.; Sinlapamongkolkul, P. Infant feeding practices in relation to iron status and other possible nutritional deﬁciencies in Pathumthani, Thailand. Asia Pac. J. Clin. Nutr. 2019, 28, 577–583. [PubMed] 30. References Rojroongwasinkul, N.; Kijboonchoo, K.; Wimonpeerapattana, W.; Purttiponthanee, S.; Yamborisut, U.; Boonpraderm, A.; Kunapan, P.; Thasanasuwan, W.; Khouw, I. SEANUTS: The nutritional status and dietary intakes of 0.5-12-year-old Thai children. Br. J. Nutr. 2013, 110 (Suppl. 3), S36–S44. [CrossRef] 31. Brouwer, I.D.; van Liere, M.J.; de Brauw, A.; Dominguez-Salas, P.; Herforth, A.; Kennedy, G.; Lachat, C.; Omosa, E.B.; Talsma, E.F.; Vandevijvere, S.; et al. Reverse thinking: Taking a healthy diet perspective towards food systems transformations. Food Secur. 2021, 13, 1497–1523. [CrossRef] 32. Popkin, B.M.; Ng, S.W. The nutrition transition to a stage of high obesity and noncommunicable disease prevalence dominated by ultra-processed foods is not inevitable. Obes. Rev. 2021, 23, e13366. [CrossRef] p 33. Michaelsen, K.F.; Larnkjaer, A.; Molgaard, C. Amount and quality of dietary proteins during the ﬁrst two years of life in relation to NCD risk in adulthood. Nutr. Metab. Cardiovasc. Dis. 2012, 22, 781–786. [CrossRef] 34. Agostoni, C.; Scaglioni, S.; Ghisleni, D.; Verduci, E.; Giovannini, M.; Riva, E. How much protein is safe? Int. J. Obes. 2005, 29, S8–S13. [CrossRef] 35. Nordic Council of Ministers. Nordic Nutrition Recommendations 2004: Integrating Nutrition and Physical Activity; Nordic Council of Ministers: Copenhagen, Denmark, 2005; p. 436. 36. Food and Agricultural Organization of the United Nations, World Health Organization and United Nations University. Protein and Amino Acids Requirements in Human Nutrition: Report of a Joint FAO/WHO/UNU Expert Consultation; World Health Organization: Geneva, Switzerland, 2007. 37. Food and Nutrition Board. Dietary Reference Intakes for Energy, Carbohydrate, Fiber, Fat, Fatty Acids, Cholesterol, Protein and Amino Acids. In Institute of Medicine, Editor; The National Academic Press: Washington, DC, USA, 2005. 38. European Food Safety Authority. Dietary Reference Values for Nutrients Summary Report. EFSA Supporting Publ. 2017, 14, e15121E. Osmond, C.; Fall, C.H.D. Conditional Growth Models: An Exposition and Some Extensions. Disease M Health, Part B. In Handbook of Statistics; Elsevier: Amsterdam, The Netherlands, 2017; pp. 275–300. 40. Benyi, E.; Savendhal, L. The physiology of childhood growth: Hormonal regulation. Horm. Res. Paediatr. 2017, 88, 6–14. [CrossRef] [PubMed] 18 of 18 18 of 18 Nutrients 2022, 14, 3948 41. Kouwenhoven, S.M.; Fleddermann, M.; Finken, M.J.; Twisk, J.W.; van der Beek, E.M.; Abrahamse-Berkeveld, M.; van de Heijning, B.J.; van Harskamp, D.; van Goudoever, J.B.; Koletzko, B.V. Early-life metabolic and hormonal markers in blood and growth until age 2 years: Results from a randomized controlled trial in healthy infants fed a modiﬁed low-protein infant formula. Nutrients 2021, 13, 1159. References [CrossRef] 42. Melnik, B.C. Milk—A Nutrient System of Mammalian Evolution Promoting mTORC1-Dependent Translation. Int. J. Mol. Sci. 2015, 16, 17048–17087. [CrossRef] 43. Melnik, B.C. Leucine signaling in the pathogenesis of type 2 diabetes and obesity. World J. Diabetes 2012, 3, 38–53. [CrossRef] [PubMed] 44. Arnesen, E.K.; Thorisdottir, B.; Lamberg-Allardt, C.; Bärebring, L.; Nwaru, B.; Dierkes, J.; Ramel, A.; Åkesson, A. Protein intake in children and growth and risk of overweight or obesity: A systematic review and meta-analysis. Food Nutr. Res. 2022, 66. [CrossRef] [ ] 45. Thai Nationa Statistical Ofﬁce (NSO). Thailand Multiple Indicator Cluster Survey 2019, Survey Findings Report; National Statistical Ofﬁce of Thailand: Bangkok, Thailand, 2019. g 46. Mo-suwan, L.; Akeplakorn, W. Nutritional Status of Thai Children; Health System Research Institute: Ba uwan, L.; Akeplakorn, W. Nutritional Status of Thai Children; Health System Research Institute: Bangkok, Tha EF; Thai NSO. Thailand Multiple Indicator Cluster Survey 2015–2016, Final Report; NSO and UNICEF: Bangkok, T 6 Mo suwa , ; kep ako , W Nutritional Status of hai Children; ea t Syste esea c st tute a gkok, a a d, 009 47. UNICEF; Thai NSO. Thailand Multiple Indicator Cluster Survey 2015–2016, Final Report; NSO and UNICEF: Bangkok, Thailand, 2016. 47. UNICEF; Thai NSO. Thailand Multiple Indicator Cluster Survey 2015–2016, Final Report; NSO and UNICEF: Bangkok, Thailand, 2016. 48. Ministry of Public Health. Thai Recommended Daily Intakes; Ministry of Public Health: Bangkok, Thailand, 2020. SO. Thailand Multiple Indicator Cluster Survey 2015–2016, Final Report; NSO and UNICEF: Bangkok, Thailand ic Health. Thai Recommended Daily Intakes; Ministry of Public Health: Bangkok, Thailand, 2020. g try of Public Health. Thai Recommended Daily Intakes; Ministry of Public Health: Bangkok, Thailand, 2020. 48. Ministry of Public Health. Thai Recommended Daily Intakes; Ministry of Public Health 49. Kittisakmontri, K. The Impact of Dietary Protein in Complementary Foods on Infant Growth and Iron Status in a Population Facing Double-Burden of Malnutrition. Doctoral Thesis, University College London, London, UK, 2022. Available online: https://discovery.ucl.ac.uk/id/eprint/10141347/ (accessed on 8 April 2022). p y p p 50. Allen, L. Comparing the value of protein sources for maternal and child nutrition. F 50. Allen, L. Comparing the value of protein sources for maternal and child nutrition. Food Nutr. Bull. 2013, 34, 263–266. References [CrossRef] 51 I tti L L L tt C K St t C P G ll Ri f í C A M l C R i h t G P l i A K C Ch i k M 50. Allen, L. Comparing the value of protein sources for maternal and child nutrition. Food Nutr. Bull. 2013, 34, 263–266. [CrossRef] 51. Iannotti, L.L.; Lutter, C.K.; Stewart, C.P.; Gallegos Riofrío, C.A.; Malo, C.; Reinhart, G.; Palacios, A.; Karp, C.; Chapnick, M.; Cox, K.; et al. Eggs in Early Complementary Feeding and Child Growth: A Randomized Controlled Trial. Pediatrics 2017, 140. [CrossRef] 51. Iannotti, L.L.; Lutter, C.K.; Stewart, C.P.; Gallegos Riofrío, C.A.; Malo, C.; Reinhart, G.; Palacios, A.; Karp, C.; Chapnick, M.; Cox, K.; et al. Eggs in Early Complementary Feeding and Child Growth: A Randomized Controlled Trial. Pediatrics 2017, 140. [CrossRef] [ ] 52. Semba, R.D.; Shardell, M.; Ashour, F.A.S.; Moaddel, R.; Trehan, I.; Maleta, K.M.; Ordiz, M.I.; Kraemer, K.; Khadeer, M.A.; Ferrucci, L.; et al. Child Stunting is Associated with Low Circulating Essential Amino Acids. EBioMedicine 2016, 6, 246–252. [CrossRef] 53. Shapiro, M.J.; Downs, S.M.; Swartz, H.J.; Parker, M.; Quelhas, D.; Kreis, K.; Kraemer, K.; West, K.P., Jr.; Fanzo, J. A Systematic Review Investigating the Relation Between Animal-Source Food Consumption and Stunting in Children Aged 6-60 Months in Low and Middle-Income Countries. Adv. Nutr. 2019, 10, 827–847. [CrossRef] 54. Koletzko, B.; Demmelmair, H.; Grote, V.; Totzauer, M. Optimized protein intakes in term infants support physiological growth and promote long-term health. Semin. Perinatol. 2019, 43, 151153. [CrossRef] [PubMed] p g 55. Tang, M.; Hendricks, A.E.; Krebs, N.F. A meat- or dairy-based complementary diet leads to distinct growth patterns in formula-fed infants: A randomized controlled trial. Am. J. Clin. Nutr. 2018, 107, 734–742. [CrossRef] [PubMed] 56. Günther, A.L.; Remer, T.; Kroke, A.; Buyken, A.E. Early protein intake and later obesity risk: Which protein sources at which time points throughout infancy and childhood are important for body mass index and body fat percentage at 7 y of age? Am. J. Clin. Nutr. 2007, 86, 1765–1772. [CrossRef] [PubMed] 57. Williams, T.C.; Bach, C.C.; Matthiesen, N.B.; Henriksen, T.B.; Gagliardi, L. Directed acyclic graphs: A tool for causal studies in paediatrics. Pediatr. Res. 2018, 84, 487–493. [CrossRef] [PubMed]
https://openalex.org/W2938447708	http://revistacmc.espm.br/index.php/revistacmc/article/download/1840/pdf	Portuguese	null	Quem fala sobre a ditadura nos jornais? Reflexões sobre as fontes de informação jornalísticas	Comunicação, Mídia e Consumo	2,019	cc-by	7,345	COMUN. MÍDIA CONSUMO, SÃO PAULO, V. 16, N. 45, P. 80-100, JAN./ABR. 2019 DOI 10.18568/CMC.V16I45.1840 COMUN. MÍDIA CONSUMO, SÃO PAULO, V. 16, N. 45, P. 80-100, JAN./ABR. 2019 DOI 10.18568/CMC.V16I45.1840 A R T I G O 2 Universidade Federal de Juiz de Fora (UFJF). Juiz de Fora, MG, Brasil. https://orcid.org/0000-0002-1714-8189. E-mail: teneves@terra.com.br 1 Universidade Federal de Minas Gerais (UFMG). Belo Horizonte, MG, Brasil. 2 Universidade Federal de Juiz de Fora (UFJF). Juiz de Fora, MG, Brasil. https://orcid.org/0000-0002-1714-8189. E-mail: teneves@terra.com.br https://orcid.org/0000-0003-4335-7926. E-mail: nandanalon@yahoo.com.br 1 Universidade Federal de Minas Gerais (UFMG). Belo Horizonte, MG, Brasil. https://orcid.org/0000-0003-4335-7926. E-mail: nandanalon@yahoo.com.br 1 Universidade Federal de Minas Gerais (UFMG). Belo Horizonte, MG, Brasil. https //orcid org/0000 0003 4335 7926 E mail nandanalon@yahoo com br Introdução A Comissão Nacional da Verdade (CNV) foi estabelecida entre 2012 e 2014 no Brasil com o propósito de investigar os crimes cometidos por agentes da ditadura militar (1964-1985). Seus atributos eram reunir documentos, encontrar provas materiais, coletar depoimentos, realizar oitivas e elaborar um relatório final com as conclusões dos trabalhos e as recomendações para mitigar danos e prevenir novas violações. Para isso, a CNV precisou desenvolver um trabalho de mobilização social, tanto para garantir o apoio da militância dos direitos humanos e dos familiares de mortos e desaparecidos políticos quanto para alcançar outros estratos sociais que ignoravam ou conheciam mal o período inves- tigado. Esse trabalho envolveu a tarefa de se comunicar com a sociedade tanto por meios diretos (fazendo uso de mídias sociais, propaganda go- vernamental, audiências públicas) quanto pela mediação do jornalismo. Sabendo-se que as narrativas jornalísticas não são construídas ape- nas pelas escolhas textuais e imagéticas, sendo também elaboradas a partir da seleção das fontes de informação e da edição das falas a serem utilizadas, este artigo pretende compreender quais fontes foram privile- giadas no noticiário sobre a ditadura durante o funcionamento da CNV. Parte-se do pressuposto de que a escolha das fontes e do que elas dizem é sempre uma decisão política. Será testada a hipótese de que, durante o funcionamento da CNV e de outras comissões de âmbito regional e local, o discurso produzido por elas foi privilegiado nos principais veí- culos jornalísticos brasileiros como narrativa oficial acerca do período ditatorial. fernanda nalon sanglard \| teresa cristina da costa neves fernanda nalon sanglard \| teresa cristina da costa neves 81 Quem fala sobre a ditadura nos jornais? Reflexões sobre as fontes de informação jornalísticas Who talks about dictatorship in the news media? Reflections on the journalistic information sources Fernanda Nalon Sanglard1 Teresa Cristina da Costa Neves2 Fernanda Nalon Sanglard1 Teresa Cristina da Costa Neves2 Resumo: O artigo analisa a cobertura jornalística sobre a ditadura no perío- do de funcionamento da Comissão Nacional da Verdade e de outras comissões de âmbito regional. O principal objetivo é verificar quais fontes de informação tiveram espaço na cobertura e identificar se as comissões da verdade impactaram nesse processo. São analisadas as citações entre aspas veiculadas em conteúdos noticiosos por sete veículos de mídia – Folha de S. Paulo, O Estado de S. Paulo, O Globo, Carta Capital, rádio CBN, TV Globo e G1 – entre julho de 2012 e dezembro de 2014. Palavras-chave: ditadura; jornalismo; fontes de informação; comissões da verdade. Abstract: This paper analyzes journalistic coverage of the Brazilian dicta- torship during the term of National Truth Commission and other local truth commissions. The main goal is to verify which journalistic information sources have had presence in the media coverage and to identify the impact of truth com- missions in that process. The quotations of sources published by seven Brazilian media outlets (Folha de S. Paulo, O Estado de S. Paulo, O Globo, Carta Capital; CBN, TV Globo and G1) between July 2012 and December 2014 are analyzed. Keywords: dictatorship; journalism; journalistic information sources; truth commissions. Fontes como instrumento político Na escolha das falas dos entrevistados a serem incorporadas ao material jornalístico, há algo em jogo que vai além das justificativas corriqueiras baseadas na ideia de interesse público e no ethos jornalístico. Conforme pontua Flávio Agnelli (2008), a escolha das fontes (e também do que elas dizem) preanuncia a orientação que a narrativa seguirá. comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 quem fala sobre a ditadura nos jornais? 82 Tal seleção envolve uma série de questões, como demonstrou Gaye Tuchman (1978), para quem as notícias são narrações de realidades possíveis e, portanto, uma construção escolhida e editada. Ao abor- dar os aspectos estruturais que envolvem as ideologias e as rotinas de produção dos jornalistas, a autora dá pistas de como tais escolhas são feitas, demonstrando que os profissionais da imprensa estão sujeitos a constrangimentos práticos – de tempo, recursos e acesso às fontes – e ideológicos – política editorial e posicionamento político do veículo e do profissional. Manuel Pinto (2000) diz ser necessário analisar a relação com as fon- tes num quadro vasto e complexo que indique as dinâmicas internas ao campo jornalístico e também as convergências e conflitualidades decor- rentes da interação no sistema social. As fontes são pessoas, são grupos, são instituições sociais ou são vestígios – falas, documentos, dados […]. Em suma, as fontes a que os jornalistas recorrem ou que procuram os jornalistas são entidades interessadas, quer dizer, estão implicadas e desenvolvem a sua atividade a partir de estraté- gias e com tácticas bem determinadas. E, se há notícias, isso deve-se, em grande medida, ao fato de haver quem esteja interessado que certos factos sejam tornados públicos (PINTO, 2000, p. 278). Todavia, Herbert Gans (1979) menciona a tendência do jornalismo de garantir preferência às fontes oficiais em detrimento das pessoas co- muns. Traquina (2008) reitera que algumas fontes são favorecidas no processo de produção da notícia: quanto mais alta é a posição do infor- mante, maior é a tendência de que seja considerado uma boa fonte de informação. Essa lógica indica que há pessoas autorizadas a falar sobre determinado tema e outras não. Considerando a interdependência entre jornalistas e formadores de opinião, bem como o fato de que nem todas as fontes são iguais na sua capacidade de ter acesso aos meios de comunicação, Traquina (2008) compreende a mídia como um bem “estratificado socialmente”. Fontes como instrumento político Segun- do ele, foi a compreensão sobre a dependência dos “canais de rotina” que levou Michael Schudson a descrever o processo de produção das comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 fernanda nalon sanglard \| teresa cristina da costa neves 83 notícias como uma questão de representantes de uma burocracia uti- lizando notícias pré-fabricadas por representantes de outra burocracia. 3 Ver Krippendorff (1990), Hansen et al. (1998) e Bardin (2008). 4 l 4 Ver Hansen et al. (1998). 5 As ONGs são consideradas por Nilson Lage (2003) como fontes independentes. Contudo, por acreditar, assim como Traquina (2008), que não existem fontes desinteressadas e por conside- rar, nesse caso, que as ONGs e outros movimentos sociais que atuam no âmbito dos direitos humanos têm expertise no assunto, optamos por tratá-las como fontes especializadas, já que são convocadas a se posicionar em razão do conhecimento de causa que detêm. Procedimento metodológico Neste artigo, recorre-se à análise de conteúdo3 como procedimento me- todológico-analítico. O clipping noticioso produzido pela assessoria da CNV com matérias jornalísticas sobre a ditadura foi utilizado para se ter acesso ao material a ser analisado: 8.422 narrativas jornalísticas sobre a ditadura veiculadas entre julho de 2012 e dezembro de 2014 em mais de 60 veículos noticiosos. Foram selecionadas as narrativas divulgadas pelos veículos do mains- tream jornalístico que mais cobriram o tema no período analisado, optando-se por incluir três jornais impressos, uma revista impressa se- manal, uma rádio, uma emissora de TV e um portal de internet: os diários Folha de S. Paulo, O Estado de S. Paulo e O Globo, a revista Carta Capital, a rádio CBN, a TV Globo e o portal G1. Essa providência, conjugada ao método da semana composta,4 per- mitiu construir uma amostra com 404 narrativas jornalísticas referentes a esses veículos. Os artigos de opinião e editoriais foram excluídos desta análise, já que o propósito era identificar as fontes acionadas pelos pro- fissionais da imprensa e utilizadas em seus textos noticiosos. A unidade de registro analisada não foi a notícia, mas a citação direta (entre aspas) das fontes. Identificou-se, então, o total de 807 citações entre aspas de fontes de informação referenciadas pelos repórteres, entre as quais 226 publicadas por O Globo, 191 pela Folha de S. Paulo, 169 por O Estado de S. Paulo, 87 pela TV Globo, 82 pelo G1, 44 pela Carta Capital e oito pela CBN. Essas 807 falas também foram classificadas conforme o tipo de fon- te. Entre as distintas classificações de fontes jornalísticas, foram eleitas, para as finalidades deste estudo, aquelas de três naturezas, conforme coligido por Nilson Lage (2003): oficiais, testemunhais e experts. comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 84 quem fala sobre a ditadura nos jornais? 84 As primeiras são “mantidas pelo Estado; por instituições que preser- vam algum poder de Estado […]; e por empresas e organizações, como sindicatos, associações, fundações etc.” (LAGE, 2003, p. 62-63). As fon- tes testemunhais (“personagens”, no jargão jornalístico) se distinguem pelo caráter emotivo e perspectivo de seus relatos, que revelam alguma experiência ou vivência capaz de contribuir para ilustrar o que é con- tado pelo repórter. Procedimento metodológico Já os experts ou especialistas se prestam a fornecer pontos de vista e análises de fatos e suas repercussões com base em co- nhecimento perito e experiência profissional (LAGE, 2003). Entre as fontes oficiais, foram identificadas na análise a CNV, os órgãos dos poderes Executivo, Legislativo e Judiciário, bem como de- mais instituições públicas ou de interesse público e seus representantes. Foram consideradas testemunhais as vítimas (ou os resistentes) do regi- me autoritário e seus familiares, as pessoas que vivenciaram o período e os violadores. Entre os experts se incluíram historiadores, jornalistas, economistas e demais profissionais que se especializaram no período ditatorial ou em temas correlatos, assim como advogados de vítimas da ditadura e de perpetradores, organizações não governamentais5 e movi- mentos sociais voltados para a defesa dos direitos humanos. 6 Disponível em: http://g1.globo.com/ma/maranhao/noticia/2013/10/comissao-da-verdade-reali- za-audiencias-em-porto-franco-ma.html. Acesso em: 8 jan. 2017. 7 Disponível em: http://internacional.estadao.com.br/noticias/geral,doi-codi-e-tao-distante-da- -embaixada-quanto-o-ceu-do-inferno-diz-dilma,1068368. Acesso em: 8 jan. 2017. As vozes que ecoam A verificação de cada citação contida nos registros jornalísticos permite afirmar que as fontes mais valorizadas são as oficiais. Das 807 citações, 395 são provenientes delas, o que representa 49% do total analisado. Entre elas, as comissões da verdade são as que mais se destacam, tendo sua voz pronunciada 173 vezes (21,5% do total de citações) em 106 das 404 notícias sobre a ditadura. Há diversos tipos de referência às comissões, que envolvem trechos de notas oficiais e de entrevistas dos comissários, falas de seus pesquisadores comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 fernanda nalon sanglard \| teresa cristina da costa neves 85 ou respostas oficiais da assessoria. Uma das citações, extraída do portal G1, se refere à fala do policial federal Daniel Lerner, que atuou como assessor da CNV. Sobre as investigações para identificação do corpo de Epaminondas Gomes de Oliveira, desaparecido no Maranhão, ele expli- ca: “Fizemos uma exumação em Brasília e agora viemos para a região dar continuidade ao trabalho”.6 Outro exemplo de citação oficial ocorre em notícia do Estadão em que a presidenta Dilma Rousseff retruca o diplomata brasileiro Eduar- do Saboia, que comparou a situação vivida por um asilado político na embaixada do Brasil em La Paz, na Bolívia, em 2013 com a de presos políticos na época da ditadura. A fala é emblemática: “Não há nenhuma similaridade. E eu estive no DOI-Codi. Eu sei o que é o DOI-Codi. E asseguro a vocês: é tão distante o DOI-Codi da embaixada brasileira lá em La Paz, como é distante o céu do inferno. Literalmente, isso”.7 Apesar de ter sido acionada como fonte oficial, autorizada a falar so- bre assuntos de política externa, nessa citação específica e em outras circunstâncias, a figura da presidenta comporta ambiguidade, visto que ela foi vítima da ditadura e se baseou na experiência pessoal para fa- lar de uma questão de Estado. Nesse caso, é tanto fonte oficial quanto testemunhal. Dilma Rousseff foi a quarta fonte oficial mais mencionada pelos veículos de comunicação pesquisados (ver Quadro 1), num total de 26 vezes (3,2%) na amostra analisada. Ainda que não tenha se referido com recorrência ao seu histórico pessoal, em datas significativas, como as da instalação e do encerramento da CNV e a dos 50 anos do golpe de 1964, observou-se que sua experiência de vida foi valorizada. comun. mídia consumo, são paulo, v. 16, n. 6 Disponível em: http://g1.globo.com/ma/maranhao/noticia/2013/10/comissao-da-verdade-reali- za-audiencias-em-porto-franco-ma.html. Acesso em: 8 jan. 2017. 7 Disponível em: http://internacional.estadao.com.br/noticias/geral,doi-codi-e-tao-distante-da- -embaixada-quanto-o-ceu-do-inferno-diz-dilma,1068368. Acesso em: 8 jan. 2017. comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 As vozes que ecoam 45, p. 80-100, jan./abr. 2019 Quadro 1 – Detalhamento das fontes acionadas conforme a frequência das citações Quadro 1 – Detalhamento das fontes acionadas conforme a frequência das citações Fontes Frequência Porcentagem (%) Oficiais CNV 120 14,9 Outras comissões da verdade 53 6,6 Ministérios/ministros 27 3,3 Dilma Rousseff 26 3,2 Ministério Público Federal 23 2,9 Judiciário 19 2,4 Clube Militar 16 2,0 Entidades internacionais (ONU, embaixadas etc.) 16 2,0 Deputados 11 1,4 Prefeituras e governos estaduais 7 0,9 Forças Armadas 7 0,9 OAB 7 0,9 Polícias 7 0,9 Senadores 6 0,7 Comissões e órgãos oficiais de defesa dos direi- tos humanos 4 0,5 Outras fontes oficiais 46 5,7 Especializadas Historiador 34 4,2 Jornalista 30 3,7 Advogado/jurista 21 2,6 Economista 21 2,6 Pesquisador/estudioso de outras áreas 20 2,4 ONGs e movimentos sociais 13 1,6 Integrante das Forças Armadas (pronunciando pessoalmente) 3 0,4 Outras fontes especializadas 51 6,3 Personagens Vítima/resistente 80 9,9 Familiar de vítima 42 5,2 Testemunha 32 4,0 Perpetrador/suspeito de violação 28 3,5 Manifestantes 7 0,9 Militar da reserva (pronunciando individualmente) 5 0,6 Outros personagens 17 2,1 Outras fontes 8 1,0 Total 807 100,0 F l b i fernanda nalon sanglard \| teresa cristina da costa neves 87 Observa-se que as comissões da verdade e seus integrantes assumi- ram a postura de voz autorizada para se pronunciar a respeito do tema ditadura, sendo acionadas com mais frequência pelos jornalistas do que outras fontes oficiais. Os integrantes da CNV com mais citações entre aspas publicadas foram Pedro Dallari (26 vezes), José Carlos Dias (23) e Rosa Cardoso (20). Outras fontes da CNV foram acionadas, mas nem sempre tiveram falas citadas entre aspas. Ao todo, foram contabilizadas 120 citações de integrantes da CNV e seus assessores (15% do total). Já as comissões estaduais ou locais da verdade se pronunciaram 53 vezes (6,6%). No caso das comissões circunscritas a estados ou municípios, as prin- cipais fontes foram Wadih Damous, da Comissão Estadual da Verdade do Rio de Janeiro (CEV-Rio), e Gilberto Natalini, da Comissão Munici- pal da Verdade Vladimir Herzog, de São Paulo. A Comissão da Verdade do Estado de São Paulo “Rubens Paiva” também foi referenciada, po- rém as fontes variaram entre o deputado estadual Adriano Diogo (PT) e o ex-preso político Ivan Seixas, que presidiram o colegiado. As vozes que ecoam Os resultados permitem confirmar que as vozes acionadas pelo jor- nalismo na cobertura da ditadura entre julho de 2012 e dezembro de 2014, período no qual a CNV estava em funcionamento, privilegiaram o discurso oficial. Todavia, houve um deslocamento no tipo de fonte oficial que ganhou mais voz, visto que, em outros momentos, a narrativa oficial concentrou-se nas Forças Armadas ou em representantes dos três poderes e das polícias. Pelo menos três razões permitem avaliar tal varia- ção como positiva: (1) tais comissões são consideradas órgãos de Estado, com autonomia para propor recomendações e investigar as violações praticadas por esse mesmo Estado; (2) comissões da verdade costumam ter, entre seus integrantes, pessoas que resistiram à repressão e que, portanto, testemunharam o processo autoritário, o que atribui às fontes oficiais a autoridade do testemunho; (3) as investigações das comissões envolvem trabalhos de pesquisadores que subsidiaram a narrativa oficial a partir de dados extraídos de seus estudos, o que introduz no relato das fontes oficiais conteúdos especializados. comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 quem fala sobre a ditadura nos jornais? quem fala sobre a ditadura nos jornais? 88 A R T I G O Cabe ainda destacar que, por ter integrado uma série de políticas pela memória e verdade inauguradas à época pelo governo federal e ter sido criada como um órgão de Estado, a CNV pôde se pronunciar em nome dele, difundindo um discurso em sintonia com as políticas de direitos humanos do governo naquele momento. Quadro 2 – Frequência de citações entre aspas por tipo de fonte Quadro 2 – Frequência de citações entre aspas por tipo de fonte Fonte Frequência Porcentagem* Oficial 395 49% Testemunhal 211 26% Expert 193 24% Outras 8 1% Fonte: elaboração própria. *Valores arredondados Apesar de a maioria das citações entre aspas advir das fontes oficiais, se considerarmos separadamente as menções daqueles que falam em nome das comissões, é possível observar que a cobertura foi quantita- tivamente equilibrada nesse aspecto (27,5% das citações são de fontes oficiais tradicionais; 26% de testemunhais; 24% de fontes especializadas; e 21,5% de representantes das comissões da verdade). É É pertinente ainda observar que, ao analisar separadamente os tipos de fontes oficiais e testemunhais, o número de citações referentes à CNV (15% do total) foi maior até mesmo que o número de vítimas que tiveram seus relatos divulgados (10%). Isso demonstra que as comissões da verdade passaram a assumir, ao menos naquele momento, o lugar de principais narradoras do período autoritário, sendo autorizadas a falar em nome do Estado e de quem testemunhou o episódio histórico, manifestando-se como autoridade no tema. Portanto, cabe a avaliação de que as comissões, mais do que assumirem o espaço convencionalmente exercido por políticos e funcionários públicos de alto escalão nos veículos de comunicação, também se expressaram em diversas situações “em nome” das vítimas e como fontes especializadas (quando os comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 fernanda nalon sanglard \| teresa cristina da costa neves 89 pesquisadores das comissões tiveram voz). Ainda que muitas outras vozes tenham sido acionadas na cobertura, elas não predominaram tanto quanto as das comissões. Entre as fontes oficiais, o Poder Legislativo (2% das citações) so- bressaiu pouco em relação às citações dos poderes Executivo (7,5%) e Judiciário (5,5%). 8 A baixa incidência não significa necessariamente o silenciamento dessas vozes nos veículos jornalísticos. É admissível a hipótese de que, deliberadamente, essas manifestações tenham se deslocado para espaços opinativos dos periódicos impressos, nos quais permaneceram mais resguardadas do confronto direto com opiniões divergentes. quem fala sobre a ditadura nos jornais? Ainda que algumas comissões tenham sido criadas no âmbito de casas legislativas – como a “Rubens Paiva” – e, por isso, parlamentares que as compunham tenham sido ouvidos pelos jorna- listas, eles foram acionados pelo fato de integrarem uma comissão da verdade – que difere em objetivo e atuação das tradicionais comissões parlamentares. Entre as fontes oficiais, as menos recorrentes foram os militares, pro- vavelmente devido à postura das Forças Armadas de evitar se pronunciar sobre os trabalhos das comissões, de não colaborar com as investigações e de não assumir formalmente que suas instalações serviram para práti- cas abusivas e violações de direitos no período ditatorial. Juntos, os pronunciamentos das Forças Armadas e do Clube Militar (associação de direito privado sem fins lucrativos constituída pela as- sociação de militares da Marinha, do Exército, da Força Aérea e seus dependentes) representam 3% do total de citações. Já as falas de mi- litares em atuação ou da reserva de forma individual e independente das corporações ocorreram em 1% do material.8 É sintomático ainda que os militares da reserva (por meio do Clube Militar ou de forma independente) tenham se pronunciado mais do que as Forças Armadas oficialmente, sempre adotando postura crítica em relação às comissões e de defesa da instituição, com ênfase na justificativa dos atos da cor- poração e de seus integrantes. As violações costumam ser vistas como “excessos” e não como prática sistemática do período autoritário por esses militares que optaram pelo “ataque” às comissões. comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 quem fala sobre a ditadura nos jornais? 90 A R T I G O Em um dos momentos da cobertura, registros jornalísticos abordaram o embate entre tais grupos e as comissões. A consideração do presidente do Clube de Aeronáutica, brigadeiro Ivan Frota, que considerou a CNV como “uma afronta à verdade”,9 publicada pelo Estado de S. Paulo, é um exemplo. Na mesma notícia, é dito que, durante cerimônia que marcava os 50 anos do golpe civil-militar, realizada a “portas fechadas” e distante do Centro do Rio de Janeiro para evitar chamar atenção de críticos, o presidente do Clube Naval, almirante Paulo Frederico Dobbin, alegou haver “quase um massacre a um episódio da história que foi a participa- ção dos militares […]. Nosso poder de comunicação é ínfimo diante da mídia, mas será a briga de Davi e Golias. 10 Disponível em: http://oglobo.globo.com/brasil/comandante-da-marinha-comissao-da- -verdade-cumpriu-papel-dela-14817117 e em: http://politica.estadao.com.br/noticias/ geral,cnv-cumpriu-papel-e-relatorio-sera-analisado-diz-comandante-da-marinha,1606074. Aces- so em: 9 jan. 2017. 9 Disponível em: http://politica.estadao.com.br/noticias/geral,para-evitar-protestos-militares- -celebram-golpe-longe-do-centro-do-rio,1147415. Acesso em: 9 jan. 2017. 9 Disponível em: http://politica.estadao.com.br/noticias/geral,para-evitar-protestos-militares- -celebram-golpe-longe-do-centro-do-rio,1147415. Acesso em: 9 jan. 2017. 10 Disponível em: http://oglobo.globo.com/brasil/comandante-da-marinha-comissao-da- -verdade-cumpriu-papel-dela-14817117 e em: http://politica.estadao.com.br/noticias/ geral,cnv-cumpriu-papel-e-relatorio-sera-analisado-diz-comandante-da-marinha,1606074. Aces- so em: 9 jan. 2017. 11 Disponível em: http://oglobo.globo.com/brasil/anos-de-chumbo-comandante-impoe-silencio- -ao-exercito-13690198. Acesso em: 9 jan. 2017. quem fala sobre a ditadura nos jornais? A contrapropaganda é o que nos resta”. Em uma das raras falas oficiais por parte das Forças Armadas, reper- cutida em vários jornais devido ao encerramento dos trabalhos da CNV, em dezembro de 2014, o então comandante da Marinha, almirante Jú- lio Soares de Moura Neto, afirmou que a CNV “cumpriu o papel dela. Fez um relatório sobre o qual nós ainda não tivemos oportunidade de nos debruçar”.10 Depois do episódio e de tempo suficiente para estudar o documento, nada mais foi dito. A percepção de Dobbin – de que os meios de comunicação passaram a divulgar outras interpretações dos episódios históricos e de que a ver- são antes defendida pelas Forças Armadas não tem mais a ressonância social almejada – contradiz, em princípio, a postura oficial da corpora- ção de se “calar”. Contudo, ela demonstra também que a estratégia de não se pronunciar ou de conceder respostas rasas é, na verdade, um ato de protesto. Diante das demandas das comissões e dos jornalistas sobre episódios investigados, as Forças Armadas optaram por estabelecer rela- ção “diplomática”, sem, contudo, contribuir de fato. comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 fernanda nalon sanglard \| teresa cristina da costa neves 91 A proibição por parte do comandante do Exército, general Enzo Peri, de que os quartéis colaborassem com investigações sobre as violências praticadas em suas dependências durante a ditadura tomou proporções quando a CNV e o Ministério Público Federal (MPF) se deram conta do ato. O procurador da República Sérgio Suiama considerou “lamen- tável que o comando atual do Exército de um Estado Democrático de Direito esteja tão empenhado em ocultar provas e proteger autores de sequestros, torturas, homicídios e ocultações de cadáver”.11 Os relatos testemunhais O detalhamento das citações por tipo de fonte acionada demonstra ain- da que, apesar de terem recebido menos espaço do que as fontes oficiais, vítimas e resistentes da ditadura, bem como seus familiares, ocupam a segunda posição se for considerado cada tipo de fonte em separado. As vítimas e seus parentes estão representados em 122 citações entre aspas, o que significa cerca de 15% da amostra analisada. O número fica atrás apenas das citações das comissões da verdade, que ocorrem, conforme mostrado, 173 vezes (21% dos casos). As reportagens com foco nas vítimas, em geral, buscam demonstrar a dor e o sofrimento por elas vividos e também a luta dos familiares em busca de direitos e justiça. Ainda que o enquadramento prioritário de algumas dessas reportagens tenha sido temático, voltado para as ações e investigações das comissões ou para a contextualização histórica, elas verbalizam o testemunho das vítimas. Esse foi o caso de reportagem do G1 focada na contextualização his- tórica e publicada por ocasião dos 50 anos do golpe de 1964. Ao resgatar as recordações de mulheres catarinenses vitimadas pela ditadura que en- contraram na militância uma forma de ajudar a preservar a memória do período, a repórter Janara Nicoletti destaca a fala de uma personagem, Derlei Catarina de Luca, que narra como foi sua tortura: “A primei- ra noite é indescritível. Arrancam minhas roupas. Sou pendurada no comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 quem fala sobre a ditadura nos jornais? 92 A R T I G O pau-de-arara, recebo choques elétricos nos dedos, vagina, ouvido. Que- bram meus dentes. A dor é lancinante. Tão intensa que nem dá para gritar […]” (NICOLETTI, 2014). Além de relatos dramáticos, as citações testemunhais também mencionaram episódios em que os resistentes conseguiram se safar da perseguição, como demonstra a fala de José Maria Rabelo – que era dono do jornal Binômio na época do golpe – em reportagem12 do tele- jornal Bom Dia Brasil, da TV Globo. “Eles foram me deter no dia 29, às 11h. […] Enquanto eles subiram por um dos elevadores do prédio, eu descia pelo outro. O porteiro disse: ‘Seu Zé Maria, cai fora porque os homens estão te procurando’. Eu digo que esse foi o conselho mais sábio que eu recebi na minha vida”. Os relatos testemunhais Quando a frequência da utilização de citações de vítimas e familiares é observada mês a mês (Gráfico 1), torna-se nítida a maior concentração em março de 2014, mês em que a maioria das séries especiais sobre os 50 anos do golpe foi editada. Gráfico 1: Variação da quantidade de citações de vítimas e familiares por mês jul2012 ago2012 set2012 out2012 nov2012 dez2012 jan2013 fev2013 mar2013 abril2013 maio2013 jun2013 jul2013 ago2013 set2013 out2013 nov2013 dez2013 jan2014 fev2014 mar2014 abril2014 maio2014 jun2014 jul2014 ago2014 set2014 out2014 nov2014 dez2014 Frequência das citações de vítimas e familiares Vítima Familiar de vítima Fonte: elaboração própria. 12 Disponível em: http://g1.globo.com/bom-dia-brasil/noticia/2014/03/nova-geracao-de-brasilei- ros-conhece-ditadura-pelas-historias-de-familia.html. Acesso em: 9 jan. 2017. Frequência das citações de vítimas e familiares Fonte: elaboração própria. 12 Disponível em: http://g1.globo.com/bom-dia-brasil/noticia/2014/03/nova-geracao-de-brasilei- ros-conhece-ditadura-pelas-historias-de-familia.html. Acesso em: 9 jan. 2017. comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 fernanda nalon sanglard \| teresa cristina da costa neves 93 Por conta dessa efeméride, todos os veículos analisados privilegia- ram, em variadas circunstâncias, as vozes daqueles que vivenciaram a ditadura e sofreram suas consequências. O jornal O Globo, por exem- plo, publicou uma série de depoimentos de artistas vítimas do regime autoritário. Um dos objetivos de se explorar as fontes testemunhais de um epi- sódio com tamanha distância temporal é permitir que revisitem suas memórias e narrem detalhes que, em outros momentos, podem ter evi- tado mencionar. Nota-se que, no cinquentenário do golpe, o aspecto do sofrimento humano foi valorizado na cobertura. Dar voz aos artistas foi a opção da reportagem13 de Mônica Sanches veiculada no Jornal Hoje, da TV Globo, em que se colocou em pauta a vivência sob censura e o recurso de protestar pelas entrelinhas, pelos não ditos. Além das vítimas de violência física, persecutória e da censura, os fa- miliares de mortos e desaparecidos políticos também mereceram espaço na cobertura como fontes testemunhais. Suas falas representaram mais de 5% do total. Contudo, justamente aqueles que seriam a principal razão para que persistam as investigações sobre o período receberam proporcionalmente menos espaço do que algumas modalidades de fon- tes oficiais. Isso não quer dizer que foram preteridos, mas pode indicar outras possibilidades, como a dificuldade de alguns em falar sobre o sofrimento, a carência de reportagens jornalísticas investigativas que en- contrem essas fontes ou o fato de que muitos familiares são igualmente vítimas diretas do autoritarismo, como é o caso de Ivan Seixas. 13 Disponível em: http://g1.globo.com/jornal-hoje/noticia/2014/03/artistas-relembram-repressao- -e-censura-na-epoca-da-ditadura-militar.html. Acesso em: 15 dez. 2016. Os relatos testemunhais Ele foi preso aos 16 anos no DOI-Codi de São Paulo junto com seus familiares e ouviu de uma sala próxima os gritos do pai nas sessões de tortura que o levariam à morte. Fonte frequentemente acionada pelos jornalistas, Seixas se tornou militante da causa, colaborou com os trabalhos das co- missões da verdade e, por isso, serve de exemplo de fonte duplamente testemunhal e oficial: foi citado tanto como vítima e familiar de mortos comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 quem fala sobre a ditadura nos jornais? 94 A R T I G O e desaparecidos quanto como coordenador da comissão “Rubens Paiva” de São Paulo. O mesmo ocorreu com Maria Amélia Teles, conhecida como Ame- linha. Em 1972, enquanto militava no Partido Comunista do Brasil (PCdoB), foi presa, junto com o marido, na Operação Bandeirante (Oban). Seus filhos, Edson e Janaína, na ocasião com quatro e cinco anos, foram sequestrados e levados para assistirem às sessões de tortura dos pais. Toda a família tem histórico de militância na defesa dos direi- tos humanos, sendo que Amelinha integra a Comissão de Familiares de Mortos e Desaparecidos Políticos e foi assessora da Comissão da Verdade do Estado de São Paulo “Rubens Paiva”. Por conta desse histórico de sofrimento e engajamento político, Amelinha foi ouvida em algumas circunstâncias como vítima, como na reportagem14 de O Globo na qual ela indica um dos nomes de quem a torturou: “[Aparecido Laertes] Calandra, que tinha o codinome de Capitão Ubirajara, me torturou de maneira física e psicológica […].” Já como militante ou fonte oficial, Amelinha costumava ser ouvida por conhecer a fundo o sistema de repressão e pela sinceridade, que fez com que não poupasse críticas ao funcionamento da CNV, como demonstra esta citação em notícia do G1:15 “A gente já vinha preocupado também com o fato de as audiências [da CNV] serem sigilosas. Entendemos que o princípio da transparência exige publicidade de todas as audiências porque a construção da verdade é coletiva”. No caso específico dos familiares, foram especialmente acionados os parentes das vítimas de casos emblemáticos, como os netos de João Gou- lart, os filhos de Rubens Paiva e a esposa e o filho de Vladimir Herzog. Enquanto G1, Folha de S. Paulo e O Estado de S. 15 Disponível em: http://g1.globo.com/sao-paulo/noticia/2013/06/ex-ministro-reconhece-falhas- -em-comissao-nacional-da-verdade.html. Acesso em: 20 jan. 2017. 14 Disponível em: http://oglobo.globo.com/brasil/comissao-da-verdade-convocara-acusado-de- -tortura-10190253. Acesso em: 15 dez. 2016. 16 “Outras fontes” são, na verdade, informações extraídas de documentos da ditadura (processos, inquéritos, relatórios e trocas de correspondência oficial), além de trechos de jornais da época. 18 Disponível em: http://politica.estadao.com.br/noticias/geral,oficio-indica-que-reitoria-da-usp- -ajudava-a-repressao,1077031. Acesso em: 20 jan. 2017. Os relatos testemunhais Paulo foram os que mais publicaram citações de fontes oficiais (cerca de 50% das ci- tações nos três casos), o noticiário da TV Globo garantiu mais voz ao testemunho das personagens. Já o jornal O Globo, apesar de também ter comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 fernanda nalon sanglard \| teresa cristina da costa neves 95 valorizado mais as fontes oficiais, distribuiu melhor o espaço destinado a elas, tendo publicado quase 30% das citações entre aspas provenientes de fontes especializadas e aproximadamente o mesmo percentual prove- niente de fontes testemunhais. Quadro 3 – Cruzamento da quantidade de citações por tipo de fonte e veículo Totais Porcentagem das citações por veículo Fontes acionadas Carta Capital Rádio CBN O Es- tado de S.Paulo Folha de S. Paulo G1 O Globo TV Globo Fontes oficiais 40,9% 25% 50,9% 52,9% 53,7% 38,5% 32,2% Fontes especializadas 18,2% 50% 16,6% 26,2% 26,8% 27,9% 20,7% Fontes testemunhais 22,7% 25% 21,3% 16,2% 19,5% 29,6% 56,3% Outras16 18,2% 0 11,2% 4,7% 0 4% 0 Fonte: elaboração própria. Quadro 3 – Cruzamento da quantidade de citações por tipo de fonte e veículo Quadro 3 – Cruzamento da quantidade de citações por tipo de fonte e veículo A surpresa nesse caso foi a revista Carta Capital. Por se autoafirmar veículo de esquerda, que tenderia a valorizar a voz dos resistentes em relação ao discurso oficial, imaginava-se que as vítimas e seus parentes ganhariam mais espaço nas narrativas, o que não ocorreu no material analisado. Nesse caso, o espaço concedido às fontes oficiais pode se jus- tificar pelo fato de a revista se alinhar às temáticas programáticas dos governos petistas. Já o número de citações na rádio CBN foi considerado, porém repre- senta resultado inexpressivo (apenas oito citações) diante do corpus de 807 menções entre aspas. Isso se deve ao fato já mencionado de haver menor quantitativo de material referente à rádio do que às outras mí- dias e também à característica percebida, nessa cobertura da CBN, de o comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 quem fala sobre a ditadura nos jornais? 96 A R T I G O assunto ser, em geral, apresentado e discutido por comentaristas, nem tanto em notícias e reportagens. 17 Disponível em: http://g1.globo.com/jornal-nacional/noticia/2014/03/golpe-militar-de-1964- -completa-50-anos-relembre.html. Acesso em: 20 jan. 2017. 17 Disponível em: http://g1.globo.com/jornal-nacional/noticia/2014/03/golpe-militar-de-1964- -completa-50-anos-relembre.html. Acesso em: 20 jan. 2017. 18 Disponível em: http://politica.estadao.com.br/noticias/geral,oficio-indica-que-reitoria-da-usp- -ajudava-a-repressao,1077031. Acesso em: 20 jan. 2017. As fontes especializadas A cobertura também lançou mão de fontes especializadas – prin- cipalmente historiadores, jornalistas, economistas e advogados – para discutir a temática. Suas citações foram acionadas 193 vezes ou 23,9% dos casos. Entre os historiadores que ajudaram na construção das narra- tivas jornalísticas sobre a ditadura estão os professores da Universidade Federal Fluminense Daniel Aarão Reis e Jorge Ferreira e o professor da Universidade Federal do Rio de Janeiro Carlos Fico. Os três são experts no assunto e suas entrevistas foram usadas como relatos de autoridade para analisar fatos e desdobramentos em reportagens de contextuali- zação histórica: “[…] Houve uma operação militar de um golpe, mas também houve um golpe do Poder Legislativo ao depor [João] Goulart, estando em território nacional. E, logo depois, o [senador] Áureo Mou- ra Andrade empossa na Presidência da República o [deputado] Ranieri Mazzilli […]”, diz, por exemplo, Jorge Ferreira em reportagem do Jor- nal Nacional.17 Em outras circunstâncias, os historiadores foram chamados a com- parar os processos históricos brasileiros com o próprio funcionamento da CNV. Em entrevista ao Estadão,18 o historiador americano Kenneth Serbin afirma que “as comissões da verdade chegaram tarde demais (ao Brasil). Deveriam ter sido feitas nos anos 90. Com o tempo, o povo começa a esquecer o que foi o regime militar”. Já a historiadora e pro- fessora da UFRJ Maria Paula Araújo avaliou, na Folha, que “a justiça de transição no Brasil deu ênfase à questão da reparação, em detrimento de outros aspectos, como a verdade e a justiça” (BRITTO, 2013). comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 fernanda nalon sanglard \| teresa cristina da costa neves 97 Majoritariamente, as citações dos historiadores fortalecem o enten- dimento segundo o qual é necessário rever algumas das versões fixadas socialmente sobre os fatos históricos e questionar as narrativas remanes- centes que ainda valorizam o autoritarismo ou minimizam seu impacto. É possível considerar que as narrativas jornalísticas que privilegiaram essas fontes contribuem para propor outros olhares sobre a ditadura, o que, ao lado dos testemunhos, enriquece o repertório e incentiva uma postura mais crítica, ainda que esse aspecto possa não ser o observado nas coberturas cotidianas. Esses olhares mais analíticos são estimulados também pela “onda” de atividades e discussões promovidas pela sociedade civil e, especialmen- te, pelos movimentos sociais na esteira das atividades das comissões. 20 Disponível em: http://oglobo.globo.com/ela/moda/trabalho-de-zuzu-angel-estilista-do-desfile- -protesto-volta-cena-nos-50-anos-do-golpe-militar-16951260. Acesso em: 20 jan. 2017. 19 Disponível em: http://oglobo.globo.com/sociedade/historia/festival-de-diamantina-mostrara- -otica-dos-derrotados-8103237. Acesso em: 8 dez. 2016. 19 Disponível em: http://oglobo.globo.com/sociedade/historia/festival-de-diamantina-mostrara- -otica-dos-derrotados-8103237. Acesso em: 8 dez. 2016. 20 Disponível em: http://oglobo.globo.com/ela/moda/trabalho-de-zuzu-angel-estilista-do-desfile- -protesto-volta-cena-nos-50-anos-do-golpe-militar-16951260. Acesso em: 20 jan. 2017. As fontes especializadas Na já citada reportagem de O Globo,19 a historiadora Pilar Lacerda, profes- sora da UFMG, reconhece esse emaranhado de novas interpretações e realizações em prol das memórias traumáticas: “Esse momento que o Brasil vive de rever a ditadura nos entusiasmou […]. A gente acha que um festival tem a obrigação de trazer novas abordagens […]. A gente quer mais do que é inédito, do que foi silenciado”. A citação de Pilar, relativa à escolha do tema do Festival de História de Diamantina em 2013, robustece a ideia de que, quando há políti- cas públicas de memória, elas incentivam práticas culturais sobre temas específicos, como a ditadura. No período de funcionamento da CNV, integrantes de movimentos sociais, ONGs, cineastas e outros artistas fo- ram acionados pelos jornalistas como fontes especializadas para falar de manifestações culturais organizadas nesse sentido. As notícias mostram que não faltaram protestos, exibições fílmicas, peças de teatro e lançamentos de livros para debater o tema do autorita- rismo. É o caso de reportagem20 do caderno de moda Ela, publicado por O Globo, em que são acionados como fontes historiador de moda, esti- lista e familiar de vítima para falar de uma exposição lançada em 2014 comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 quem fala sobre a ditadura nos jornais? 98 A R T I G O sobre a trajetória artística e política de Zuzu Angel, vítima da ditadura morta na década de 1970 e mãe do desaparecido político Stuart Angel. Contudo, apesar de muitos registros jornalísticos sobre manifes- tações em favor da rememoração da ditadura, em geral, tais temas foram tratados de forma episódica na cobertura. Quando enquadrados de modo temático, privilegiaram o uso de citações das fontes oficiais e especializadas em detrimento de integrantes de movimentos sociais independentes, organizações não governamentais de defesa dos direitos humanos e manifestantes, cujas citações identificadas correspondem apenas a 2,5% do total. Conclusão O estudo comprovou a hipótese de que as fontes oficiais são privilegiadas, à exceção da TV Globo, que deu preferência às narrativas testemunhais. Todavia, mesmo recorrendo majoritariamente ao discurso oficial, tais veículos divulgaram, durante o funcionamento da CNV, narrativas por- tadoras de novos ângulos de abordagem sobre o período da ditadura, tendo sido as comissões da verdade e seus integrantes as principais fontes dos jornalistas no período. A análise permitiu verificar certa pluralidade de pontos de vista nas informações colhidas junto às comissões da verdade, revelando- -as como fontes não estritamente oficiais, embora assim tenham sido essencialmente consideradas pela cobertura jornalística. Além de se pronunciarem em nome do Estado, os integrantes das comissões se ex- pressaram, não raro, também como quem testemunhou o período ou como experts. Isso se deve à composição das comissões, que tiveram en- tre seus integrantes pessoas que resistiram à repressão e testemunharam o processo autoritário, bem como pesquisadores especialistas no tema ditadura. Nesse caso, portanto, vozes não oficiais ganharam status de fonte oficial. Essa convivência de modalidades diversas de fontes na imprensa bra- sileira durante a atuação da CNV abriu horizontes de significação sobre o período ditatorial. A oficialização das fontes (muitas vezes provenientes comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 fernanda nalon sanglard \| teresa cristina da costa neves 99 de ambientes e funções não oficiais) que atuaram numa comissão com status de “política de Estado” autorizou e estimulou outras vozes de fon- tes testemunhais e especializadas até então desprezadas pelos veículos jornalísticos. A exposição e confrontação dessas informações provenientes de fon- tes variadas quanto à sua relação com a temática, mas majoritariamente convergentes no modo de compor uma narrativa de abusos, transgres- sões e impunidade, fez silenciar algumas das vozes que tradicionalmente tiveram preponderância na narração dos momentos mais duros da histó- ria brasileira no século XX. Os porta-vozes das Forças Armadas brasileiras praticamente recusaram a prerrogativa de se pronunciar como fonte ofi- cial na cobertura ao preferirem a estratégia da privação voluntária da palavra à disputa discursiva com outras fontes, oficiais e não oficiais, o que só não ocorreu em raríssimos momentos. Embora seu caráter circunstancial as tornem uma espécie de fonte oficial não convencional, as comissões da verdade representaram efe- tivamente a inserção de uma nova voz nas narrativas. Conclusão Por mais que se tenha valorizado o discurso oficial, abriu-se espaço, ao menos durante a vigência dos trabalhos de investigação, à disseminação de variantes na produção de conteúdo jornalístico sobre a ditadura. Referências Dissertação (Mestrado em Comunicação) – Faculdade de Arquitetura, Artes e Comuni- cação, Universidade Estadual Paulista. São Paulo, 2008. Dissertação (Mestrado em Comunicação) – Faculdade de Arquitetura, Artes e Comuni- cação, Universidade Estadual Paulista. São Paulo, 2008. NICOLETTI, J. 50 anos após o golpe militar, presas de SC relatam tortura e de- tenção. G1, 31 mar. 2014. Disponível em: http://g1.globo.com/sc/santa-catarina/ noticia/2014/03/50-anos-apos-o-golpe-militar-presas-de-sc-relatam-tortura-e-detencao. html. Acesso em: 19 jan. 2017. PINTO, M. Fontes jornalísticas: contributos para o mapeamento do campo. Comunica- ção e Sociedade 2, Cadernos do Noroeste, Série Comunicação, v. 14, n. 1-2, p. 277-294, 2000. TRAQUINA, N. Teorias do jornalismo. v. II. A tribo jornalística – uma comunidade interpretativa transnacional. Florianópolis: Insular, 2008. TUCHMAN, G. Making news: a study in the construction of reality. Nova York: The Free Press, 1978. Referências BARDIN, L. Análise de conteúdo. Lisboa: Edições 70, 2008. BARDIN, L. Análise de conteúdo. Lisboa: Edições 70, 2008. BRITTO, P. Justiça de transição priorizou indenizações, mas não a reconciliação, diz historiadora. Folha de S. Paulo, 2 jul. 2013. Disponível em: http://www1.folha.uol.com. br/poder/2013/07/1304812-justica-de-transicao-priorizou-indenizacoes-mas-nao-a-re- conciliacao-diz-historiadora.shtml. Acesso em: 8 dez. 2014. GANS, H. Deciding what news is: a study of CBS Evening News, NBC Nightly News, Newsweek and Time. Northwestern University Press, 1979. HANSEN, A.; COTTLE, S.; NEGRINE, R.; NEWBOLD, C. Mass communication BRITTO, P. Justiça de transição priorizou indenizações, mas não a reconciliação, diz historiadora. Folha de S. Paulo, 2 jul. 2013. Disponível em: http://www1.folha.uol.com. br/poder/2013/07/1304812-justica-de-transicao-priorizou-indenizacoes-mas-nao-a-re- conciliacao-diz-historiadora.shtml. Acesso em: 8 dez. 2014. GANS, H. Deciding what news is: a study of CBS Evening News, NBC Nightly News, BRITTO, P. Justiça de transição priorizou indenizações, mas não a reconciliação, diz historiadora. Folha de S. Paulo, 2 jul. 2013. Disponível em: http://www1.folha.uol.com. br/poder/2013/07/1304812-justica-de-transicao-priorizou-indenizacoes-mas-nao-a-re- conciliacao diz historiadora shtml Acesso em: 8 dez 2014 BRITTO, P. Justiça de transição priorizou indenizações, mas não a reconciliação, diz historiadora. Folha de S. Paulo, 2 jul. 2013. Disponível em: http://www1.folha.uol.com. b / d /2013/07/1304812 j ti d t i i i i d i br/poder/2013/07/1304812-justica-de-transicao-priorizou-indenizacoes-mas-nao-a-re- conciliacao-diz-historiadora.shtml. Acesso em: 8 dez. 2014. GANS, H. Deciding what news is: a study of CBS Evening News, NBC Nightly News, Newsweek and Time. Northwestern University Press, 1979. conciliacao-diz-historiadora.shtml. Acesso em: 8 dez. 2014. GANS, H. Deciding what news is: a study of CBS Evening News, NBC Nightly News, Newsweek and Time. Northwestern University Press, 1979. HANSEN, A.; COTTLE, S.; NEGRINE, R.; NEWBOLD, C. Mass communication research methods. Nova York: New York University Press, 1998. KRIPPENDORFF, K. Metodología de análisis de contenido. Barcelona: Paidós, 1990. comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019 100 quem fala sobre a ditadura nos jornais? A R T I G O LAGE, N. A reportagem: teoria e técnica de entrevista e pesquisa jornalística. Rio de Janeiro: Record, 2003. LAGE, N. A reportagem: teoria e técnica de entrevista e pesquisa jornalística. Rio de Janeiro: Record, 2003. MESQUITA, Flávio Agnelli. As fontes jornalísticas no Caso Dossiê – uma análise de enquadramento da cobertura das revistas Veja, Época, IstoÉ e Carta Capital. 2008. 144f. MESQUITA, Flávio Agnelli. As fontes jornalísticas no Caso Dossiê – uma análise de enquadramento da cobertura das revistas Veja, Época, IstoÉ e Carta Capital. 2008. 144f. Data de submissão: 08/08/2018 Data de aceite: 11/02/2019 Sobre as autoras Fernanda Nalon Sanglard – Jornalista, MBA em Marketing, mestre e doutora em Comunicação. Atualmente, é pesquisadora de pós-doutorado do Grupo de Pesquisa em Mídia e Esfera Pública (EME/UFMG). Fernanda Nalon Sanglard – Jornalista, MBA em Marketing, mestre e doutora em Comunicação. Atualmente, é pesquisadora de pós-doutorado do Grupo de Pesquisa em Mídia e Esfera Pública (EME/UFMG). Teresa Cristina da Costa Neves – Mestre em Comunicação e Cultura, doutora em Estudos Literários e professora associada do Departamento de Fundamen- tos, Teorias e Contextos da Faculdade de Comunicação da UFJF. Data de submissão: 08/08/2018 Data de aceite: 11/02/2019 comun. mídia consumo, são paulo, v. 16, n. 45, p. 80-100, jan./abr. 2019
https://openalex.org/W4200263915	https://escholarship.org/content/qt3r805645/qt3r805645.pdf?t=rwx3ww	English	null	Effectiveness of mRNA Vaccines Against COVID-19 Hospitalization by Age and Chronic Medical Conditions Burden Among Immunocompetent US Adults, March-August 2021	The journal of infectious diseases (Online. University of Chicago Press)/The Journal of infectious diseases	2,021	public-domain	7,139	Permalink https://escholarship.org/uc/item/3r805645 Title Effectiveness of mRNA Vaccines Against COVID-19 Hospitalization by Age and Chronic Medical Conditions Burden Among Immunocompetent US Adults, March-August 2021 UCLA UCLA Previously Published Works UCLA UCLA Previously Published Works UCLA Previously Published Works Title Effectiveness of mRNA Vaccines Against COVID-19 Hospitalization by Age and Chronic Medical Conditions Burden Among Immunocompetent US Adults, March-August 2021 Effectiveness of mRNA Vaccines Against COVID-19 Hospitalization by Age and Chronic Medical Conditions Burden Among Immunocompetent US Adults, March-August 2021 similar between those aged 18–64 years versus ≥65 years (P > .05). VE against severe COVID-19 was very high among adults without chronic conditions and lessened with increasing co- morbidity burden. similar between those aged 18–64 years versus ≥65 years (P > .05). VE against severe COVID-19 was very high among adults without chronic conditions and lessened with increasing co- morbidity burden. y Keywords. chronic medical conditions; COVID-19; preex- isting conditions; vaccine effectiveness. Through October 2021, the coronavirus disease 2019 (COVID- 19) pandemic caused by severe acute respiratory syndrome co- ronavirus 2 (SARS-CoV-2) resulted in more than 2.3 million hospitalizations and 690 000 deaths in the United States [1]. In December 2020, the US Food and Drug Administration granted emergency use authorization for 2 messenger RNA (mRNA) COVID-19 vaccines, BNT162b2 from Pfizer-BioNTech and mRNA-1273 from Moderna [1]. Nathaniel M. Lewis,1, Eric A. Naioti,1 Wesley H. Self,2 Adit A. Ginde,3 David J. Douin,3 H. Keipp Talbot,2 Jonathan D. Casey,2 Nicholas M. Mohr,4 Anne Zepeski,4 Manjusha Gaglani,5,6, Shekhar A. Ghamande,5 Tresa A. McNeal,5 Nathan I. Shapiro,7 Kevin W. Gibbs,8 D. Clark Files,8 David N. Hager,9 Arber Shehu,9 Matthew E. Prekker,10 Heidi L. Erickson,10 Michelle N. Gong,11 Amira Mohamed,11 Daniel J. Henning,12 Jay S. Steingrub,13 Ithan D. Peltan,14 Samuel M. Brown,14 Emily T. Martin,15 Kinsley Hubel,16 Catherine L. Hough,16 Laurence W. Busse,17 Caitlin C. ten Lohuis,17 Abhijit Duggal,18 Jennifer G. Wilson,19 Alexandra J. Gordon,19 Nida Qadir,20 Steven Y. Chang,20 Christopher Mallow,21 Carolina Rivas,21 Hilary M. Babcock,22 Jennie H. Kwon,22 Matthew C. Exline,23 Natasha Halasa,2 James D. Chappell,2 Adam S. Lauring,24 Carlos G. Grijalva,2 Todd W. Rice,2, Jillian P. Rhoads,2 William B. Stubblefield,2 Adrienne Baughman,2 Kelsey N. Womack,2 Christopher J. Lindsell,2 Kimberly W. Hart,2 Yuwei Zhu,2 Stephanie J. Schrag,1 Miwako Kobayashi,1 Jennifer R. Verani,1 Manish M. Patel,1,a and Mark W. Tenforde1,a; IVY Network Collaborators mRNA COVID-19 vaccines elicit immunity against the SARS- CoV-2 spike protein [2] and have been shown in clinical trials [3, 4] and observational studies [5–7] to be effective in preventing severe COVID-19 requiring hospitalization. Real-world evalu- ations of vaccine effectiveness (VE) against COVID-19 hos- pitalization frequently pool multiple age groups and persons with and without preexisting chronic medical conditions. VE estimates by age group and comorbidity burden can comple- ment large pooled VE analyses using heterogenous populations. Effectiveness of mRNA Vaccines Against COVID-19 Hospitalization by Age and Chronic Medical Conditions Burden Among Immunocompetent US Adults, March-August 2021 Identifying the heterogeneity of VE against severe COVID-19 can also potentially inform decisions about targeting preventive measures to populations most likely to benefit from them.i 1Centers for Disease Control and Prevention COVID-19 Response Team, Atlanta, Georgia, USA, 2Vanderbilt University Medical Center, Nashville, Tennessee, USA, 3University of Colorado School of Medicine, Aurora, Colorado, USA, 4University of Iowa, Iowa City, Iowa, USA, 5Baylor Scott and White Health, Temple, Texas, USA, 6Texas A&M University College of Medicine, Temple, Texas, USA, 7Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA, 8Wake Forest University Baptist Medical Center, Winston-Salem, North Carolina, USA, 9Johns Hopkins Hospital, Baltimore, Maryland, USA, 10Hennepin County Medical Center, Minneapolis, Minnesota, USA, 11Montefiore Healthcare Center, Albert Einstein College of Medicine, Bronx, New York, USA, 12University of Washington School of Medicine, Seattle, Washington, USA, 13Baystate Medical Center, Springfield, Massachusetts, USA, 14Intermountain Medical Center and University of Utah, Salt Lake City, Utah, USA, 15University of Michigan School of Public Health, Ann Arbor, Michigan, USA, 16Oregon Health and Science University Hospital, Portland, Oregon, USA, 17Emory University School of Medicine, Atlanta, Georgia, USA, 18Cleveland Clinic, Cleveland, Ohio, USA, 19Stanford University School of Medicine, Palo Alto, California, USA, 20David Geffen School of Medicine at University of California Los Angeles, Ronald Reagan-University of California Los Angeles Medical Center, Los Angeles, California, USA, 21University of Miami, Miami, Florida, USA, 22Washington University, St Louis, Missouri, USA, 23Ohio State University Wexner Medical Center, Columbus, Ohio, USA, and 24University of Michigan School of Medicine, Ann Arbor, Michigan, USA Prior work has identified heterogeneity of VE by immuno- compromised state, age, variants, and time since vaccination [5–7], leading to decisions in the United States to provide a third vaccine dose to immunocompromised persons and a booster dose to adults generally [8]. Despite some evidence that mRNA VE could differ by factors such as burden of chronic medical conditions, race/ethnicity, sex, and obesity (body mass index [BMI] ≥ 30 kg/m2) [9], these factors have not often been studied in detail. Therefore, we evaluated the effectiveness of mRNA vaccines against COVID-19 hospitalizations stratified by burden of chronic conditions, age, and other demographics. Vaccine effectiveness (VE) against COVID-19 hospitalization was evaluated among immunocompetent adults (≥18 years) during March–August 2021 using a case-control design. Received 9 November 2021; editorial decision 16 December 2021; accepted 16 December 2021; published online 21 December 2021. aM. M. P. and M. W. T. contributed equally as senior authors. Correspondence: Nathaniel M. Lewis, PhD, Influenza Prevention and Control Team, Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Rd NE, Mailstop 24/7, Atlanta, GA 30329 (pha6@cdc.gov). The Journal of Infectious Diseases® 2022;225:1694–700 Published by Oxford University Press for the Infectious Diseases Society of America 2021. This work is written by (a) US Government employee(s) and is in the public domain in the US. https://doi.org/10.1093/infdis/jiab619 B R I E F R E P O R T Effectiveness of mRNA Vaccines Against COVID-19 Hospitalization by Age and Chronic Medical Conditions Burden Among Immunocompetent US Adults, March-August 2021 Effectiveness of mRNA Vaccines Against COVID-19 Hospitalization by Age and Chronic Medical Conditions Burden Among Immunocompetent US Adults, March-August 2021 Among 1669 hospitalized COVID-19 cases (11% fully vaccinated) and 1950 RT-PCR–negative controls (54% fully vaccinated), VE was 96% (95% confidence interval [CI], 93%–98%) among patients with no chronic medical conditions and 83% (95% CI, 76%– 88%) among patients with ≥ 3 categories of conditions. VE was Powered by the California Digital Library University of California eScholarship.org Received 9 November 2021; editorial decision 16 December 2021; accepted 16 December 2021; published online 21 December 2021. METHODS The Centers for Disease Control and Prevention (CDC) collab- orates with the Influenza and Other Viruses in the Acutely Ill (IVY) Network to monitor effectiveness of COVID-19 vaccines against COVID-19 hospitalization. As previously described [5, 6], we prospectively enrolled adults ≥ 18 years of age at 21 IVY Network hospitals during 11 March to 15 August 2021. We have reported VE during this surveillance period [5, 6], but did not assess VE by burden of chronic conditions or other 1694 • JID 2022:225 (15 May) • BRIEF REPORT intervals), age (continuous), sex, self-reported race and ethnicity, burden of underlying conditions (0, 1, 2, or ≥ 3 categories), and US Health and Human Services region of the admitting hospital. VE was calculated as: VE = (1 − adjusted odds ratio) × 100. characteristics presented here. Using a case-control design, we assessed mRNA VE against COVID-19 hospitalization by com- paring odds of prior vaccination in hospitalized case patients with COVID-19 versus control patients without COVID-19. We restricted analysis to immunocompetent persons because our objective was to inform VE within the general population. Interaction terms were introduced into the main VE model to evaluate VE by primary characteristics of interest: age group (18–64 years or ≥ 65 years) and number of chronic medical con- dition categories (0, 1, 2, or ≥ 3). Additional exploratory models were constructed with interaction terms between vaccination status and race/ethnicity (non-Hispanic white, non-Hispanic black, Hispanic, all other races non-Hispanic, unknown), sex (female or male), and obesity (obese or not obese). To estimate VE by condition category within each age category, an addi- tional regression model was fit to include the 3-way interaction among vaccination status, age group, and conditions category along with each 2-way interaction. Likelihood ratio χ2 tests were used to estimate whether differences in VE across groups were significant at a threshold of P < .05. Case or control status was determined by clinical testing re- sults as well as centralized reverse transcription polymerase chain reaction (RT-PCR) testing of upper respiratory sam- ples at Vanderbilt University Medical Center (Nashville, TN). Cases were hospitalized with a COVID-19-like illness (≥1 of fever, cough, shortness of breath, loss of taste or smell, use of respiratory support for the acute illness, pulmonary findings on chest imaging consistent with pneumonia) and had a pos- itive RT-PCR or antigen test for SARS-CoV-2 within 10 days following symptom onset. METHODS Two hospitalized control groups that tested negative for SARS-CoV-2 by RT-PCR were enrolled: (1) test-negative controls with COVID-19-like illness and (2) syndrome-negative controls without COVID-19-like illness. The second control group was included due to imperfect diag- nostic accuracy of SARS-CoV-2 tests, which could lead to mis- classification of cases. Vaccination coverage and VE estimates were highly similar for individual control groups [7], and there- fore these groups were combined to improve statistical power. Sites attempted a 1:1 enrollment ratio of cases to controls in each group, with controls admitted within 2 weeks of cases, al- though individual matching was not performed. i Older adults and those with certain chronic medical condi- tions were targeted for early priority vaccination after mRNA vaccines became available and may be affected more by waning effectiveness than other groups that were vaccinated later, re- sulting in potentially lower VE. Therefore, we performed a sen- sitivity analysis that restricted vaccinated patients to those with a reference date within 120 days of their second vaccine dose, a period during which significant waning against severe COVID- 19 is not expected [6]. An additional sensitivity analysis was included to explore differences in VE by conditions burden re- stricted to cases with hypoxemia (O2 saturation < 92% or supple- mental oxygen support) within 24 hours of admission to account for the possibility of patients with multimorbidity being more likely to be hospitalized for mild illness (ie, admission bias). Vaccination status was determined primarily by review of elec- tronic medical records (EMRs), state vaccine registries, provider or pharmacy records, and available CDC vaccination cards, with patient or proxy self-report of vaccination (including known date and location) during enrollment interviews considered as vaccin- ated in the absence of source documentation. Vaccination status was classified based on vaccine receipt before a reference date, defined as the date of symptom onset for cases and test-negative controls and days prior to admission for syndrome-negative con- trols. Participants were either unvaccinated or were fully vaccin- ated if 2 doses of a mRNA vaccine were received ≥ 14 days before the reference date; patients were excluded if they received ≥ 1 vac- cine doses but were not fully vaccinated, received a non-mRNA vaccine, or received multiple COVID-19 vaccine products. This activity was conducted as a public health surveillance activity, with waiver of informed consent, consistent with ap- plicable federal law and CDC policy, eg, 45 C.F.R. METHODS part 46.102(l) (2), 21 C.F.R. part 56; 42 U.S.C. §241(d); 5 U.S.C. §552a; 44 U.S.C. §3501 et seq. RESULTS of categories of underlying medical conditionsh 0 546 (32.7) 332 (17.0) 727 (30.6) 151 (12.2) 1 489 (29.3) 498 (25.5) 686 (28.8) 301 (24.3) 2 365 (21.9) 540 (27.7) 532 (22.4) 373 (30.1) ≥3 269 (16.1) 580 (29.7) 434 (18.2) 415/1240 (33.5) ecific underlying medical conditions Cardiovascular disease 848 (50.8) 1307 (67.0) 1224 (51.5) 931 (75.1) Hypertension 774 (46.4) 1141 (58.5) 1098 (46.2) 817 (65.9) Endocrine disease (including diabetes) 500 (30.0) 757 (38.8) 715 (30.1) 542 (43.7) Diabetes 435 (26.1) 638 (32.7) 620 (26.1) 453 (36.5) Pulmonary disease 332 (19.9) 565 (29.0) 524 (22.0) 373 (30.1) COPD 117 (7.0) 293 (15.0) 210 (8.8) 200 (16.1) Renal disease 154 (9.2) 342 (17.5) 236 (9.9) 260 (21.0) Hematologic disease 114 (6.8) 244 (12.5) 209 (8.8) 149 (12.0) Neurological disease 127 (7.6) 217 (11.1) 193 (8.1) 151 (12.2) Table 1. RESULTS Characteristics of Participants by Cases (Hospitalized With COVID-19) vs Control (Hospitalized Without COVID-19) Status and by COVID-19 Vaccination Status—21 Hospitalsa in 18 US States, March– August 2021 Characteristicb Case Patients (n = 1669) Control Patients (n = 1950) Unvaccinated Participants (n = 2379) Vaccinatedc Participants (n = 1240) COVID-19 case 1669 (100.0) 0 (0.0) 1481 (62.3) 188 (15.2) Fully vaccinated 188 (11.3) 1052 (53.9) 0 (0.0) 1240 (100.0) Hypoxemiad 1239/1662 (74.5) 617/1049 (58.8) 1398/1980 (70.6) 458/731 (62.7) Admitted to ICU 970/1645 (59.0) 823/1930 (42.6) 1246/2345 (53.1) 547/1230 (44.5) Median age, y (IQR) 54 (41–66) 62 (47–72) 53 (40–64) 67 (57–76) Age group, ≥65 y 462 (27.7) 845 (43.3) 580 (24.4) 727 (58.6) Sex, female 815 (48.8) 931 (47.7) 1133 (47.6) 613 (49.4) Race/ethnicitye White, non-Hispanic 733 (43.9) 1188 (60.9) 1135 (47.7) 786 (63.4) Black, non-Hispanic 427 (25.6) 425 (21.8) 624 (26.2) 228 (18.4) Any race, Hispanic 391 (23.4) 242 (12.4) 468 (19.7) 165 (13.3) All other races, non-Hispanic 82 (4.9) 76 (3.9) 108 (4.5) 50 (4.0) Unknown 36 (2.2) 19 (1.0) 44 (1.8) 11 (0.9) US census regionf Northeast 245 (14.7) 291 (14.9) 343 (14.4) 193 (15.6) South 737 (44.2) 761 (39.0) 1035 (43.5) 463 (37.3) Midwest 305 (18.3) 505 (25.9) 489 (20.6) 321 (25.9) West 382 (22.9) 393 (20.2) 512 (21.5) 263 (21.2) Residence in long-term care facilityg 34/1602 (2.1) 118/1861 (6.3) 54/2276 (2.4) 98/1187 (8.3) Has health insurance 1473/1668 (88.3) 1807/1950 (92.7) 2074/2379 (87.2) 1206/1239 (97.3) Employed 611/1300 (47.0) 480/1651 (29.1) 794/1885 (42.1) 297/1066 (27.9) Health care worker 83/1300 (6.4) 92/1651 (5.6) 108/1885 (5.7) 67/1066 (6.3) Attended some college or more 557/1147 (48.6) 758/1494 (50.7) 740/1673 (44.2) 575/968 (59.4) ≥1 hospital admission in past year 371/1506 (24.6) 955/1831 (52.2) 764/2172 (35.2) 562/1165 (48.2) Self-reported prior laboratory-confirmed SARS-CoV-2 infection 51 (3.1) 163 (8.4) 136 (5.7) 78 (6.3) No. RESULTS After excluding participants who did not meet inclusion cri- teria, 3619 immunocompetent adults (1669 case patients, 11% fully vaccinated; 1950 control patients, 54% fully vaccinated) were included. Fully vaccinated patients (compared with un- vaccinated) were older (median age, 67 vs 53 years), and more likely to report being non-Hispanic white (63% vs 48%), have been hospitalized ≥ 1 time during the past year (48% vs 35%), and have ≥ 1 chronic medical condition (88% vs 69%), in- cluding cardiovascular disease (75% vs 52%), endocrine disease including diabetes (44% vs 30%), pulmonary disease (30% vs 22%), and renal disease (21% vs 10%), but were less commonly obese (45% vs 53%) (Table 1). A similar proportion of cases Trained personnel obtained information on participants’ preexisting chronic medical conditions associated with severe COVID-19 [10] through EMR review [7] using a standardized case report form (Supplementary Table 1). Individual condi- tions were grouped into 7 categories: cardiovascular, neurologic, pulmonary, gastrointestinal, endocrine, renal, and hematologic. Participants were classified by the number of categories in which conditions were documented (0, 1, 2 or ≥ 3). Logistic regression models, with COVID-19 case status as the outcome and vaccination status as the predictor, were used to estimate overall VE adjusted for date of admission (biweekly BRIEF REPORT • JID 2022:225 (15 May) • 1695 ble 1. RESULTS Characteristics of Participants by Cases (Hospitalized With COVID-19) vs Control (Hospitalized Without COVID-19) Status and by COVID-19 Vaccination Status—21 Hospitalsa in 18 US States, March– ugust 2021 haracteristicb Case Patients (n = 1669) Control Patients (n = 1950) Unvaccinated Participants (n = 2379) Vaccinatedc Participants (n = 1240) OVID-19 case 1669 (100.0) 0 (0.0) 1481 (62.3) 188 (15.2) lly vaccinated 188 (11.3) 1052 (53.9) 0 (0.0) 1240 (100.0) ypoxemiad 1239/1662 (74.5) 617/1049 (58.8) 1398/1980 (70.6) 458/731 (62.7) dmitted to ICU 970/1645 (59.0) 823/1930 (42.6) 1246/2345 (53.1) 547/1230 (44.5) edian age, y (IQR) 54 (41–66) 62 (47–72) 53 (40–64) 67 (57–76) ge group, ≥65 y 462 (27.7) 845 (43.3) 580 (24.4) 727 (58.6) x, female 815 (48.8) 931 (47.7) 1133 (47.6) 613 (49.4) ce/ethnicitye White, non-Hispanic 733 (43.9) 1188 (60.9) 1135 (47.7) 786 (63.4) Black, non-Hispanic 427 (25.6) 425 (21.8) 624 (26.2) 228 (18.4) Any race, Hispanic 391 (23.4) 242 (12.4) 468 (19.7) 165 (13.3) All other races, non-Hispanic 82 (4.9) 76 (3.9) 108 (4.5) 50 (4.0) Unknown 36 (2.2) 19 (1.0) 44 (1.8) 11 (0.9) S census regionf Northeast 245 (14.7) 291 (14.9) 343 (14.4) 193 (15.6) South 737 (44.2) 761 (39.0) 1035 (43.5) 463 (37.3) Midwest 305 (18.3) 505 (25.9) 489 (20.6) 321 (25.9) West 382 (22.9) 393 (20.2) 512 (21.5) 263 (21.2) esidence in long-term care facilityg 34/1602 (2.1) 118/1861 (6.3) 54/2276 (2.4) 98/1187 (8.3) as health insurance 1473/1668 (88.3) 1807/1950 (92.7) 2074/2379 (87.2) 1206/1239 (97.3) mployed 611/1300 (47.0) 480/1651 (29.1) 794/1885 (42.1) 297/1066 (27.9) ealth care worker 83/1300 (6.4) 92/1651 (5.6) 108/1885 (5.7) 67/1066 (6.3) tended some college or more 557/1147 (48.6) 758/1494 (50.7) 740/1673 (44.2) 575/968 (59.4) hospital admission in past year 371/1506 (24.6) 955/1831 (52.2) 764/2172 (35.2) 562/1165 (48.2) lf-reported prior laboratory-confirmed SARS-CoV-2 infection 51 (3.1) 163 (8.4) 136 (5.7) 78 (6.3) o. 1696 • JID 2022:225 (15 May) • BRIEF REPORT RESULTS gLong-term care facility included reporting living in a nursing home, assisted living home, or rehabilitation hospital, or other subacute or chronic facility before the hospital admission. hChronic medical conditions were obtained through medical chart review by trained personnel and classified by condition category specified in the table; a full list of conditions is included in Supplementary Table 1. VE differed by number of condition categories in a gradient, from persons with 0 categories of conditions (VE, 96%; 95% CI, 93%–98%) to persons with ≥ 3 (VE, 83%; 95% CI, 76%–88%; P < .001) (Figure 1); results were similar when restricted to hypoxemic cases (Supplementary Figure 1). VE was not statis- tically different for patients aged ≥ 65 years compared with pa- tients aged 18–64 years (VE, 88% [95% CI, 84%–91%] vs 91% [95% CI, 88%–93%]; P = .142). The relationship between VE and number of chronic medical condition categories did not vary by age group (P = .903). Exploratory analyses showed that VE differed by sex (male, 88% [95% CI, 84%–91%] vs female, 92% [95% CI, 89%–94%]; P = .037) but not across race/ethnicity groups (P = .415) or by obesity status (obese, 91% [95% CI, 89%–94%] vs not obese, 88% [95% CI, 85%–91%]; P = .105; Supplementary Table 2). Results were similar in a sensitivity analysis limiting the analyt- ical population to patients with illness onset within 120 days of full vaccination. RESULTS aHospitals by region were Northeast: Baystate Medical Center (Springfield, MA), Beth Israel Deaconess Medical Center (Boston, MA), Montefiore Medical Center (Bronx, NY); South: Vanderbilt University Medical Center (Nashville, TN), University of Miami Medical Center (Miami, FL), Emory University Medical Center (Atlanta, GA), Johns Hopkins Hospital (Baltimore, MD), Wake Forest University Baptist Medical Center (Winston-Salem, NC), Baylor Scott and White Health (Temple, TX); Midwest: University of Iowa Hospitals and Clinics (Iowa City, IA), University of Michigan Hospital (Ann Arbor, MI), Hennepin County Medical Center (Minneapolis, MN), Barnes-Jewish Hospital (St Louis, MO), Cleveland Clinic (Cleveland, OH), Ohio State University Wexner Medical Center (Columbus, OH); West: Stanford University Medical Center (Stanford, CA), UCLA Medical Center (Los Angeles, CA), UCHealth University of Colorado Hospital (Aurora, CO), Oregon Health and Science University Hospital (Portland, OR), Intermountain Medical Center (Murray, UT), University of Washington (Seattle, WA). bData are not complete for all characteristics in the table; denominators are included in the table for characteristics in which data is missing. cFully vaccinated with mRNA COVID-19 vaccines defined as ≥ 14 days from dose 2. dHypoxemia defined as needing O2 support or having blood O2 levels below 92% within first 24 hours of admission. Information to assess hypoxemia was collected for case patients and test-negative controls but not for patients in the syndrome-negative control group. eRacial and ethnic groups were reported by the patient or proxy. fNortheast: Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, and Vermont; Midwest: Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, Ohio, South Dakota, and Wisconsin; South: Alabama, Arkansas, Delaware, District of Columbia, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, Oklahoma, South Carolina, Tennessee, Texas, Virginia, and West Virginia; West: Alaska, Arizona, California, Colorado, Hawaii, Idaho, Montana, Nevada, New Mexico, Oregon, Utah, Washington, and Wyoming. gLong-term care facility included reporting living in a nursing home, assisted living home, or rehabilitation hospital, or other subacute or chronic facility before the hospital admission. hChronic medical conditions were obtained through medical chart review by trained personnel and classified by condition category specified in the table; a full list of conditions is included in Supplementary Table 1. met our definition of hypoxemia across underlying condition categories (77%, 73%, 77%, and 71% for 0, 1, 2, and ≥ 3 condi- tion categories, respectively). RESULTS Characteristicb Case Patients (n = 1669) Control Patients (n = 1950) Unvaccinated Participants (n = 2379) Vaccinatedc Participants (n = 1240) Gastrointestinal disease 52 (3.1) 139 (7.1) 114 (4.8) 77 (6.2) BMI-based obesity (≥30 kg/m2) 970/1645 (59.0) 823/1930 (42.6) 1246/2345 (53.1) 547/1230 (44.5) Data are No./total No. (%) except where indicated. Abbreviations: BMI, body-mass index; COPD, chronic obstructive pulmonary disease; COVID-19, coronavirus disease 2019; ICU, intensive care unit; IQR, interquartile range; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. aHospitals by region were Northeast: Baystate Medical Center (Springfield, MA), Beth Israel Deaconess Medical Center (Boston, MA), Montefiore Medical Center (Bronx, NY); South: Vanderbilt University Medical Center (Nashville, TN), University of Miami Medical Center (Miami, FL), Emory University Medical Center (Atlanta, GA), Johns Hopkins Hospital (Baltimore, MD), Wake Forest University Baptist Medical Center (Winston-Salem, NC), Baylor Scott and White Health (Temple, TX); Midwest: University of Iowa Hospitals and Clinics (Iowa City, IA), University of Michigan Hospital (Ann Arbor, MI), Hennepin County Medical Center (Minneapolis, MN), Barnes-Jewish Hospital (St Louis, MO), Cleveland Clinic (Cleveland, OH), Ohio State University Wexner Medical Center (Columbus, OH); West: Stanford University Medical Center (Stanford, CA), UCLA Medical Center (Los Angeles, CA), UCHealth University of Colorado Hospital (Aurora, CO), Oregon Health and Science University Hospital (Portland, OR), Intermountain Medical Center (Murray, UT), University of Washington (Seattle, WA). bData are not complete for all characteristics in the table; denominators are included in the table for characteristics in which data is missing. cFully vaccinated with mRNA COVID-19 vaccines defined as ≥ 14 days from dose 2. dHypoxemia defined as needing O2 support or having blood O2 levels below 92% within first 24 hours of admission. Information to assess hypoxemia was collected for case patients and test-negative controls but not for patients in the syndrome-negative control group. eRacial and ethnic groups were reported by the patient or proxy. fNortheast: Connecticut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, and Vermont; Midwest: Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, Ohio, South Dakota, and Wisconsin; South: Alabama, Arkansas, Delaware, District of Columbia, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, Oklahoma, South Carolina, Tennessee, Texas, Virginia, and West Virginia; West: Alaska, Arizona, California, Colorado, Hawaii, Idaho, Montana, Nevada, New Mexico, Oregon, Utah, Washington, and Wyoming. RESULTS of categories of underlying medical conditionsh 0 546 (32.7) 332 (17.0) 727 (30.6) 151 (12.2) 1 489 (29.3) 498 (25.5) 686 (28.8) 301 (24.3) 2 365 (21.9) 540 (27.7) 532 (22.4) 373 (30.1) ≥3 269 (16.1) 580 (29.7) 434 (18.2) 415/1240 (33.5) Specific underlying medical conditions Cardiovascular disease 848 (50.8) 1307 (67.0) 1224 (51.5) 931 (75.1) Hypertension 774 (46.4) 1141 (58.5) 1098 (46.2) 817 (65.9) Endocrine disease (including diabetes) 500 (30.0) 757 (38.8) 715 (30.1) 542 (43.7) Diabetes 435 (26.1) 638 (32.7) 620 (26.1) 453 (36.5) Pulmonary disease 332 (19.9) 565 (29.0) 524 (22.0) 373 (30.1) COPD 117 (7.0) 293 (15.0) 210 (8.8) 200 (16.1) Renal disease 154 (9.2) 342 (17.5) 236 (9.9) 260 (21.0) Hematologic disease 114 (6.8) 244 (12.5) 209 (8.8) 149 (12.0) Neurological disease 127 (7.6) 217 (11.1) 193 (8.1) 151 (12.2) s needing O2 support or having blood O2 levels below 92% within first 24 hours of admission. Information to assess hypoxemia was collected for case patients and test-negative controls but not for patients in the syndrome-negative ups were reported by the patient or proxy. ut, Maine, Massachusetts, New Hampshire, New Jersey, New York, Pennsylvania, Rhode Island, and Vermont; Midwest: Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, Ohio, South Dakota, and bama, Arkansas, Delaware, District of Columbia, Florida, Georgia, Kentucky, Louisiana, Maryland, Mississippi, North Carolina, Oklahoma, South Carolina, Tennessee, Texas, Virginia, and West Virginia; West: Alaska, Arizona, California, ho, Montana, Nevada, New Mexico, Oregon, Utah, Washington, and Wyoming. ty included reporting living in a nursing home, assisted living home, or rehabilitation hospital, or other subacute or chronic facility before the hospital admission. ditions were obtained through medical chart review by trained personnel and classified by condition category specified in the table; a full list of conditions is included in Supplementary Table 1. Characteristicb Case Patients (n = 1669) Control Patients (n = 1950) Unvaccinated Participants (n = 2379) Vaccinatedc Participants (n = 1240) Gastrointestinal disease 52 (3.1) 139 (7.1) 114 (4.8) 77 (6.2) BMI-based obesity (≥30 kg/m2) 970/1645 (59.0) 823/1930 (42.6) 1246/2345 (53.1) 547/1230 (44.5) Data are No./total No. (%) except where indicated. Abbreviations: BMI, body-mass index; COPD, chronic obstructive pulmonary disease; COVID-19, coronavirus disease 2019; ICU, intensive care unit; IQR, interquartile range; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. DISCUSSION Models were additionally adjusted for date of hospital admission (biweekly intervals), US Department of Health and Human Services region of hospital, age (continuous), sex, and race/ethnicity (non-Hispanic white, non-Hispanic black, Hispanic of any race, non-Hispanic other, or unknown), and number of condition categories. VE point estimates are rounded to the nearest whole number. Error bars represent 95% confidence intervals. 100 96 93 87 83 97 93 86 84 95 91 86 84 Underlying conditions 0 1 2 3+ 90 80 70 Overall 18–64 years Age group Adjusted vaccine efectiveness (%) 65+ years Figure 1. Vaccine effectiveness by age group and number of chronic medical conditions. Adjusted vaccine effectiveness (VE) was estimated using logistic regression com- paring odds of being fully vaccinated with an mRNA COVID-19 vaccine versus being unvaccinated, in case patients and control patients, using the equation VE = 100 × (1 − odds ratio). Overall VE by number of condition categories documented (0, 1, 2, or ≥ 3) was calculated by including an interaction term between vaccination status and number of condition categories. An additional model including a 3-way interaction between age, conditions, and vaccination status was included to calculate VE by number of condition categories within age groups. Models were additionally adjusted for date of hospital admission (biweekly intervals), US Department of Health and Human Services region of hospital, age (continuous), sex, and race/ethnicity (non-Hispanic white, non-Hispanic black, Hispanic of any race, non-Hispanic other, or unknown), and number of condition categories. VE point estimates are rounded to the nearest whole number. Error bars represent 95% confidence intervals. hospitals. While the number of categories of chronic conditions was considered, the severity of conditions within each category was not; for example, hypertension and severe heart failure were both quantified as 1 cardiovascular condition. We had in- complete capture of data for a few variables, such as height and weight used to determine BMI-based obesity (captured for 99% of patients). However, missing information was minimal and unlikely to bias VE estimates. Furthermore, persons with more chronic medical conditions may be more likely to be hospital- ized even with milder COVID-19 illness, resulting in lower VE; however, VE was similar restricting analysis to cases with hy- poxemia who would have an indication for admission. Multiple comparisons were made in this analysis, with the possibility of type I or type II error. DISCUSSION This analysis suggests COVID-19 mRNA vaccines are highly effective for preventing COVID-19 hospitalizations for het- erogeneous immunocompetent adults, with some decline in VE with increasing burden of chronic medical conditions. VE was very high (96%), including among adults ≥ 65 years old (95%), among people without conditions, which make up half the US population [11]. In addition, adults aged 18–64 years have accounted for the majority of cumulative US COVID-19 hospitalizations, with hospitalization rates among the unvac- cinated > 10 times those of the fully vaccinated [1]; they also tend to have lower vaccination coverage [12] and fewer con- ditions and compared with adults aged ≥ 65 years. As efforts continue to increase vaccination coverage in the United States, these data suggest that vaccinating younger persons without chronic medical conditions will substantially reduce COVID- 19 hospitalizations. Our findings also suggest that VE against COVID-19–asso- ciated hospitalization decreases roughly proportionally to the number of chronic medical conditions, both overall and strati- fied by age. The conditions included in our analysis align closely with those identified as risk factors for severe COVID-19 in the prevaccination era [10], suggesting that the same chronic conditions placing a person at high risk for severe COVID- 19 are also associated with COVID-19 hospitalization among vaccinated persons. Prior studies have suggested that anti- body response and immune protection after vaccination could be attenuated for persons with severe chronic medical condi- tions, including both immunocompromising conditions [13] BRIEF REPORT • JID 2022:225 (15 May) • 1697 100 96 93 87 83 97 93 86 84 95 91 86 84 Underlying conditions 0 1 2 3+ 90 80 70 Overall 18–64 years Age group Adjusted vaccine efectiveness (%) 65+ years Figure 1. Vaccine effectiveness by age group and number of chronic medical conditions. Adjusted vaccine effectiveness (VE) was estimated using logistic regression com- paring odds of being fully vaccinated with an mRNA COVID-19 vaccine versus being unvaccinated, in case patients and control patients, using the equation VE = 100 × (1 − odds ratio). Overall VE by number of condition categories documented (0, 1, 2, or ≥ 3) was calculated by including an interaction term between vaccination status and number of condition categories. An additional model including a 3-way interaction between age, conditions, and vaccination status was included to calculate VE by number of condition categories within age groups. Notes Valesano, and Julie Gilbert, University of Michigan; Akram Khan, Olivia Krol, Zachary Zouyed, Emma Silverman, Genesis Briceno, and Emmanuel Mills, Oregon Health and Science University; Christine D. Crider, Kyle A. Steinbock, Thomas C. Paulson, and Layla A. Anderson, University of Washington; Christy IVY Network Collaborators. Tresa McNeal, Shekhar Ghamande, Nicole Calhoun, Kempapura Murthy, Judy Herrick, Amanda McKillop, Eric Hoffman, Martha Zayed, Michael Smith, Natalie Settele, Jason Ettlinger, Elisa Priest, Jennifer Thomas, Alejandro Arroliga, and Madhava Beeram, Baylor Scott and White Health; Ryan Kindle, Lori-Ann Kozikowski, Lesley De Souza, Scott Ouellette, and Sherell Thornton-Thompson, Baystate Medical Center; Patrick Tyler, Beth Israel Deaconess Medical Center; Omar Mehkri, Meg Mitchell, Connery Brennan, Kiran Ashok, and Bryan Poynter, Cleveland Clinic; Nicholas Stanley, Emory University; Audrey Hendrickson, Sean Caspers, and Tyler Scharber, Hennepin County Medical Center; Jeffrey Jorgensen, Robert Bowers, Jennifer King, Valerie Aston, and Brent Armbruster, Intermountain Medical Center; Richard E. Rothman, Johns Hopkins University; Rahul Nair and Jen- Ting (Tina) Chen, Montefiore Medical Center; Sarah Karow, Emily Robart, Paulo Nunes Maldonado, Maryiam Khan, and Preston So, Ohio State University; Joe Levitt, Cynthia Perez, Anita Visweswaran, and Jonasel Roque, Stanford University; Trevor Frankel, Omai Garner, and Sukantha Chandrasekaran, University of California, Los Angeles; Jennifer Goff, David Huynh, Adit Ginder, David Douin, Kelly Jensen, Conner Driver, Michael Carricato, and Ian Chambers, UCHealth University of Colorado Hospital; Paul Nassar, Lori Stout, Zita Sibenaller, Alicia Walter, Jasmine Mares, Logan Olson, and Bradley Clinansmith, University of Iowa; Carolina Rivas and Hayley Gershengorn, University of Miami; E. J. McSpadden, Rachel Truscon, Anne Kaniclides, Lara Thomas, Ramsay Bielak, Weronika Damek Valvano, Rebecca Fong, William J. Fitzsimmons, Christopher Blair, Andrew L. Valesano, and Julie Gilbert, University of Michigan; Akram Khan, Olivia Krol, Zachary Zouyed, Emma Silverman, Genesis Briceno, and Emmanuel Mills, Oregon Health and Science University; Christine D. Crider, Kyle A. Steinbock, Thomas C. Paulson, and Layla A. Anderson, University of Washington; Christy Potential conflict of interest. W. H. S. reports grants and consultant fees from Merck, and consultant fees from Aerpio Pharmaceuticals, all outside this work. A. A. G. reports grant support from NIH, Department of Defense (DoD), and investigator-initiated grant support from AbbVie and Faron Pharmaceuticals, all outside this work. J. D. C. reports a grant from the National Institutes of Health (NIH). D. C. F. reports consultant fees from Cytovale and membership on a Medpace Data Safety Monitoring Board (DSMB). D. N. H. DISCUSSION Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Supplementary Data S. L. reports consultant fees from Sanofi and fees from Roche for membership on a trial steering committee. C. G. G. reports consultant fees from Pfizer, Merck, and Sanofi-Pasteur; and grants from Campbell Alliance/Syneos Health, NIH, the Food and Drug Administration, AHRQ, and Sanofi. T. W. R. reports personal fees from Cumberland Pharmaceuticals, Inc as the Director of Medical Affairs; consultant fees from Cytovale, Inc; and DSMB membership fees from Sanofi. C. J. L. reports grants from NIH, DoD, and the Marcus Foundation; organizational contract fees from bioMerieux, Endpoint LLC, and Entegrion, Inc; and a patent issued to Cincinnati Children’s Hospital Medical Center for risk stratification in sepsis and septic shock. All other authors report no potential conflicts. Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or com- ments should be addressed to the corresponding author. Notes Disclaimer. The findings and conclusions in this report are those of the authors and do not necessarily represent the offi- cial position of the Centers for Disease Control and Prevention. Financial support. This work was supported by the US Centers for Disease Control and Prevention (grant number 75D30121F00002) to W.H.S.; and the National Center for Advancing Translational Sciences (grant number UL1 TR002243 Clinical and Translational Science Award for the REDCap data tool). J. H. K. is supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (grant number 1K23AI137321). All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. IVY Network Collaborators. Tresa McNeal, Shekhar Ghamande, Nicole Calhoun, Kempapura Murthy, Judy Herrick, Amanda McKillop, Eric Hoffman, Martha Zayed, Michael Smith, Natalie Settele, Jason Ettlinger, Elisa Priest, Jennifer Thomas, Alejandro Arroliga, and Madhava Beeram, Baylor Scott and White Health; Ryan Kindle, Lori-Ann Kozikowski, Lesley De Souza, Scott Ouellette, and Sherell Thornton-Thompson, Baystate Medical Center; Patrick Tyler, Beth Israel Deaconess Medical Center; Omar Mehkri, Meg Mitchell, Connery Brennan, Kiran Ashok, and Bryan Poynter, Cleveland Clinic; Nicholas Stanley, Emory University; Audrey Hendrickson, Sean Caspers, and Tyler Scharber, Hennepin County Medical Center; Jeffrey Jorgensen, Robert Bowers, Jennifer King, Valerie Aston, and Brent Armbruster, Intermountain Medical Center; Richard E. Rothman, Johns Hopkins University; Rahul Nair and Jen- Ting (Tina) Chen, Montefiore Medical Center; Sarah Karow, Emily Robart, Paulo Nunes Maldonado, Maryiam Khan, and Preston So, Ohio State University; Joe Levitt, Cynthia Perez, Anita Visweswaran, and Jonasel Roque, Stanford University; Trevor Frankel, Omai Garner, and Sukantha Chandrasekaran, University of California, Los Angeles; Jennifer Goff, David Huynh, Adit Ginder, David Douin, Kelly Jensen, Conner Driver, Michael Carricato, and Ian Chambers, UCHealth University of Colorado Hospital; Paul Nassar, Lori Stout, Zita Sibenaller, Alicia Walter, Jasmine Mares, Logan Olson, and Bradley Clinansmith, University of Iowa; Carolina Rivas and Hayley Gershengorn, University of Miami; E. J. McSpadden, Rachel Truscon, Anne Kaniclides, Lara Thomas, Ramsay Bielak, Weronika Damek Valvano, Rebecca Fong, William J. Fitzsimmons, Christopher Blair, Andrew L. DISCUSSION and nonimmunocompromising conditions [14]; however, few show clear associations with lower VE. Our analysis shows that mRNA VE against severe disease is also lessened by chronic medical conditions among immunocompetent adults, inde- pendent of age. Booster vaccines for recipients of COVID-19 vaccines are now recommended for persons aged ≥ 18 years [15]. Our findings suggests that persons with a higher burden of chronic medical conditions may experience greater incremental benefit from additional vaccine doses. Finally, our findings may have implications for interpretation of data from observational COVID-19 VE studies. Our finding of 96% VE against COVID-19 hospitalizations for healthy adults (ie, immunocompetent patients with no preexisting con- ditions) was similar to VE against severe disease observed in mRNA COVID-19 vaccine clinical trials [3, 4]. This similarity suggests that efficacy estimates from phase 3 COVID-19 clin- ical trials successfully approximated real-world VE for people without chronic medical conditions but overestimated effec- tiveness for people with chronic medical comorbidities. In conclusion, this analysis provides insight into the heteroge- neity of VE by overall health status, beyond immunocompetency alone. Vaccination reduced the risk of COVID-19 hospitaliza- tion by > 95% in healthy adults without chronic medical condi- tions regardless of age, sex, obesity, or race/ethnicity. Continued efforts to vaccinate US adults, aided by the evaluation and re- porting of VE for specific subpopulations, will likely have a marked impact on severe COVID-19. Our findings are subject to some limitations. Enrolling sites were academic medical centers and may have inpatient popula- tions with a higher burden of chronic diseases than community 1698 • JID 2022:225 (15 May) • BRIEF REPORT S. L. reports consultant fees from Sanofi and fees from Roche for membership on a trial steering committee. C. G. G. reports consultant fees from Pfizer, Merck, and Sanofi-Pasteur; and grants from Campbell Alliance/Syneos Health, NIH, the Food and Drug Administration, AHRQ, and Sanofi. T. W. R. reports personal fees from Cumberland Pharmaceuticals, Inc as the Director of Medical Affairs; consultant fees from Cytovale, Inc; and DSMB membership fees from Sanofi. C. J. L. reports grants from NIH, DoD, and the Marcus Foundation; organizational contract fees from bioMerieux, Endpoint LLC, and Entegrion, Inc; and a patent issued to Cincinnati Children’s Hospital Medical Center for risk stratification in sepsis and septic shock. All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Notes reports salary support from Incyte Corporation, EMPACT Precision Medicine, and the Marcus Foundation. M. N. G. reports grant support from NIH and the Agency for Healthcare Research and Quality (AHRQ); and fees for participating on a DSMB for Regeneron and for participating on a scientific advisory panel for Philips Healthcare. D. J. H. reports consulting fees from Cytovale and Opticyte. I. D. P. reports grants from NIH and Janssen Pharmaceuticals; and institutional fees from Asahi Kasei Pharma and Regeneron. S. M. B. reports fees from Hamilton for chairing a DSMB, and institutional fees from Faron, Sedana, and Janssen; grants from Sedana, Janssen, NIH, and the DoD; book royalties from Oxford University and Brigham Young University; and personal fees from New York University for service on a DSMB. E. T. M. reports personal fees from Pfizer and a grant from Merck, all for unrelated work. A. K. reports grants from United Therapeutics, Johnson & Johnson, 4D Medical, Lung LLC, and Reata Pharmaceuticals. A. S. M. reports consulting fees from Sanofi-Pasteur and Seqirus. S. Y. C. was a speaker for La Jolla Pharmaceuticals and a Consultant for PureTech Health. J. H. K. reports grant support from NIH. M. C. E. reports talks on nutrition in COVID pneumonia at APEN conference sponsored by Abbott Labs. N. H. reports grants from Sanofi and Quidel. J. D. C. reports a grant from the National Center for Advancing Translational Sciences, NIH. A. BRIEF REPORT • JID 2022:225 (15 May) • 1699 of print 6 August 2021]. Clin Infect Dis doi: 10.1093/cid/ ciab687. Kampe, Jakea Johnson, Rendie McHenry, Marcia Blair, and Laura Short, Vanderbilt University Medical Center; Mary LaRose, Leigha Landreth, Madeline Hicks, and Lisa Parks, Wake Forest University; and Jahnavi Bongu, David McDonald, Candice Cass, Sondra Seiler, David Park, Tiffany Hink, Meghan Wallace, Carey-Ann Burnham, and Olivia G. Arter, Washington University. 8. Centers for Disease Control and Prevention. Vaccine re- commendations and guidelines of the ACIP. https://www. cdc.gov/vaccines/hcp/acip-recs/vacc-specific/covid-19. html. Accessed 12 October 2021. 9. Pellini R, Venuti A, Pimpinelli F, et al. Initial observations on age, gender, BMI, and hypertension in antibody responses to SARS-CoV-2 BNT162b2 vaccine. EClinicalMedicine 2021; 36:100928. 1700 • JID 2022:225 (15 May) • BRIEF REPORT References 1. Centers for Disease Control and Prevention. COVID data tracker. https://covid.cdc.gov/covid-data-tracker/#datatracker- home. Accessed 28 September 2021. 1. Centers for Disease Control and Prevention. COVID data tracker. https://covid.cdc.gov/covid-data-tracker/#datatracker- home. Accessed 28 September 2021. 10. Centers for Disease Control and Prevention. COVID-19. People with certain medical conditions. https://www.cdc. gov/coronavirus/2019-ncov/need-extra-precautions/people- with-medical-conditions.html. Accessed 6 October 2021. 2. Sadarangani M, Marchant A, Kollmann TR. Immunological mechanisms of vaccine-induced protection against COVID- 19 in humans. Nat Rev Immunol 2021; 21:475–84. 11. Diesel J, Sterrett N, Dasgupta S, et al. COVID-19 vaccination coverage among adults—United States, December 14, 2020– May 22, 2021. MMWR Morb Mortal Wkly Rep 2021; 70:922–7. 3. Baden LR, El Sahly HM, Essink B, et al. Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl J Med 2021; 384:403–16. 12. Boersma P, Black LI, Ward BW. Prevalence of multiple chronic conditions among US adults, 2018. Prev Chronic Dis 2020; 17:200130. 4. El Sahly H, Baden LR, Essink B, et al. Efficacy of the mRNA- 1273 SARS-CoV-2 vaccine at completion of blinded phase. N Engl J Med 2021; 385:1774–85. 4. El Sahly H, Baden LR, Essink B, et al. Efficacy of the mRNA- 1273 SARS-CoV-2 vaccine at completion of blinded phase. N Engl J Med 2021; 385:1774–85. 13. Chodick G, Tene L, Rotem RS, et al. The effectiveness of the two-dose BNT162b2 vaccine: analysis of real-world data [published online ahead of print 17 May 2021]. Clin Infect Dis doi: 10.1093/cid/ciab438. 5. Self WH, Tenforde MW, Rhoads JP, et al. Comparative ef- fectiveness of Moderna, Pfizer-BioNTech, and Janssen (Johnson & Johnson) vaccines in preventing COVID-19 hos- pitalizations among adults without immunocompromising conditions—United States, March–August 2021. MMWR Morb Mortal Wkly Rep 2021; 70:1337–43. 14. Ruddy JA, Connolly CM, Boyarsky BJ, et al. High antibody response to two-dose SARS-CoV-2 messenger RNA vacci- nation in patients with rheumatic and musculoskeletal dis- eases. Ann Rheum Dis 2021; 80:1351–2. 6. Tenforde MW, Self WH, Adams K, et al. Association be- tween mRNA vaccination and COVID-19 hospitalization and disease severity. JAMA 2021; 326:2043–54. 15. Centers for Disease Control and Prevention. Interim clin- ical considerations for use of COVID-19 vaccines cur- rently authorized in the United States. https://www.cdc. gov/vaccines/covid-19/clinical-considerations/covid-19- vaccines-us.html#footnote-01. Accessed 4 October 2021. 7. Tenforde MW, Patel MM, Ginde AA, et al. Effectiveness of SARS-CoV-2 mRNA vaccines for preventing Covid-19 hos- pitalizations in the United States [published online ahead 1700 • JID 2022:225 (15 May) • BRIEF REPORT
https://openalex.org/W4390668530	https://www.e3s-conferences.org/articles/e3sconf/pdf/2024/03/e3sconf_i-core2024_03007.pdf	English	null	Change Of Pvdf Ultrafiltration Membranes For Humus Acid Removal Applications In Water By Adding Fe2o3/Zeolite Additives	E3S web of conferences	2,024	cc-by	3,280	E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 https://doi.org/10.1051/e3sconf/202447303007 Change Of Pvdf Ultrafiltration Membranes For Humus Acid Removal Applications In Water By Adding Fe2o3/Zeolite Additives Zuhriah Mumtazah 1,, Reva Edra Nugraha 2, Arif Priyangga3, Maktum Muharja1, Rizki Fitria Darmayanti4, Ditta Kharisma Yolanda Putri1 1Department of Chemical Engineering, Faculty of Engineering, Universitas Jember, Jl. Kalimantan 37 Jember 68121, East Java, Indonesia 2Department of Chemical Engineering, Faculty of Engineering, Universitas Pembangunan Nasional “Veteran” Jawa Timur, Surabaya 60294, East Java, Indonesia 3Department of Chemistry, Institut Teknologi Sepuluh Nopember, ITS Sukolilo, Surabaya 60111, Jember 68121, East Java, Indonesia 2Department of Chemical Engineering, Faculty of Engineering, Universitas Pembangunan Nasional “Veteran” Jawa Timur, Surabaya 60294, East Java, Indonesia 3D t t f Ch i t I tit t T k l i S l h N b ITS S k lil S b 60111 3Department of Chemistry, Institut Teknologi Sepuluh Nopember, ITS Sukolilo, Surabaya 60111, Indonesia 4Department of Agro-industrial Technology, Faculty of Agriculture, Universitas Muhammadiyah Jember, Jalan Karimata 49, Jember, 68121, Indonesia Abstract. One of the most often used polymers as the primary component of membranes is polyvinylidene fluoride or PVDF. Nonetheless, its hydrophobic characteristic remains a significant barrier to this material's utilization. This study aims to reduce the likelihood of fouling by adding Fe2O3/Zeolite additions to the PVDF membrane. Fe2O3/Zeolite was used to modify the membrane through surface coating. Compared to the pure PVDF membrane, the results demonstrated that adding additives to the membrane polymer solution increased the purified water and humic acid fluxes. The best results in this study were obtained by modifying the PVDF membrane and adding Fe2O3/Zeolite additions in a ratio of 1 gr: 0.5 gr (M2). Based on these findings, it can be said that. 1 1 Introduction Due to its strong chemical resistance, thermal stability, and capacity for membrane production, PVDF is a frequently employed material [1]. PVDF membranes have extensive application in ultrafiltration and microfiltration procedures[2]. However, PVDF membranes are more prone to clogging and have fewer uses since they are a semi-crystalline polymer with –CH2-CF2– repeating units that produce a hydrophobic structure[3]. Fluids containing hydrophobic species are the source of blockages because they reduce membrane permeability and lead to the formation of activated sludge, which can shorten membrane life and raise operating expenses[4]. Both reversible and irreversible blockages are possible[5]. Foulants that adhere firmly to the membrane pores induce irreversible fouling, whereas foulants that stick to the membrane surface cause reversible fouling[6]. Antifouling membranes must be Corresponding author: zuhriahmumtazah@unej.ac.id © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (https://creativecommons.org/licenses/by/4.0/). E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 https://doi.org/10.1051/e3sconf/202447303007 developed and modified for more effective MBR applications by adding compounds to improve their hydrophilic qualities[2]. developed and modified for more effective MBR applications by adding compounds to improve their hydrophilic qualities[2]. By engineering the membrane surface to be more hydrophilic, the membrane modification technique seeks to improve the membrane's hydrophilicity, antibacterial qualities, and performance while producing more effective wastewater treatment outcomes[7]. Grafting, covalent coupling, irradiation, plasma treatment, layer adsorption, and coating are a few alteration procedures[8]. The coating technology is the most adaptable, has a less complicated process and is reasonably priced[7]. The dip-coating method involves applying a liquid phase coating solution to the substrate's surface, allowing the solution to cover the surface before it dries [9]. The most excellent permeate flow readings and a hydrophilic surface on PVDF were achieved using the dip-coating technique. The dip-coating method doesn't require particular conditions (high pressure and temperature), is simple to use, and is highly efficient for industrial applications [8]. Iron oxide nanoparticles can be added to PES and CA membranes to lessen their poor flux [10]. Iron oxide is biocompatible, has low toxicity, and functions as an adsorbent for ionic pollutants while also improving the mechanical stability of membranes [11]. Compared to a pure PVDF membrane, the mixed matrix membrane has more holes and is more apparent with the addition of Fe2O3 [12]. Higher flux and FRR are produced when Fe2O3 is added to PVC instead of when it is not [13]. Zeolite is an inorganic crystal with high adsorption qualities that contains silica, oxygen, and aluminum. It also enhances the surface area available to produce biofilms [14]. Zeolites are extensively employed in industry to eliminate heavy metals, lessen surplus ammonium, adsorb gas, separate linear from non-linear hydrocarbons, and soften water [15]. One alternative for creating membranes with superoleophobicity and influential heavy metal ion adsorption is natural zeolite, a porous aluminosilicate mineral with high hydrophilicity and ion exchange capabilities [16]. Compared to when 4A zeolite is not added, the PSf matrix with 4A zeolite added yields flux, F7RR, RIrr, and RRev [17]. The present work aimed to investigate the impact of surface modification on PVDF membranes by adding Fe2O3/Zeolite via dip-coating. * Corresponding author: zuhriahmumtazah@unej.ac.id Functional group, hydrophilicity, morphological, and hydrophilicity tests are used to characterize membranes and tested humus acid selectivity and pure water flow of humus acid to assess membrane performance. 2. 2. Distilled water, 70% alcohol, Fe2O3, hollow fiber membrane (PVDF), and zeolite are the materials employed in this study. We weighed 0.15 grams of PVA and got 100 milliliters of purified water ready. PVA is a substance that dissolves in water, is environmentally friendly, and is frequently used in producing membranes [18]. Next, at a temperature of 120 C and a speed of 200–300 rpm, the ingredients are combined and stirred with a hot plate stirrer until they are homogenous [19]. Added 0.3 grams of Fe2O3 and Zeolite were added to the solution and mixed using a hot plate stirrer for 30 minutes. The solution was sonicated for 30 minutes using an ultrasonicator to obtain a homogeneous Fe2O3/Zeolite suspension [3]. After soaking in the dope solution for five hours, the membrane was allowed to dry at ambient temperature. The steps for membrane preparation are shown in Figure 1. Table 1 displays the composition of the membrane. 2 2 https://doi.org/10.1051/e3sconf/202447303007 E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 Fig. 1. Procedures for preparing membranes. Fig. 1. Procedures for preparing membranes. Table 1. Fe2O3/Zeolite Composition. Membrane Type Membrane Name Ratio Fe2O3 Zeolit PVDF P0 0 0 PVDF/ Fe2O3-Zeolit P1 1.0 0.5 PVDF/ Fe2O3-Zeolit P2 0.5 1.0 PVDF/ Fe2O3-Zeolit P3 1.0 1.0 Table 1. Fe2O3/Zeolite Composition. The functional groups of the membrane were tested using an FTIR Spectrophotometer both before and after modification. Attenuated Total Reflection (ATR-FTIR) Thermo Scientific iD5 ATR-Nicolet iS5 Japan is the apparatus's specs. The membrane is dried for a few hours before being placed in the sample holder. Infrared spectra were recorded between 400 and 4000 cm-1 in the wavenumber range. Test for Contact Angle, The Drop Master 300 from Kyowa Interface Science Co. in Japan, was used to assess the degree of hydrophilicity of the membrane. Data is recorded at least five times for each membrane sample, and the average value is utilized. Fig. 2. Tool Kit for Ultrafiltration. Water and humic acid flux in a performance test experiment to examine the impact of change on membrane performance. The filtration test was employed using a set of ultrafiltration cells at a pressure of 1.5 bar. The experiment used two kinds of bait: humic acid (50 ppm) and pure water (aquadest). With a dead-end ultrafiltration module powered by gas pressure, the amount of feed that flows through the membrane may be detected. 2. In Figure 2, the ultrafiltration apparatus is displayed. A volume of pure water (for measuring the flow of pure Fig. 2. Tool Kit for Ultrafiltration. Fig. 2. Tool Kit for Ultrafiltration. Water and humic acid flux in a performance test experiment to examine the impact of change on membrane performance. The filtration test was employed using a set of ultrafiltration cells at a pressure of 1.5 bar. The experiment used two kinds of bait: humic acid (50 ppm) and pure water (aquadest). With a dead-end ultrafiltration module powered by gas pressure, the amount of feed that flows through the membrane may be detected. In Figure 2, the ultrafiltration apparatus is displayed. A volume of pure water (for measuring the flow of pure Water and humic acid flux in a performance test experiment to examine the impact of change on membrane performance. The filtration test was employed using a set of ultrafiltration cells at a pressure of 1.5 bar. The experiment used two kinds of bait: humic acid (50 ppm) and pure water (aquadest). With a dead-end ultrafiltration module powered by gas pressure, the amount of feed that flows through the membrane may be detected. In Figure 2, the ultrafiltration apparatus is displayed. A volume of pure water (for measuring the flow of pure Water and humic acid flux in a performance test experiment to examine the impact of change on membrane performance. The filtration test was employed using a set of ultrafiltration cells at a pressure of 1.5 bar. The experiment used two kinds of bait: humic acid (50 ppm) and pure water (aquadest). With a dead-end ultrafiltration module powered by gas pressure, the amount of feed that flows through the membrane may be detected. In Figure 2, the ultrafiltration apparatus is displayed. A volume of pure water (for measuring the flow of pure 3 E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 https://doi.org/10.1051/e3sconf/202447303007 water) or humic acid solution (for evaluating the rejection and flux of humic acid) is fed into the ultrafiltration module, which is equipped with a membrane that varies in pressure relief to perform the measurements. Retentate is the solution that remains on the membrane surface, and permeate is the fluid that gets through the membrane. The permeability coefficient (Lp) for pure water, selectivity tests for humic acid samples, and flux (J) for pure water are all determined by the membrane's permeability. 3.1 Membrane Chemical Structure The results of an FTIR examination show changes in the chemical composition of the PVDF membrane following treatment with Fe2O3/Zeolite. The PVDF membrane combined with Fe2O3/Zeolite and the pure PVDF membrane are very different, as Figure 3 illustrates. The presence of asymmetric C=O, C-C, and C-H groups, which signify the presence of Fe2O3/Zeolite bound to the membrane surface, characterizes this distinction. Because of their strong affinity for water, these two groups make Fe2O3/Zeolite very hydrophilic. It is clear from the FTIR data in Figure 3 that the Fe2O3/Zeolite change in the membrane system was effective. Fig. 3. PVDF Membrane Infrared Spectra with and Without Modification. Fig. 3. PVDF Membrane Infrared Spectra with and Without Modification. 2. water) or humic acid solution (for evaluating the rejection and flux of humic acid) is fed into the ultrafiltration module, which is equipped with a membrane that varies in pressure relief to perform the measurements. Retentate is the solution that remains on the membrane surface, and permeate is the fluid that gets through the membrane. The permeability coefficient (Lp) for pure water, selectivity tests for humic acid samples, and flux (J) for pure water are all determined by the membrane's permeability. 3. Results and discussion 3. 3.2 Hydrophilicity of Membranes Using a contact angle meter to measure the angle of contact between the membrane surface and the water droplets, the hydrophilicity of the membrane was examined. The hydrophilicity of a membrane is positively correlated with its contact angle with water. Figure 4 illustrates how the degree of hydrophilicity of the membrane printing fluid changes when additives are added. Figure 4 shows how adding Fe2O3/Zeolite results in a more hydrophilic PVDF membrane with a reduced contact angle [20]. 4 4 4 https://doi.org/10.1051/e3sconf/202447303007 E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 Fig. 4. PVDF membrane contact angle before and after modification. Fig. 4. PVDF membrane contact angle before and after modification. 4. Conclusion Fe2O3/Zeolite additions improve the hydraulic characteristics and overall performance of PVDF-based membranes. Pure water and humic acid flux increase when Fe2O3/Zeolite is added to the membrane because it increases pore size and hydrophilicity. Based on the overall results, Fe2O3/Zeolite is a suitable additive that may be utilized to improve the properties of PVDF-based membranes, particularly regarding hydrophilicity, which directly affects the membrane's filtration performance. 3.3 Pure Water Flux and Humic Acid Flux The pure water flow values from pure PVDF and PVDF modified with Fe2O3/Zeolite are displayed in Figure 5. The purified water flux a virgin PVDF membrane (P0) generates is just 9.96 L/m2.h. In the meantime, the pure water flow values generated by the modified membranes (P1, P2, and P3) reached 15.77, 38.16, and 21.98 L/m2.h. Following alteration, the membrane's hydrophilic characteristics and pore size both increased, increasing the membrane's pure water flux value [21]. Fig. 5. Pure Water Flux of PVDF membrane before and after modification. Fig. 5. Pure Water Flux of PVDF membrane before and after modification. Figure 6 illustrates this same tendency in the flow performance of filtration employing humic acid solution as input. With Fe2O3/Zeolite-modified membranes, the amount of humic acid the membrane can hold increases. The size of the membrane pore increases with an increase in the additive concentration. The P2 membrane yielded the most flux. Figure 7 displays the humus acid selectivity test findings. This graphic also shows how adding the Fe2O3/Zeolite additive influences the humus acid rejection. The selectivity value is generally inversely related to all membranes measured humic acid flow value. This is because of the membrane's pore size. Water may flow through the membrane more quickly due to the larger pore size, increasing the permeate. Conversely, selectivity will diminish due to more humus acid particles entering the permeate due to the presence of pores, especially those with larger diameters. Because the PVDF membrane's surface is covered in relatively dense, small-sized, and few-numbered holes, it 5 E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 https://doi.org/10.1051/e3sconf/202447303007 possesses the highest selectivity of any membrane, measuring 89.15%. Pure PVDF's surface properties allow water ions and humus acid particles to flow through, resulting in a meager flux value [22]. Fig. 6. Humic Acid Flux of PVDF membrane before and after modification. Fig. 6. Humic Acid Flux of PVDF membrane before and after modification. Acknowledgement The author would like to thank the Institute for Research and Community Service at Jember University. University. References 1. D. Rahmadi, S. Mulyati, C. Meurah Rosnelly, A. Ambarita, and dan Yanna Syamsuddin, “Syeh Abdul Rauf No.7 Darussalam, Banda Aceh Indonesia 23111 Jurusan Teknik Kimia, Fakultas Teknik,” 2021. 1. D. Rahmadi, S. Mulyati, C. Meurah Rosnelly, A. Ambarita, and dan Yanna Syamsuddin, “Syeh Abdul Rauf No.7 Darussalam, Banda Aceh Indonesia 23111 Jurusan Teknik Kimia, Fakultas Teknik,” 2021. 2. R. S. Silitonga et al., “The modification of PVDF membrane via crosslinking with chitosan and glutaraldehyde as the crosslinking agent,” Indonesian Journal of Chemistry, vol. 18, no. 1, pp. 1– 6, 2018, doi: 10.22146/ijc.25127. 2. R. S. Silitonga et al., “The modification of PVDF membrane via crosslinking with chitosan and glutaraldehyde as the crosslinking agent,” Indonesian Journal of Chemistry, vol. 18, no. 1, pp. 1– 6, 2018, doi: 10.22146/ijc.25127. 3. K. Samree et al., “Enhancing the antibacterial properties of PVDF membrane by hydrophilic surface modification using titanium dioxide and silver nanoparticles,” Membranes (Basel), vol. 10, no. 10, pp. 1–18, Oct. 2020, doi: 10.3390/membranes10100289. 3. K. Samree et al., “Enhancing the antibacterial properties of PVDF membrane by hydrophilic surface modification using titanium dioxide and silver nanoparticles,” Membranes (Basel), vol. 10, no. 10, pp. 1–18, Oct. 2020, doi: 10.3390/membranes10100289. 4. M. Fathizadeh et al., “Antifouling UV-treated GO/PES hollow fiber membranes in a membrane bioreactor (MBR),” Environ Sci (Camb), vol. 5, no. 7, pp. 1244–1252, Jul. 2019, doi: 10.1039/c9ew00217k. 4. M. Fathizadeh et al., “Antifouling UV-treated GO/PES hollow fiber membranes in a membrane bioreactor (MBR),” Environ Sci (Camb), vol. 5, no. 7, pp. 1244–1252, Jul. 2019, doi: 10.1039/c9ew00217k. 6 E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 https://doi.org/10.1051/e3sconf/202447303007 5. K. Poojamnong, K. Tungsudjawong, W. Khongnakorn, and P. Jutaporn, “Characterization of reversible and irreversible foulants in membrane bioreactor (MBR) for eucalyptus pulp and paper mill wastewater treatment using fluorescence regional integration,” J Environ Chem Eng, vol. 8, no. 5, Oct. 2020, doi: 10.1016/j.jece.2020.104231. 6. P. Sittisom, O. Gotore, R. Ramaraj, G. Tran Van, Y. Unpaprom, and T. Itayama1, “Energy and Environmental Communication Research Review Membrane fouling issues in anaerobic membrane bioreactors (AnMBRs) for biogas production Article Infor,” 2019. 7. S. Lotfikatouli et al., “Enhanced anti-fouling performance in Membrane Bioreactors using a novel cellulose nanofiber-coated membrane,” Sep Purif Technol, vol. 275, Nov. 2021, doi: 10.1016/j.seppur.2021.119145. 8. H. B. Madalosso, R. Machado, D. Hotza, and C. Marangoni, “Membrane Surface Modification by Electrospinning, Coating, and Plasma for Membrane Distillation Applications: A State-of-the-Art Review,” Advanced Engineering Materials, vol. References 23, no. 6. John Wiley and Sons Inc, Jun. 01, 2021. doi: 10.1002/adem.202001456. 9. X. Tang and X. Yan, “Dip-coating for fibrous materials: mechanism, methods and applications,” Journal of Sol-Gel Science and Technology, vol. 81, no. 2. Springer New York LLC, pp. 378–404, Feb. 01, 2017. doi: 10.1007/s10971-016-4197-7. 10. C. Evangeline et al., “Iron oxide modified polyethersulfone/cellulose acetate blend membrane for enhanced defluoridation application,” Desalination Water Treat, vol. 156, pp. 177–188, Jul. 2019, doi: 10.5004/dwt.2018.23174. 11. N. Said et al., “Enhanced hydrophilic polysulfone hollow fiber membranes with addition of iron oxide nanoparticles,” Polym Int, vol. 66, no. 11, pp. 1424–1429, Nov. 2017, doi: 10.1002/pi.5401. 12. H. Ismail et al., “PVDF/Fe2O3 mixed matrix membrane for oily wastewater treatment,” 2019. 13. E. Demirel, B. Zhang, M. Papakyriakou, S. Xia, and Y. Chen, “Fe2O3 nanocomposite PVC membrane with enhanced properties and separation performance,” J Memb Sci, vol. 529, pp. 170– 184, 2017, doi: 10.1016/j.memsci.2017.01.051. 14. X. Zhuang et al., “Novel TiO2/GO-Al2O3 Hollow Fiber Nanofiltration Membrane for Desalination and Lignin Recovery,” Membranes (Basel), vol. 12, no. 10, Oct. 2022, doi: 10.3390/membranes12100950. 15. C. Algieri and E. Drioli, “Zeolite membranes: Synthesis and applications,” Separation and Purification Technology, vol. 278. Elsevier B.V., Jan. 01, 2022. doi:10.1016/j.seppur.2021.119295. 16. C. Wang, H. Guo, J. Yu, K. Feng, and J. Huang, “Micro/nanostructural silica/alkali- treated natural zeolite coated fabrics for oil-water separation and heavy metal ions removal,” Microporous and Mesoporous Materials, vol. 327, Nov. 2021, doi: 10.1016/j.micromeso.2021.111430. 17. T. Anjum, R. Tamime, and A. L. Khan, “Mixed-matrix membranes comprising of polysulfone and porous uio-66, zeolite 4a, and their combination: Preparation, removal of humic acid, and antifouling properties,” Membranes (Basel), vol. 10, no. 12, pp. 1– 21, Dec. 2020, doi: 10.3390/membranes10120393. 18. D. Harpaz, T. Axelrod, A. L. Yitian, E. Eltzov, R. S. Marks, and A. I. Y. Tok, “Dissolvable polyvinyl-alcohol film, a time-barrier to modulate sample flow in a 3D- printed holder for capillary flow paper diagnostics,” Materials, vol. 12, no. 3, Jan. 2019, doi: 10.3390/ma12030343. 7 7 E3S Web of Conferences 473, 03007 (2024) I-CORE 2023 https://doi.org/10.1051/e3sconf/202447303007 19. I. G. Wenten, K. Khoiruddin, A. K. Wardani, P. T. P. Aryanti, D. I. Astuti, and A. A. I. A. S. Komaladewi, “Preparation of antifouling polypropylene/ZnO composite hollow fiber membrane by dip-coating method for peat water treatment,” Journal of Water Process Engineering, vol. 34, Apr. 2020, doi: 10.1016/j.jwpe.2020.101158. 20. S. Muchtar, M. Y. Wahab, S. Mulyati, N. Arahman, and M. References Riza, “Superior fouling resistant PVDF membrane with enhanced filtration performance fabricated by combined blending and the self- polymerization approach of dopamine,” Journal of Water Process Engineering, vol. 28, pp. 293– 299, Apr. 2019, doi: 10.1016/j.jwpe.2019.02.012. 21. F. Liu, N. A. Hashim, Y. Liu, M. R. M. Abed, and K. Li, “Progress in the production and modification of PVDF membranes,” Journal of Membrane Science, vol. 375, no. 1–2. pp. 1–27, Jun. 15, 2011. doi: 10.1016/j.memsci.2011.03.014. 22. B. P. Tripathi, P. Das, F. Simon, and M. Stamm, “Ultralow fouling membranes by surface modification with functional polydopamine,” Eur Polym J, vol. 99, pp. 80–89, Feb. 2018, doi: 10.1016/j.eurpolymj.2017.12.006. 8 8
https://openalex.org/W3046910519	https://bib-pubdb1.desy.de/record/463168/files/Flenner-2020-Pushing%20the%20temporal%20resolution%20i.pdf	English	null	Pushing the temporal resolution in absorption and Zernike phase contrast nanotomography: enabling fast <i>in situ</i> experiments	Journal of synchrotron radiation	2,020	cc-by	6,571	research papers research papers Pushing the temporal resolution in absorption and Zernike phase contrast nanotomography: enabling fast in situ experiments ISSN 1600-5775 Silja Flenner,a* Malte Storm,b Adam Kubec,c Elena Longo,a Florian Do¨ring,c Danie¨l M. Pelt,d Christian David,c Martin Mu¨llera and Imke Grevinga* Received 14 January 2020 Accepted 3 June 2020 aInstitute of Materials Research, Helmholtz-Zentrum Geesthacht, Max-Planck-Strasse 1, 21502 Geesthacht, Germany, bDiamond Light Source Ltd, Didcot, Oxfordshire OX11 0DE, United Kingdom, cPaul Scherrer Institut, Forschnungsstrasse 111, 5232 Villingen, Switzerland, and dCentrum Wiskunde and Informatica, Science Park 123, 1098 XG Amsterdam, The Netherlands. *Correspondence e-mail: silja.flenner@hzg.de, imke.greving@hzg.de Edited by A. Momose, Tohoku University, Japan Hard X-ray nanotomography enables 3D investigations of a wide range of samples with high resolution (<100 nm) with both synchrotron-based and laboratory-based setups. However, the advantage of synchrotron-based setups is the high ﬂux, enabling time resolution, which cannot be achieved at laboratory sources. Here, the nanotomography setup at the imaging beamline P05 at PETRA III is presented, which offers high time resolution not only in absorption but for the ﬁrst time also in Zernike phase contrast. Two test samples are used to evaluate the image quality in both contrast modalities based on the quantitative analysis of contrast-to-noise ratio (CNR) and spatial resolution. High-quality scans can be recorded in 15 min and fast scans down to 3 min are also possible without signiﬁcant loss of image quality. At scan times well below 3 min, the CNR values decrease signiﬁcantly and classical image-ﬁltering techniques reach their limitation. A machine-learning approach shows promising results, enabling acquisition of a full tomography in only 6 s. Overall, the transmission X-ray microscopy instrument offers high temporal resolution in absorption and Zernike phase contrast, enabling in situ experiments at the beamline. Keywords: nanotomography; full-field X-ray microscopy; in situ experiments; image quality; time resolution; Zernike phase contrast. Supporting information: this article has supporting information at journals.iucr.org/s Supporting information: this article has supporting information at journals.iucr.org/s research papers al., 2006)], whereas absorption contrast imaging can be used in combination with highly efﬁcient capillary condensers. (Ge et al., 2018) Other full-ﬁeld techniques like holotomography have the potential to be very fast (Villanova et al., 2017) but often require at least three distances to reconstruct specimen of arbitrary composition, which limits possible in situ appli- cation. Helmholtz-Zentrum Geesthacht at the PETRA III storage ring at DESY, where a full-ﬁeld X-ray microscope has been installed (Ogurreck et al., 2013; Greving et al., 2017, 2018; Flenner et al., 2018). A schematic of the setup is displayed in Fig. 1. The beamshaping condenser (Jeﬁmovs et al., 2008; Vartiainen et al., 2015) has a diameter of 1.8 mm with 50 nm ﬁnest structure size. Structures are made with HSQ (hydrogen silsesquioxane) resist on an Si3N4 membrane with an Ir ALD (atomic layer deposition) coating (Vila-Comamala et al., 2011), providing an illumination at the sample plane of 50 mm 50 mm. A Fresnel zone plate (FZP) made from gold on an Si3N4 membrane of 250 nm thickness with outermost zone width dr = 50 nm and a diameter of 100 mm (i.e. number of zones N = 500) is used as an objective lens (Gorelick et al., 2011), resulting in a focal distance of 44 mm at 11 keV. The Zernike phase rings are fabricated from gold on an Si3N4 membrane of 250 nm thickness and the structure height of 1.1 mm was chosen to give a phase shift of /2 at an energy of 11 keV. Phase rings with line widths between 0.5 mm and 1.3 mm were used. All optics were designed and manufactured at the Paul Scherrer Institut. To reduce coherence effects from the source, a rotating paper (standard printing paper) is used as a decoherer. Improvements in X-ray optics [e.g. higher aspect ratio of zone plates, blazed zone plates (Mohacsi et al., 2014)] and detectors and higher ﬂux at new generation sources will reduce acquisition times for full-ﬁeld nanotomography even further. There is, however, always a trade-off between scan time and contrast-to-noise ratio (CNR). The image quality strongly depends on the count rate at the detector and therefore on the exposure time (Waske et al., 2010). research papers Shorter exposure times reduce the image quality but allow for a higher sample throughput, reduction of sample movement caused by environmental factors and by long-term drifts, and a dose reduction, the latter being especially important for biological samples. Here, we present a hard X-ray nanotomography setup based at a third-generation source, which offers high temporal resolution, not only for absorption but also for phase contrast methods. Tomographic scan times down to 6 s were achieved and the advantages and disadvantages of different scanning times are compared. The standard X-ray absorption micro- scopy as well as Zernike phase contrast are evaluated in terms of contrast and spatial resolution. The high-Z material nano- porous gold (NPG) was chosen as a test case for the absorp- tion contrast tomography. This material has been proven to be well suited as a test object for evaluating TXM performance before (Larsson et al., 2019). For the Zernike phase contrast we chose a low-Z phase object with nano-sized grains, namely a magnesium alloy (Ghasemi et al., 2018; Penther et al., 2018). An X-ray sCMOS camera (Hamamatsu C12849-101U, 6.5 mm pixel size, 2048 2048 pixels, 16 bit image depth) with a 10 mm Gadox scintillator was used as a detector. The scin- tillation layer is directly coupled to the sCMOS chip and results in a high photon efﬁciency. The detector was placed 20.45 m behind the sample. Because of this large sample-to- detector distance possible at this instrument, no light optical magniﬁcation is necessary, enabling a high photon efﬁciency. With this setup, a pixel size of down to 13 nm and a spatial resolution of 50 nm have been achieved at 11 keV in 2D. In addition to standard absorption microscopy, Zernike phase contrast can be performed by adding phase rings in the back focal plane of the FZP. During the tomographic scan, the sample was rotated continuously at a constant speed (ﬂy scan mode) with a high-precision air-bearing rotation axis (PI miCos custom design; the motion errors are given in Table S1 2. Materials and methods 1. Introduction Nanotomography is a widely used tool for 3D evaluation in materials science, for biological as well as medical sample systems. Using synchrotron radiation, full-ﬁeld transmission X-ray microscopy (TXM) tomograms with high spatial reso- lution (<100 nm) are routinely recorded in time frames of 15 min to 1 h (Andrews et al., 2010; Yuan et al., 2018). Benchtop devices are evolving rapidly in terms of resolution, scanning time and image quality (Patterson et al., 2016). TXMs have been implemented in benchtop machines and even phase contrast modalities are available, e.g. Zernike phase contrast (Zernike, 1934; Schmahl et al., 1994). However, one major drawback of benchtop machines will not be resolved easily: the ﬂux density at the sample is limited and therefore the time resolution cannot compete with synchrotron-based systems. Nanotomography setups at synchrotrons can offer fast scan- ning times and/or high image quality as well as phase-contrast modes thanks to the highly brilliant source. Recently, the ﬁrst fast TXM experiments in absorption mode with scanning times of 1 min were reported at NSLS II using a highly efﬁ- cient capillary condenser (Ge et al., 2018). However, phase contrast methods such as Zernike phase contrast are currently much slower, since usually less efﬁcient optics are used [beamshaping condenser, Koehler-like illumination (Vogt et https://doi.org/10.1107/S1600577520007407 1 of 8 J. Synchrotron Rad. (2020). 27 J. Synchrotron Rad. (2020). 27 research papers research papers of the supporting information). This reduces the total scanning time and eliminates sample movements induced by the repe- titive acceleration and deceleration. Table 1 Parameters of the different scan modes. The scans highlighted in bold have only been measured in absorption. The exposure time has to be adapted so that blurring during the exposure is prevented. On the other hand, the efﬁciency decreases with more images because of the dead-time of the detector. A compromise between these two parameters has to be found. For faster scan times, smearing of more than one pixel was allowed to keep the efﬁciency reasonably high. However, a continuous rotation has a drawback: the sample will move during each image. As the rotation is an angular motion, the absolute linear motion in the projection is largest for the outermost parts of the sample. If this motion is limited to a value of b pixels (with b 1 for sub-pixel blurring), the exposure time texp per projection and the total scan time tscan (which is determined by the rotation speed and therefore deﬁnes how fast the sample moves) are linked by the following formula, Total scan time Total exposure time (s) Exposure time (s) Number of images Binning Efﬁciency 53 min 2472 1 2472 1 0.77 15 min 811.3 0.55 1475 2 0.90 6 min 288.9 0.22 1313 2 0.80 3 min 149.8 0.22 681 4 0.83 1.5 min 65.0 0.11 591 4 0.72 1 min 42.4 0.05 848 4 0.70 36 s 16.9 0.04 423 4 0.47 18 s 8.1 0.033 245 4 0.44 6 s 2.7 0.033 81 4 0.44 texp ¼ 2b NoPixel tscan: texp ¼ 2b NoPixel tscan: The number of images acquired in one scan does not only depend on the exposure time and rotation speed but also on the overhead required by the camera. The Hamamatsu camera can bin the image directly on the chip, therefore the overhead time (camera readout time) with the current infrastructure can be reduced from 0.1 s (no binning) to 0.05 s (binning 2) and 0.04 s (binning 4). The binning and the number of images inﬂuence the ratio between the total exposure time t and the total scan time tscan (efﬁciency E = t/tscan). 2.2. Reconstruction and analysis The resolution of the 2D/3D volume was estimated via Fourier ring/shell correlation (FRC, FSC). In the 3D case, the projections were divided into two stacks and the two recon- structed volumes were then used to estimate the FSC. Here, the original binning level for each scan was used (see Table 1). We used the half-bit threshold criterion to calculate the resolution from the FRC/FSC curves (van Heel & Schatz, 2005). All reconstructions were performed with the Gridrec algo- rithm (Dowd et al., 1999) and a Shepp–Logan ﬁlter using the TomoPy package (Gu¨rsoy et al., 2014). The observed drifts of up to 500 nm during 1 h in the vertical direction can be easily corrected for (Storm et al., 2017; Guizar-Sicairos et al., 2011), which leads to signiﬁcant reduction in ring artifacts (Pelt & Parkinson, 2018). However, the movements in the x direction in the range of 200 nm during 1 h are much more difﬁcult to correct for. Several alignment methods are available for absorption contrast, but most of them fail for Zernike phase contrast because of artifacts induced by the phase ring. Drift correction in the vertical direction was performed for the 15 min scans, usually in the range of 3–4 pixels (unbinned). For shorter scans, this is not necessary. The segmentation of the reconstructed volumes was carried out by automated thresh- olding based on the minimum method (Prewitt & Mendel- J. Synchrotron Rad. (2020). 27 research papers A higher binning increases the signal-to-noise ratio, reduces the overhead time (higher E) and allows for a longer exposure time without blurring (because of fewer pixels), and therefore increases the efﬁciency. sohn, 1966). For the fast scans, ﬁlters were applied and compared. A fast Fourier transform bandpass ﬁlter (low pass) and a median 3D ﬁlter with a of 2 pixels turned out to be most efﬁcient while keeping edges for the shortest scan times (van der Walt et al., 2014). For longer scan times, a median 3D ﬁlter in combination with a non-local means ﬁlter gave the best results. The CNR was calculated by the following equa- tion (Muhogora et al., 2008), written for gold and air, for example, CNR ¼ IAu Iair ð1=2Þ 2 Au þ 2 air 1=2 ; Scans were performed with total scan times from 53 min (absorption) and 15 min (Zernike phase contrast) down to 6 s. In total, we performed nine scans for absorption and six scans for Zernike phase contrast with different parameters (e.g. exposure time, number of projections, binning), which can be found in Table 1. For the very short scans of 18 s and 6 s, a maximum shift b was allowed to be larger than one pixel in order to keep a reasonably high efﬁciency, i.e. keeping the total dead-time of the detector as small as possible. Since the detector has a point spread function of 2.5 pixels, this is acceptable. where IAu and Iair are the mean grey values in gold and air, and Au and air are the standard deviations for these materials. The ratio is calculated on the unﬁltered data using the mask of the segmented volume. All data were binned to the same effective pixel size before reconstruction to eliminate the inﬂuence of binning on the CNR. Since the magnesium sample consists of two different materials (SiC particles and Mg matrix) and voids, the CNR between each material and the voids, as well as the CNR between these two materials, can be calculated. 2.2. Reconstruction and analysis Flenner et al. Pushing the temporal resolution in nanotomography 3 of 8 2.1. X-ray microscopy setup 2.1. X-ray microscopy setup The experiment was performed at the nanotomography endstation at the imaging beamline P05 operated by the Figure 1 Schematic of the Zernike phase contrast setup at the imaging beamline P05. Figure 1 Schematic of the Zernike phase contrast setup at the imaging beamline P05. 2 of 8 Flenner et al. Pushing the temporal resolution in nanotomography J. Synchrotron Rad. (2020). 27 research papers 3.1. Standard X-ray absorption microscope The image quality of the reconstructed slices strongly depends on the total exposure time (Fig. 2). In the 15 min scan, all features can be clearly resolved with high contrast. The 53 min scan is less noisy but artifacts from long-term sample movements can be recognized. In the 3 min scan, the features are clearly resolved and after applying ﬁlters the noise can be removed. In the 36 s scan the overall structure can be determined but the noise becomes a critical factor. Because of the high noise level, more ﬁltering is needed so the ﬁltered image looks more blurred and smaller details are lost (Fig. 2). In the unﬁltered 6 s scan, no inner structures can be resolved, so intensive ﬁltering is necessary. Nevertheless, the segmentation after ﬁltering clearly shows similarities with the other segmentations and key features can be recognized. g Reconstructed slices of NPG (absorption contrast) without any alignment or ﬁltering (left). Slices after ﬁlters have been applied (middle): non-local means ﬁlter (53 min, 15 min), median 3D and non-local means (3 min), low-pass ﬁlter and non-local means (36 s), and low-pass ﬁlter and median 3D (6 s). Automated threshold segmentation of slices (right). a qualitative reference to compare the similarity of different scans. After ﬁltering the 3 min scan, smaller particles (<300 nm) are no longer segmented correctly in the auto- mated segmentation. In the short scans, the overall shape of the voids is clearly visible but only larger particles (>500 nm) are segmented correctly. In the 6 s scan, most of the infor- mation about the SiC particles is lost, and when using the automated segmentation the wrong particles are segmented. Nevertheless, the voids inside the magnesium are still resolved. Small features, like a gap of 150 nm in width between two individual ligaments indicated by the red arrows in Fig. 2, are resolved in the 15 min scan. Although the gap is clearly visible in the ﬁltered 3 min image, the automated segmentation fails to resolve the gap completely. 3.2. Zernike phase-contrast X-ray microscope The inner structure of the low-absorbing magnesium composite can be determined using Zernike phase contrast (Fig. 3). In the 15 min scan, SiC particles of different sizes from 1.5 mm down to 150 nm, as well as voids inside the magnesium, are resolved. Because of the automated segmentation based on grey value thresholds, the segmentation of the 15 min scan does not represent the perfect sample structure but is used as 4 of 8 Flenner et al. Pushing the temporal resolution in nanotomography research papers Figure 2 Reconstructed slices of NPG (absorption contrast) without any alignment or ﬁltering (left). Slices after ﬁlters have been applied (middle): non-local means ﬁlter (53 min, 15 min), median 3D and non-local means (3 min), low-pass ﬁlter and non-local means (36 s), and low-pass ﬁlter and median 3D (6 s). Automated threshold segmentation of slices (right). experiment is 250 nm (Larsson et al., 2019). Gold is a strongly absorbing material at 11 keV, with a / of only 6.25 (optical material parameter given in Table S2). The calculated trans- mittance of a sample of 10 mm diameter is 18% at 11 keV, so a good contrast in absorption mode is expected. As a test sample for Zernike phase contrast, we used a low- absorbing magnesium composite with 10 vol% SiC particles of sub-micrometre size (Penther et al., 2018; Ghasemi et al., 2018). This allowed testing the resolution by analyzing larger particles and smaller particles. The absorption of magnesium, as well as SiC, is very low. The calculated absorption for a sample of 20 mm diameter is only 5.7% at 11 keV and the / ratios are >100 for Mg and SiC. Therefore, this sample is well suited as a test sample for Zernike phase contrast. The magnesium composite and NPG sample do have different natures: the NPG sample has a binary structure (gold–air) which yields hard boundaries with many high- frequency contributions, while the magnesium composite has fewer very ﬁne structures and the contrast between its main constituents (SiC grains and Mg) is weaker than between gold and air. Therefore, a direct comparison regarding the spatial resolution and CNR for these two samples is misleading and should not be made. Choosing these different samples, however, offers a guideline for a wide range of samples. 2.3. Materials NPG has recently received increasing interest (Lilleodden & Voorhees, 2018; Weissmu¨ller & Sieradzki, 2018; Qi & Weissmu¨ller, 2013) and is an ideal test sample for the char- acterization of nanotomography setups. The size of the gold ligaments can be tailored depending on the expected resolu- tion of the setup. The ligament size in the sample used in this 3 of 8 research papers 3. Results 3.1. Standard X-ray absorption microscope 3.3. Contrast-to-noise ratio The calculated CNR increases, because of the Poisson photon statistics, with the number of photons and therefore with the total exposure time t (Fig. 4). The quality improves rapidly when scan times are increased and the effect levels J. Synchrotron Rad. (2020). 27 research papers depend on the setup and scan time but also on the contrast of the sample itself. off towards higher average count rates. On the other hand, sample- and optics-induced drifts can decrease the CNR, the resolution and the overall image quality of the reconstruction, when scan times are increased. The CNR does not only p The following function was ﬁtted to the calculated CNR, a 1 exp ﬃﬃt p b : Figure 3 Reconstructed slices of magnesium (Zernike phase contrast) without any ﬁltering (left). Filtering (middle) allows automatic segmentation (right). White spots depict SiC crystals, black areas indicate voids inside the material and grey areas depict the Mg matrix. Features of 150 nm size can be detected in the 15 min scan while already in the 3 min scan all features smaller than 300 nm are not extracted by the segmentation. Only rough structures are resolved in the 36 s and 6 s scans. Here, a represents an upper limit of the achievable CNR while b describes how fast the CNR approaches the experimental limit. The maximum achievable CNR is limited. It is deﬁned by the intrinsic material properties, like the variation in mean grey values and their standard deviation (Lovric et al., 2013). Furthermore, the used instrument setup can limit the CNR, e.g. artifacts from sample movement caused by thermal drifts during long scan times. For NPG in absorption, after 15 min a CNR of already 95% of the upper limit is reached, while it does not increase much with respect to the 53 min scan (97%). However, in the latter, artifacts, e.g. from long-term sample drift, can already be recognized and decrease the overall image quality (Fig. 2, upper left). At 3 min, it still has a CNR of 65% of the limit value. In this case, a good compromise between the CNR and short scan time for absorption is 15 min. For the magnesium sample in Zernike phase contrast, the CNRs calculated for the different materials in the sample show different curve progressions. J. Synchrotron Rad. (2020). 27 Flenner et al. Pushing the temporal resolution in nanotomography 5 of 8 3.3. Contrast-to-noise ratio The highest CNR is recorded between SiC and air, which is expected because of the largest differences in and . Here, 92% of the limit a is reached after 15 min. For the magnesium (CNRs for Mg–air and Mg–SiC), values of 75% of the CNR limit are reached after a scan time of 15 min. The halo effect observed in the Zernike reconstruction can lead to a wider range of grey values inside one material and therefore a larger, i.e. smaller, CNR. The slower increase in CNR for the magnesium phases might be because of its heterogeneity in grey values (see also Fig. 3, upper row): regions of brighter and darker magnesium can be identiﬁed, suggesting that the SiC particles are not completely immersed in the magnesium phase. This suggests that an extension of the scan time towards 30 min could result Figure 3 Figure 3 Reconstructed slices of magnesium (Zernike phase contrast) without any ﬁltering (left). Filtering (middle) allows automatic segmentation (right). White spots depict SiC crystals, black areas indicate voids inside the material and grey areas depict the Mg matrix. Features of 150 nm size can be detected in the 15 min scan while already in the 3 min scan all features smaller than 300 nm are not extracted by the segmentation. Only rough structures are resolved in the 36 s and 6 s scans. Figure 4 CNRs for different total exposure times t for absorption contrast (black) and Zernike phase contrast (colour). The CNR increases with a ½1 expð ﬃﬃt p =b Þ (the ﬁt is shown as dotted lines). For more details about the ﬁt see Table S3. ure 4 g CNRs for different total exposure times t for absorption contrast (black) and Zernike phase contrast (colour). The CNR increases with a ½1 expð ﬃﬃt p =b Þ (the ﬁt is shown as dotted lines). For more details about the ﬁt see Table S3. re times t for absorption contrast (black) and Zernike phase contrast (colour). The CNR increases with wn as dotted lines). For more details about the ﬁt see Table S3. 5 of 8 J. Synchrotron Rad. (2020). 27 research papers Figure 5 (a) Absorption contrast and (b) Zernike phase contrast of a Siemens star. The <50 nm lines (third inner ring, red arrow) are clearly resolved in both images. 3.5. Improving image quality by machine learning A mixed-scale dense convolutional neural network (msdnet; Pelt et al., 2018; Pelt & Sethian, 2018) was used to improve the image quality. The training was performed on 100 slices of the reconstructed 6 s scan (absorption) and 36 s scan (Zernike phase contrast), while the 15 min scan was used as a ground truth (target). The network was validated on 20 slices and tested on 20 slices (Fig. 7). Training the network on individual slices in the x–y direction leads to artifacts in the other direction (z direction). Therefore, for each slice the four closest adjacent slices were used as additional input channels, which eliminates the artifacts and improves the image quality in all directions. Fig. 7 shows that the noise is completely eliminated since the network only learns the ‘real’ structures and does not reproduce the random noise, so no additional noise-reduction ﬁlter is necessary. The structures of the NPG do match very well with the structures visible in Fig. 2. For the magnesium sample, the noise is reduced signiﬁcantly, larger grain structures become clearer and smaller grains become visible. Altogether, machine learning is a very powerful 3.3. Contrast-to-noise ratio The half-period resolution (as determined by FRC) for (a) is 48.1 nm 1.6 nm and for (b) is 47.2 nm 2.5 nm. this is expected because the number of photons is proportional to the scan time, N / t. In this case, the 3D resolution does not reach the optical resolution limit as it is limited by noise. A half-period resolution of 64.0 nm 1.2 nm in 3D was achieved in the 15 min absorption scan with a binning of 2 and a resulting effective pixel size of 29.8 nm. A half-period reso- lution of 83.5 nm 1.6 nm was achieved for the 3 min scan. However, reducing the scan time further below 3 min leads to a signiﬁcant decrease in spatial resolution. This is expected since the noise level is increasing because of the limited ﬂux. In addition, the limited number of angles for the tomographic reconstruction decreases the reconstruction quality further. The spatial resolution in the Zernike phase contrast appears to be lower in the 3D volume (81.9 nm 1.6 nm for 15 min and 107.6 nm 4.3 nm for 3 min). (a) Absorption contrast and (b) Zernike phase contrast of a Siemens star. The <50 nm lines (third inner ring, red arrow) are clearly resolved in both images. The half-period resolution (as determined by FRC) for (a) is 48.1 nm 1.6 nm and for (b) is 47.2 nm 2.5 nm. in a further increase of the CNR and lead to better distin- guishable magnesium phases. 3.5. Improving image quality by machine learning 6 of 8 Flenner et al. Pushing the temporal resolution in nanotomography 3.4. Spatial resolution The resolution in 2D was estimated using a Siemens star test pattern with smallest feature sizes of 25 nm (Fig. 5). The lines of the third innermost ring (line widths between 48 nm and 37 nm) can clearly be resolved. The highest achievable half- period resolution estimated from the FRC using the half-bit resolution criterion is [Fig. 6(a)] 48.1 nm 1.6 nm for absorption and 47.2 nm 2.5 nm for Zernike phase contrast. This value is larger than the half-period optical resolution limit of the FZP of 30.5 nm (= 0.5 1.22 50 nm) corresponding to half of the Rayleigh criterion. This shows that the setup is limited by either the mechanics or the detector system. The calculated resolution values correspond to 3 pixels on the detector and are in line with the manufacturer speciﬁcation (30 line-pairs mm1 which equates to a half-period resolution of 2.5 pixels). The resolution in 3D calculated for the absorption sample by the FSC improves when extending the scan times [Fig. 6(b)]; Figure 6 (a) Estimation of the resolution via FSC of a 2D Siemens star test pattern for absorption (black) and Zernike phase contrast (red). The best achieved half-period resolution in 2D is 47.2 nm 2.5 nm. (b) An overview of the calculated resolution for different scan times using the half-bit criterion. The best half-period resolution in 3D of 64.0 nm was achieved in the 15 min absorption scan using a binning of 2. Figure 6 g (a) Estimation of the resolution via FSC of a 2D Siemens star test pattern for absorption (black) and Zernike phase contrast (red). The best achieved half-period resolution in 2D is 47.2 nm 2.5 nm. (b) An overview of the calculated resolution for different scan times using the half-bit criterion. The best half-period resolution in 3D of 64.0 nm was achieved in the 15 min absorption scan using a binning of 2. 6 of 8 Flenner et al. Pushing the temporal resolution in nanotomography J. Synchrotron Rad. (2020). 27 research papers times have to be considered more carefully. Here, a good compromise could be a 15 min scan with additional ML denoising. Improvements in spatial resolution are not expected for scan times longer than 15 min for both contrast methods. 3.4. Spatial resolution Figure 7 The result of training with the machine learning network msdnet (Pelt et al., 2018; Pelt & Sethian, 2018) for short scans. (a) In the x–y direction and (b) in the y–z direction of a 6 s absorption scan of NPG. (c, d) Slices of a 36 s Zernike phase contrast scan of magnesium alloy in (c) the x–y direction and (d) the x–z direction. The noise is eliminated and the structures show great similarities to the structures observed in longer scans. For the original slice in the x–y direction, see Figs. 2 and 3, lower left. For these two reference samples, the ideal scan time for the current setup at the P05 nanotomography endstation is 15– 30 min for high-quality scans and 3 min for fast scans. Even though this is dependent on sample contrast and structure, these values can be used as a good guideline for other samples as well. The fast scan mode can, for example, be used when performing in situ experiments. Another approach to proﬁt from both high image quality and short scan times is to carry out a long high-quality scan before the in situ experiment and short scans during the in situ experiment. The image quality of the short time scans can then later be improved using machine learning (Yang et al., 2018; Pelt et al., 2018). First tests with msdnet showed that even for very short scan times the structure of the sample can be extracted after training the network using the 15 min scan as reference. Figure 7 Time-resolved TXM with short scan times is feasible at the current setup of the nanotomography endstation at P05 for both absorption and Zernike phase contrast with high spatial resolution. This enables fast in situ experiments. It is to be expected that the overall scan time can be further decreased in the future, once the new double multilayer monochromator (DMM) at P05 is fully commissioned. The DMM can provide one order of magnitude more ﬂux than the double-crystal monochromator used in this study. The result of training with the machine learning network msdnet (Pelt et al., 2018; Pelt & Sethian, 2018) for short scans. (a) In the x–y direction and (b) in the y–z direction of a 6 s absorption scan of NPG. alternative to classical ﬁltering to reduce the noise and extract features in low-quality scans. Fast nanotomography using an X-ray microscope has the potential to bridge the gap between ultra-fast micro- tomography with time resolution down to a few milliseconds (Garcı´a-Moreno et al., 2019) and spatial resolution of down to 1 mm, and high-resolution imaging techniques such as focused ion-beam tomography and ptychography with spatial resolu- tion down to a few tenths of a nanometre but several hours of acquisition time. J. Synchrotron Rad. (2020). 27 4. Discussion The ideal scan time of a speciﬁc sample in a nanotomography setup can be determined by analysing different parameters like CNR and spatial resolution. Using the nanotomography setup at P05, we can extract information about the samples reliably down to scan times of 3 min without loss of spatial resolution. Although the image quality of the unﬁltered short scans appears to be very poor, ﬁltering can enhance the image quality drastically so that even threshold segmentation is possible, but with some loss of detail. Acknowledgements We thank Sepideh Kamrani and Erica Lilleodden for providing the test samples used in this study. This research was supported in part through the Maxwell computational resources operated at Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany. Open access funding enabled and organized by Projekt DEAL. A good compromise between the best CNR and short scan time is found to be at 15 min for both contrast methods. At 15 min, the CNR in the absorption scan is already close to the experimental limit and does not increase signiﬁcantly when extending the scan time. In the case of Zernike phase contrast, an increase in scan time might result in a further improvement of the CNR of this MgSiC material, in particular the CNRs of Mg–air and Mg– SiC. However, it has to be taken into account that at long scan times other factors, e.g. thermal drifts, become predominant and might lead to a reduction in the spatial resolution as well as the CNRs. For weakly absorbing biological samples, the dose becomes an important factor and therefore the scan 3.4. Spatial resolution (c, d) Slices of a 36 s Zernike phase contrast scan of magnesium alloy in (c) the x–y direction and (d) the x–z direction. The noise is eliminated and the structures show great similarities to the structures observed in longer scans. For the original slice in the x–y direction, see Figs. 2 and 3, lower left. alternative to classical ﬁltering to reduce the noise and extract features in low-quality scans. Flenner et al. Pushing the temporal resolution in nanotomography 7 of 8 References ( ) y Greving, I., Flenner, S., Larsson, E., Storm, M., Wilde, F., Lilleodden, Storm, M., Beckmann, F. & Rau, C. (2017). Opt. Lett. 42, 4982. E., Dose, T., Burmester, H., Lottermoser, L., David, C. & Beckmann, F. (2018). Microsc. Microanal. 24, 228–229. Vartiainen, I., Holzner, C., Mohacsi, I., Karvinen, P., Diaz, A., Pigino, G. & David, C. (2015). Opt. Express, 23, 13278. ( ) Greving, I., Ogurreck, M., Marschall, F., Last, A., Wilde, F., Dose, Vila-Comamala, J., Gorelick, S., Fa¨rm, E., Kewish, C. M., Diaz, A., Barrett, R., Guzenko, V. A., Ritala, M. & David, C. (2011). Opt. Express, 19, 175. T., Burmester, H., Lottermoser, L., Mu¨ller, M., David, C. & Beckmann, F. (2017). J. Phys. Conf. Ser. 849, 012056. Barrett, R., Guzenko, V. A., Ritala, M. & David, C. (2011). Opt. Express, 19, 175. Guizar-Sicairos, M., Diaz, A., Holler, M., Lucas, M. S., Menzel, A., Wepf, R. A. & Bunk, O. (2011). Opt. Express, 19, 21345. Villanova, J., Daudin, R., Lhuissier, P., Jauffre`s, D., Lou, S., Martin, C. L., Laboure´, S., Tucoulou, R., Martı´nez-Criado, G. & Salvo, L. (2017). Mater. Today, 20, 354–359. Gu¨rsoy, D., De Carlo, F., Xiao, X. & Jacobsen, C. (2014). J. Synchrotron Rad. 21, 1188–1193. Vogt, U., Lindblom, M., Charalambous, P., Kaulich, B. & Wilhein, T. (2006). Opt. Lett. 31, 1465. y Heel, M. van & Schatz, M. (2005). J. Struct. Biol. 151, 250–262. Walt, S. van der, Scho¨nberger, J. L., Nunez-Iglesias, J., Boulogne, F., Warner, J. D., Yager, N., Gouillart, E., Yu, T. & scikit-image contributors (2014). PeerJ. 2, e453. Jeﬁmovs, K., Vila-Comamala, J., Stampanoni, M., Kaulich, B. & David, C. (2008). J. Synchrotron Rad. 15, 106–108. Larsson, E., Gu¨rsoy, D., De Carlo, F., Lilleodden, E., Storm, M., Wilde, F., Hu, K., Mu¨ller, M. & Greving, I. (2019). J. Synchrotron Rad. 26, 194–204. ( ) Waske, A., Rahn, H. & Odenbach, S. (2010). AIP Conf. Proc. 1221, 124. Weissmu¨ller, J. & Sieradzki, K. (2018). MRS Bull. 43, 14–19. Lilleodden, E. T. & Voorhees, P. W. (2018). MRS Bull. 43, 20–26. Lilleodden, E. T. & Voorhees, P. W. (2018). MRS Bull. 43, 20–26. Lovric, G., Barre´, S. F., Schittny, J. C., Roth-Kleiner, M., Stampanoni, M. & Mokso, R. (2013). J. Appl. Cryst. 46, 856–860. Lovric, G., Barre´, S. F., Schittny, J. C., Roth-Kleiner, M., Stampanoni, M. & Mokso, R. (2013). J. Appl. Cryst. 46, 856–860. Yang, X., De Andrade, V., Scullin, W., Dyer, E. References Muhogora, W. E., Devetti, A., Padovani, R., Msaki, P. & Bonutti, F. (2008). Radiat. Prot. Dosimetry, 129, 231–236. Ogurreck, M., Wilde, F., Herzen, J., Beckmann, F., Nazmov, V., Mohr, J., Haibel, A., Mu¨ller, M. & Schreyer, A. (2013). J. Phys. Conf. Ser. 425, 182002. Andrews, J. C., Pianetta, P., Meirer, F., Chen, J., Almeida, E., van der Meulen, M. C., Alwood, J. S., Lee, C., Zhu, J. & Cui, Y. (2010). AIP Conf. Proc. 1234, 79. Patterson, B. M., Cordes, N. L., Henderson, K., Mertens, J. C. E., Clarke, A. J., Hornberger, B., Merkle, A., Etchin, S., Tkachuk, A., Leibowitz, M., Trapp, D., Qiu, W., Zhang, B., Bale, H., Lu, X., Hartwell, R., Withers, P. J. & Bradley, R. S. (2016). Exp. Mech. 56, 1585–1597. Dowd, B. A., Campbell, G. H., Marr, R. B., Nagarkar, V. V., Tipnis, S. V., Axe, L. & Siddons, D. P. (1999). Proc. SPIE, 3772, 224– 236. Flenner, S., Larsson, E., Furlan, K., Laipple, D., Storm, M., Wilde, F., Blick, R., Schneider, G. A., Zierold, R., Janssen, R., David, C., Beckmann, F., Muller, M. & Greving, I. (2018). Microsc. Microanal. 24, 148–149. Pelt, D., Batenburg, K. & Sethian, J. (2018). J. Imaging, 4, 128. D. M. & Parkinson, D. Y. (2018). Meas. Sci. Technol. 29 Pelt, D. M. & Sethian, J. A. (2018). Proc. Natl Acad. Sci. USA, 115, 254–259. Garcı´a-Moreno, F., Kamm, P. H., Neu, T. R., Bu¨lk, F., Mokso, R., Schlepu¨tz, C. M., Stampanoni, M. & Banhart, J. (2019). Nat. Commun. 10, 1–9. Penther, D., Ghasemi, A., Riedel, R., Fleck, C. & Kamrani, S. (2018). Mater. Sci. Eng. A, 738, 264–272. Ge, M., Coburn, D. S., Nazaretski, E., Xu, W., Gofron, K., Xu, H., Yin, Z. & Lee, W.-K. (2018). Appl. Phys. Lett. 113, 083109. Prewitt, J. M. S. & Mendelsohn, M. L. (1966). Ann. N. Y. Acad. Sci. 128, 1035–1053. 128, 1035–1053. Qi, Z. & Weissmu¨ller, J. (2013). ACS Nano, 7, 5948–5954. , Qi, Z. & Weissmu¨ller, J. (2013). ACS Nano, 7, 5948–5954. Ghasemi, A., Penther, D. & Kamrani, S. (2018). Mater. Charact. 142, 137–143. Schmahl, G., Rudolph, D., Guttmann, P., Schneider, G., Thieme, J., Niemann, B. & Wilhein, T. (1994). Synchrotron Radiat. News, 7, 19–22. Gorelick, S., Vila-Comamala, J., Guzenko, V. A., Barrett, R., Salome´, M. & David, C. (2011). J. Synchrotron Rad. 18, 442–446. Funding information The authors gratefully acknowledge the ﬁnancial support from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project number 192346071, SFB 986 (project Z2); DMP is ﬁnancially supported by The Nether- lands Organization for Scientiﬁc Research (NWO), project number 016.Veni.192.235. 7 of 8 research papers 8 of 8 Flenner et al. Pushing the temporal resolution in nanotomography References L., Kasthuri, N., De Carlo, F. & Gu¨rsoy, D. (2018). Sci. Rep. 8, 2575. Yuan, K., De Andrade, V., Feng, Z., Sturchio, N. C., Lee, S. S. & F P (2018) J Ph Ch C 122 2238 2247 Yuan, K., De Andrade, V., Feng, Z., Sturchio, N. C., Lee, S. S. & Fenter, P. (2018). J. Phys. Chem. C, 122, 2238–2247. Zernike, F. (1934). Physica, 1, 689–704. Yuan, K., De Andrade, V., Feng, Z., Sturchio, N. C., Lee Fenter, P. (2018). J. Phys. Chem. C, 122, 2238–2247. Z ik F (1934) Ph i 1 689 704 Mohacsi, I., Karvinen, P., Vartiainen, I., Guzenko, V. A., Somogyi, A., Kewish, C. M., Mercere, P. & David, C. (2014). J. Synchrotron Rad. 21, 497–501. g Fenter, P. (2018). J. Phys. Chem. C, 122, 2238–2247. Z ik F (1934) Ph i 1 689 704 Zernike, F. (1934). Physica, 1, 689–704. J. Synchrotron Rad. (2020). 27
https://openalex.org/W2904975284	https://europepmc.org/articles/pmc6320773?pdf=render	English	null	Roco Proteins and the Parkinson’s Disease-Associated LRRK2	International journal of molecular sciences	2,018	cc-by	12,086	Received: 15 November 2018; Accepted: 14 December 2018; Published: 17 December 2018 Abstract: Small G-proteins are structurally-conserved modules that function as molecular on-off switches. They function in many different cellular processes with differential speciﬁcity determined by the unique effector-binding surfaces, which undergo conformational changes during the switching action. These switches are typically standalone monomeric modules that form transient heterodimers with speciﬁc effector proteins in the ‘on’ state, and cycle to back to the monomeric conformation in the ‘off’ state. A new class of small G-proteins called “Roco” was discovered about a decade ago; this class is distinct from the typical G-proteins in several intriguing ways. Their switch module resides within a polypeptide chain of a large multi-domain protein, always adjacent to a unique domain called COR, and its effector kinase often resides within the same polypeptide. As such, the mechanisms of action of the Roco G-proteins are likely to differ from those of the typical G-proteins. Understanding these mechanisms is important because aberrant activity in the human Roco protein LRRK2 is associated with the pathogenesis of Parkinson’s disease. This review provides an update on the current state of our understanding of the Roco G-proteins and the prospects of targeting them for therapeutic purposes. Keywords: Roc; Intramolecular mechanism; Small GTPase; LRRK2 Review Roco Proteins and the Parkinson’s Disease-Associated LRRK2 Jingling Liao 1,2,* and Quyen Q. Hoang 2,3,4,* Jingling Liao 1,2,* and Quyen Q. Hoang 2,3,4,* 1 Department of Public Health, Wuhan University of Science and Technology School of Medicine Wuhan 430081, China 1 Department of Public Health, Wuhan University of Science and Technology School of Medicine, W h 430081 Chi 3 Department of Neurology, Indiana University School of Medicine, Indianapolis, IN 46202, USA 4 Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN 46202, * Correspondence: jingling.liao@gmail.com (J.L.); qqhoang@gmail.com (Q.Q.H.); Tel.: +86-177-8646-6520 (J.L.); +1-317-274-4371 (Q.Q.H.) International Journal of Molecular Sciences International Journal of Molecular Sciences 1. Introduction Ank, ankyrin domain; LRR, leucine- rich repeats; ROC, Ras of complex proteins; COR, C-terminal of Roc; Kinase, kinase domain; WD40, WD40-like beta-propeller repeat domain; cNB, cyclic nucleotide binding; DEP, Dishevelled, EGL-10, pleckstrin domain; GEF, guanine nucleotide exchange factor; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins domain; Death, death domain; PTP, protein tyrosine phosphatase domain; RhoGEF/RhoGAP, RhoGEF/GAP domain. The red dotted-lines highlight the features that differentiate the three groups of Roco proteins. Figure 1. Schematic depiction of the Roco proteins. A common feature among them is the central Roc-COR domains, which are ﬂanked by various functional domains. Ank, ankyrin domain; LRR, leucine-rich repeats; ROC, Ras of complex proteins; COR, C-terminal of Roc; Kinase, kinase domain; WD40, WD40-like beta-propeller repeat domain; cNB, cyclic nucleotide binding; DEP, Dishevelled, EGL-10, pleckstrin domain; GEF, guanine nucleotide exchange factor; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins domain; Death, death domain; PTP, protein tyrosine phosphatase domain; RhoGEF/RhoGAP, RhoGEF/GAP domain. The red dotted-lines highlight the features that differentiate the three groups of Roco proteins. rich repeats; ROC, Ras of complex proteins; COR, C-terminal of Roc; Kinase, kinase domain; WD40 WD40-like beta-propeller repeat domain; cNB, cyclic nucleotide binding; DEP, Dishevelled, EGL-10 pleckstrin domain; GEF, guanine nucleotide exchange factor; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins domain; Death, death domain; PTP, protein tyrosine phosphatase domain; RhoGEF/RhoGAP, RhoGEF/GAP domain. The red dotted-lines highlight the features that differentiate the three groups of Roco proteins. leucine-rich repeats; ROC, Ras of complex proteins; COR, C-terminal of Roc; Kinase, kinase domain; WD40, WD40-like beta-propeller repeat domain; cNB, cyclic nucleotide binding; DEP, Dishevelled, EGL-10, pleckstrin domain; GEF, guanine nucleotide exchange factor; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins domain; Death, death domain; PTP, protein tyrosine phosphatase domain; RhoGEF/RhoGAP, RhoGEF/GAP domain. The red dotted-lines highlight the features that differentiate the three groups of Roco proteins. Additionally, P.J.M. Van Haastert’s group discovered the region between the Roc and the kinase domain COR (C-terminus of Roc), and called proteins consisting of Roc and COR “Roco proteins” for their unique structures [1]. The group used the amino acid sequence encompassing the Roc-COR tandem domain of GbpC to search for other Roco proteins from various databases. They found Roco proteins in prokaryotes, Dictyostelium, plants, and animals, each consisting of a juxtaposition of the Roc and the COR domains; there are no proteins containing either Roc or COR domain alone. 1. Introduction The Ras/GTPase superfamily consists of monomeric small GTPases with molecular masses of 20–30 kDa. Small GTPases act as molecular switches that cycle between the active GTP-bound and inactive GDP-bound states. The functional cycle is regulated mainly by two different groups of regulatory proteins: (1) The Guanine nucleotide exchange factors (GEFs), which facilitate GDP dissociation from the small GTPases and exchanging with GTP leading to the transmission of various upstream signals; and (2) GTPase-activating proteins (GAPs), which accelerate the speed of the intrinsic GTP hydrolysis of the GTPase, thereby resulting in the inactivation of the molecular switch. The Ras/GTPase superfamily perform their cellular function by activating downstream protein kinase cascades. The Ras signaling pathway is a classic example in the mitogen-activated protein kinase (MAPK) pathway. In the past decade, a novel group of Ras/GTPase superfamily has been discovered, which is called the Ras of complex proteins (Roc). These Roc GTPase exist as a domain within a large multi-domain protein [1]. Roc was ﬁrst described in the slim mold Dictyostelium discoideum which Int. J. Mol. Sci. 2018, 19, 4074; doi:10.3390/ijms19124074 www.mdpi.com/journal/ijms 2 of 15 Int. J. Mol. Sci. 2018, 19, 4074 possesses a complex protein termed GbpC (cGMP binding protein C) [2]. P.J.M. Van Haastert’s and J.L. Smith’s group identiﬁed that GbpC contains a number of different domains, including a leucine-rich repeats domain (LRR), a Ras domain, MEK kinase domain, a Ras guanine nucleotide exchange factor N-terminal (RasGEF-N) domain, a DEP domain, a RasGEF domain, a cGMP-binding domain, a GRAM domain (Glucosyltransferases Rab-like GTPase activators and Myotubularins), and a second cGMP-binding domain. In this study, the authors proposed that the MEKK activity of GbpC is intramolecularly regulated by its upstream Ras domain, and that the MEKK activity of GbpC depends on the cGMP transmitted from the cGMP-binding domains through the RasGEF and Ras domains, all occurring within the same protein (Figure 1). This Ras domain is unique with respect to the other canonical Ras-family GTPases, in that both its upstream regulator and its downstream effector kinase are contained within the same polypeptide chain. Int. J. Mol. Sci. 1. Introduction 2018, 19, x 2 of 15 group identified that GbpC contains a number of different domains, including a leucine-rich repeats domain (LRR), a Ras domain, MEK kinase domain, a Ras guanine nucleotide exchange factor N- terminal (RasGEF-N) domain, a DEP domain, a RasGEF domain, a cGMP-binding domain, a GRAM domain (Glucosyltransferases Rab-like GTPase activators and Myotubularins), and a second cGMP- binding domain. In this study, the authors proposed that the MEKK activity of GbpC is intramolecularly regulated by its upstream Ras domain, and that the MEKK activity of GbpC depends on the cGMP transmitted from the cGMP-binding domains through the RasGEF and Ras domains, all occurring within the same protein (Figure 1). This Ras domain is unique with respect to the other canonical Ras-family GTPases, in that both its upstream regulator and its downstream effector kinase are contained within the same polypeptide chain. Figure 1. Schematic depiction of the Roco proteins. A common feature among them is the central Roc- COR domains, which are flanked by various functional domains. Ank, ankyrin domain; LRR, leucine- rich repeats; ROC, Ras of complex proteins; COR, C-terminal of Roc; Kinase, kinase domain; WD40, WD40-like beta-propeller repeat domain; cNB, cyclic nucleotide binding; DEP, Dishevelled, EGL-10, pleckstrin domain; GEF, guanine nucleotide exchange factor; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins domain; Death, death domain; PTP, protein tyrosine phosphatase domain; RhoGEF/RhoGAP, RhoGEF/GAP domain. The red dotted-lines highlight the features that differentiate the three groups of Roco proteins. Figure 1. Schematic depiction of the Roco proteins. A common feature among them is the central Roc-COR domains, which are ﬂanked by various functional domains. Ank, ankyrin domain; LRR, leucine-rich repeats; ROC, Ras of complex proteins; COR, C-terminal of Roc; Kinase, kinase domain; WD40, WD40-like beta-propeller repeat domain; cNB, cyclic nucleotide binding; DEP, Dishevelled, EGL-10, pleckstrin domain; GEF, guanine nucleotide exchange factor; GRAM, glucosyltransferases, Rab-like GTPase activators and myotubularins domain; Death, death domain; PTP, protein tyrosine phosphatase domain; RhoGEF/RhoGAP, RhoGEF/GAP domain. The red dotted-lines highlight the features that differentiate the three groups of Roco proteins. Figure 1. Schematic depiction of the Roco proteins. A common feature among them is the central Roc- COR domains, which are flanked by various functional domains. 1. Introduction Based on their domain architecture, the identified Roco proteins are categorized into three groups. The largest group is found in Dictyostelium and metazoan, which is characterized by the central LRR-Roc- COR-Kinase tandem domains (Figure 1). In these proteins, the central architecture comprises the Roc- COR tandem domains, which are always preceded by a leucine-rich repeat domain (LRR) and followed by a kinase domain belonging to the MAPKKK subfamily. The N- and C-terminus flanking the Roc-COR domain consists of remarkably diverse structures, including WD-40-like beta-propeller repeats (WD40 and Kelch motif), and domains which interact with small GTPases (RasGEF, RhoGEF, and RhoGAP) [1] (Figure 1). The second group of Roco proteins is characterized by lacking of the kinase domain is found in mammals (MASL1), the plant Arabidopsis, and prokaryotes [3] (Figure 1). Additionally, P.J.M. Van Haastert’s group discovered the region between the Roc and the kinase domain COR (C-terminus of Roc), and called proteins consisting of Roc and COR “Roco proteins” for their unique structures [1]. The group used the amino acid sequence encompassing the Roc-COR tandem domain of GbpC to search for other Roco proteins from various databases. They found Roco proteins in prokaryotes, Dictyostelium, plants, and animals, each consisting of a juxtaposition of the Roc and the COR domains; there are no proteins containing either Roc or COR domain alone. Based on their domain architecture, the identiﬁed Roco proteins are categorized into three groups. The largest group is found in Dictyostelium and metazoan, which is characterized by the central LRR-Roc-COR-Kinase tandem domains (Figure 1). In these proteins, the central architecture comprises the Roc-COR tandem domains, which are always preceded by a leucine-rich repeat domain (LRR) and followed by a kinase domain belonging to the MAPKKK subfamily. The N- and C-terminus ﬂanking the Roc-COR domain consists of remarkably diverse structures, including WD-40-like beta-propeller repeats (WD40 and Kelch motif), and domains which interact with small GTPases (RasGEF, RhoGEF, and RhoGAP) [1] (Figure 1). The second group of Roco proteins is characterized by lacking of the kinase domain is found in mammals (MASL1), the plant Arabidopsis, and prokaryotes [3] (Figure 1). The proteins from this Int. J. Mol. Sci. 2018, 19, 4074 3 of 15 group only contain the central domain architecture, lacking a kinase domain. The Death-associated protein kinase (DAPK1) represent the third group of Roco proteins. 2. Biological Functions of Roco Proteins The Roco family of proteins have been identiﬁed in a wide phylogenetic range, from bacteria to humans [1]. This family of proteins has drawn intense interest because it has been linked with human disease. For example, mutations in LRRK2 have been associated with PD [9,10], and DAPK1 has been associated with cancer [6,7]. The diversity of domains ﬂanking either side of the central core GTPase and kinase domains, including a variety of regulatory and protein-protein interaction domains, indicate that these proteins have diverse functions, and that the Roc domains appear to serve as the molecular switches that regulate their activities. 1. Introduction DAPK1 is characterized by its death domain, which is found in proteins with apoptotic functions [4–6]. The central domains architecture of DAPK is followed by the death domain, whereas the kinase domain is located in the N-terminus, and is the only one that lacks an LRR domain in the group. DAPK1 was ﬁrst identiﬁed in the 1990s [7] as a serine/threonine kinase that is a positive mediator of programmed cell death induced by IFN-γ. p p g y Although the Roco proteins, such as DAPK1 and a few from Dictyostelium and prokaryotes described above, have been studied for some time [1,8], they were not widely studied, and the features of Roc-COR had not been well characterized until two research groups linking mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2), a human Roco protein, with autosomal dominant Parkinson’s disease (PD) in 2004 [9,10]. Since then, there has been intense interest and research activity in the quest to understand the details of Roco proteins. Due to the association of LRRK2 with both familial and sporadic PD, it has become the most intensively-studied protein among the Roco family. Abnormally high kinase activity of LRRK2 is associated with PD pathogenesis; therefore, the majority of studies have been mainly focused on understanding the role of the kinase activity in relation to the biological function of LRRK2 and in disease. A unique feature of LRRK2 is that it catalyses two distinct biochemical activities, phosphotransfer via its kinase domain and GTP hydrolysis via its Roc domain [11,12]. Although the Roc domains of Roco proteins are annotated as belonging to the small Ras GTPase superfamily, P.J.M. Van Haastert’s group utilized Cluster Analysis for 21 Roc domains and 34 other small GTPases to reveal that the Roc domains were clearly distinguished from the other groups of Ras/GTPase proteins [1,13]. This can explain why the Roc domain of DAPK1 had remained unidentiﬁed for decades, despite having been well-studied since the 1990s [7]. In recent years, there has been increasing interests in understanding the role of the GTPase activity of Roco proteins in an effort to understand the pathogenesis mechanisms LRRK2 in PD [8,14]. In this review, we will focus on discussing the potentially unique mechanism of action of the Roc domain compared to the classic Ras/GTPase, and the roles of GTPase activity in the functioning of Roco proteins and in disease. 2.2. Human Roco Proteins: DAPK1, LRRK1/2, MASL1 2.2. Human Roco Proteins: DAPK1, LRRK1/2, MASL1 The four human Roco proteins identified so far comprise DAPK1, LRRK1 (leucine-rich repeats kinase1), LRRK2 (leucine-rich repeats kinase 2), and MASL1 (malignant fibrous histiocytoma amplified sequence 1). A common feature among these four proteins is that they all contain a central Roc-COR domain characteristic of Roco proteins; however, the variety of the different surrounding domains illustrates the diverse functions of these proteins. The smallest of the group, MASL1, is composed of only three domains, an LRR domain followed by the Roc-COR tandem domains. It lacks a kinase domain, thereby leaving GTPase as its sole catalytic activity, indicating that most likely the Roc domain is the main functional domain to carry out its cellular functions [8,14]. DAPK1 consists of a kinase domain at the N-terminus followed by an ankyrin repeat (ANK), then the central Roc- COR domain, which in turn is followed by its death domain (which is a protein-protein interaction mediating domain that exists in many apoptosis-promoting proteins) [20]. LRRK2 and LRRK1 are closely related in vertebrates [21]. However, LRRK1 has rarely been reported to be associated with a human disease except that one family was found to carry LRRK1 mutations linked to the osteosclerotic metaphyseal dysplasia (OSMD) [22], while LRRK2 has been strongly associated with PD, Alzheimer’s disease (AD), and immune disorders [23,24]. As such, LRRK2 has been an important The four human Roco proteins identiﬁed so far comprise DAPK1, LRRK1 (leucine-rich repeats kinase1), LRRK2 (leucine-rich repeats kinase 2), and MASL1 (malignant ﬁbrous histiocytoma ampliﬁed sequence 1). A common feature among these four proteins is that they all contain a central Roc-COR domain characteristic of Roco proteins; however, the variety of the different surrounding domains illustrates the diverse functions of these proteins. The smallest of the group, MASL1, is composed of only three domains, an LRR domain followed by the Roc-COR tandem domains. It lacks a kinase domain, thereby leaving GTPase as its sole catalytic activity, indicating that most likely the Roc domain is the main functional domain to carry out its cellular functions [8,14]. DAPK1 consists of a kinase domain at the N-terminus followed by an ankyrin repeat (ANK), then the central Roc-COR domain, which in turn is followed by its death domain (which is a protein-protein interaction mediating domain that exists in many apoptosis-promoting proteins) [20]. LRRK2 and LRRK1 are closely related in vertebrates [21]. 2.1. GbpC and Pats1 in D. Discoideum Some of the most studied Roco proteins have been found from D. discoideum. Although 11 Roco genes have been identiﬁed in D. discoideum so far [1], GbpC and Pats1 (protein associated with transduction of signal 1) have been the most thoroughly studied. GbpC plays an important role in chemotaxis in slime mold [15,16], a process that involves the phosphorylation of myosin II and its assembly in the cytoskeleton, which is regulated by cGMP binding [16]. In addition to the Roc-COR tandem domain and kinase domain, GbpC has a peculiar C-terminal region that contains a Ras guanine exchange factor (Ras-GEF) domain, a GRAM domain, and 2 cGMP-binding domains. The binding of cGMP to GpbC induces an intramolecular activation mechanism by sequentially activating the Ras-GEF, Roc, and kinase domains [17] (Figure 2). GbpC translocation is induced by cAMP and depends on its GRAM domain to associate with the cellular membrane and cell cortex [18]. At the membrane and cell cortex, GbpC would phosphorylate its substrates via its kinase domain, which in turn is regulated by the upstream signals from Roc [18]. It appears that the Roc domain of GbpC functions by undergoing a G-protein cycle that is reminiscent of the canonical G-proteins, except that 4 of 15 Int. J. Mol. Sci. 2018, 19, 4074 the upstream regulator and the downstream effector happen to reside in the same polypeptide chain. It is still unclear whether or not these interesting ﬁndings are inferable to all other Roco family proteins. Int. J. Mol. Sci. 2018, 19, x 4 of 15 Figure 2. Schematic depiction of the regulatory event that occurs in GbpC. cGMP binding results in the activation of Roc, which in turns activate the kinase domain. Figure 2. Schematic depiction of the regulatory event that occurs in GbpC. cGMP binding results in the activation of Roc, which in turns activate the kinase domain. Figure 2. Schematic depiction of the regulatory event that occurs in GbpC. cGMP binding results in the activation of Roc, which in turns activate the kinase domain. Figure 2. Schematic depiction of the regulatory event that occurs in GbpC. cGMP binding results in the activation of Roc, which in turns activate the kinase domain. Pats1 was first identified as a novel gene linked to the cytokinesis by using a phenotype mutagenesis screen [19]. In addition to the GTP-binding domain and kinase domain, Jonathan C. 2.1. GbpC and Pats1 in D. Discoideum Abysalh and colleagues also identified a protein tyrosine phosphatase domain in the N-terminus of Pats1 (Figure 1). Overexpression of the kinase domain of Pats1 resulted in a severe cytokinesis defect in attached culture cells, and knockout of the Pats1 gene also caused a cytokinesis deficit phenotype [19]. Overexpression of the kinase domain alone was sufficient to rescue the cytokinesis deficit in the Pats1-null cell. In this study, the results suggested that Pats1 has an important role in cytokinesis, but the details of the mechanism involved remained unclear. The GTP binding domain of Pats1 shares sequence homology with both Rho and Ras small GTPases [19]; however, its GTPase activity and its GTP binding kinetics have not been fully characterized. Thus, the role of the GTP binding domain in Pats1-regulated cytokinesis in cell division remains unclear. Interestingly, Pats1 consists of a Roc- COR domain and a kinase domain, but it lacks other regulatory domains that are found in GbpC, such as RasGEF, thus suggesting that Pats1 might require a yet unknown extramolecular regulatory protein to modulate its GTPase activity. Pats1 was ﬁrst identiﬁed as a novel gene linked to the cytokinesis by using a phenotype mutagenesis screen [19]. In addition to the GTP-binding domain and kinase domain, Jonathan C. Abysalh and colleagues also identiﬁed a protein tyrosine phosphatase domain in the N-terminus of Pats1 (Figure 1). Overexpression of the kinase domain of Pats1 resulted in a severe cytokinesis defect in attached culture cells, and knockout of the Pats1 gene also caused a cytokinesis deﬁcit phenotype [19]. Overexpression of the kinase domain alone was sufﬁcient to rescue the cytokinesis deﬁcit in the Pats1-null cell. In this study, the results suggested that Pats1 has an important role in cytokinesis, but the details of the mechanism involved remained unclear. The GTP binding domain of Pats1 shares sequence homology with both Rho and Ras small GTPases [19]; however, its GTPase activity and its GTP binding kinetics have not been fully characterized. Thus, the role of the GTP binding domain in Pats1-regulated cytokinesis in cell division remains unclear. Interestingly, Pats1 consists of a Roc-COR domain and a kinase domain, but it lacks other regulatory domains that are found in GbpC, such as RasGEF, thus suggesting that Pats1 might require a yet unknown extramolecular regulatory protein to modulate its GTPase activity. 2.2. Human Roco Proteins: DAPK1, LRRK1/2, MASL1 2.2. Human Roco Proteins: DAPK1, LRRK1/2, MASL1 However, LRRK1 has rarely been reported to be associated with a human disease except that one family was found to carry LRRK1 mutations linked to the osteosclerotic metaphyseal dysplasia (OSMD) [22], while LRRK2 has been strongly associated with PD, Alzheimer’s disease (AD), and immune disorders [23,24]. As such, LRRK2 has been an important potential drug target for PD. 5 of 15 Int. J. Mol. Sci. 2018, 19, 4074 DAPK1 was ﬁrst discovered as a protein involved in the process of cell death [7], and it was subsequently determined to be a positive regulator of both apoptotic and type-II autophagic cell death [7,25]. DAPK1 might also function as a tumor suppressor by triggering apoptosis. Mutations leading to a reduced expression of DAPK1 have been associated with inheritable predisposition to chronic lymphocytic leukemia [26]. Additionally, DAPK1 may also be involved in neuronal cell death, such as epilepsy and Alzheimer’s disease [27]. The Roc domain negatively regulates DAPK1 activity through binding GTP to the P-loop motif of its Roc domain. The Roc domain of DAPK1 has also been implicated in mediating cellular functions through the formation of a DAPK1 homodimer, as well as by interacting with other Roco family proteins and cytoskeleton, including LRRK2 protein [25,28,29]. y g y p y g p The biological function of MASL1 is the least understood among the four human Roco proteins. MASL1 was originally identified as a novel gene which was amplified from a malignant fibrous histiocytoma, implying that MASL1 was involved in cell fate and division [30]. Later cellular studies of MASL1 suggested that it plays a role in the regulation of erythroid differentiation of CD34 (+) cells and necrotic cell death through the ERK pathway [31]. The Roc domain of MASL1 has been demonstrated to physically interact with Raf1, which is upstream of ERK signal cascade [31]. Because MASL1 lacks a kinase domain, its Roc domain acts on extramolecular effectors similar to the classic Ras/small GTPase family. LRRK1 is closely related to LRRK2, and it was believed that they derived from the same ancient gene via DNA duplication [32]. Both LRRK1 and LRRK2 are expressed widely in human tissues and organs, including the brain. Although they share a very similar multi-domains architecture, mutations in LRRK2 are strongly associated with familial and sporadic PD, whereas mutations in LRRK1 are found in a family with OSMD [22]. Recent ﬁndings from Weirong R. 2.2. Human Roco Proteins: DAPK1, LRRK1/2, MASL1 2.2. Human Roco Proteins: DAPK1, LRRK1/2, MASL1 Xing’s group suggest that LRRK1 regulates actin assembly in mature osteoclasts by phosphorylating L-plastin [32,33]. g y y p p y g p Among the human Roco proteins, LRRK2 has been the most studied. Human genetics has linked LRRK2 to PD, Crohn’s disease, multibacillary leprosy, and cancer [34]. LRRK2 has been implicated in a wide range of cellular processes including mitochondrial maintenance, synaptic vesicle cycling, autophagy, lysosomal biology, cytoskeletal regulation, neurite outgrowth regulation, and translational control [35,36]. Mice having LRRK2 knocked out showed kidney, lung, and liver abnormalities, including the accumulation of vesicles within cells and alterations in markers for autophagy [37,38]. A more recent study showed that alteration in LRRK2 could also result in defective chaperone-mediated autophagy (CMA) [39]. LRRK2 has been shown to interact with many cytoskeletal proteins, including β-tubulin [40], actin [41] and moesin [42], thus suggesting that LRRK2 might play a role in the control of cytoskeletal remodeling and regulating neurite outgrowth [43–45]. An increasing number of studies are implicating LRRK2 functioning at membranous structures [46,47]. In 2014, Mark Cookson’s group performed a chip-based LRRK2 interaction screening assay and found a number of interactors known to be involved in the vesicular recycling process, including Rab7L1, which is also involved in intracellular protein sorting [48,49]. Further investigation by Dario Alessi’s group showed that Rab7L1 recruits LRRK2 to the trans-Golgi network, and that this interaction activates the kinase activity of LRRK2 [50]. Using a phosphoproteomics approach, the same team also identiﬁed a subset of Rab GTPases as potential substrates of LRRK2 [51], then they used systematic proteomic analysis to identify ten speciﬁc Rabs that were endogenously phosphorylated by LRRK2 [52], thereby identifying a potential role for LRRK2 in ciliogenesis. Subsequently, the same team in collaboration with Susan Pfeffer’s group showed that LRRK2 blocks primary cilium formation through the Sonic hedgehog signaling pathway [53]. Taken together, it appears that LRRK2 plays an important role in a number of different cellular processes involving membranous structures; however, its precise function(s) and mechanism in disease pathogenesis remain elusive. 3.1. Roco Proteins are Functional GTPases G-proteins are characterized by two main functions: guanosine nucleotides (GDP and GTP) binding and GTP hydrolysis. The guanosine nucleotide-binding property of the Roco proteins has been widely reported for proteins from prokaryotic to human [25,28,54]. Wouter N. van Egmond and colleagues used GTP-agarose pull down to assay for the GTP binding ability of D. discoideum GbpC, and they demonstrated that the nucleotide exchange process of Roc domain of Gbpc was speciﬁcally activated by its C-terminus GbpC-RasGEF domain [17]. Katja Gotthardt and colleagues quantiﬁed the nucleotide binding ability of C. tepidum Roco in vitro by using puriﬁed recombinant Roc-COR domain, which showed that Roco has the low binding afﬁnity for both GDP and GTP (in the micromolar range) [55]. Guanosine nucleotides binding of the human Roco proteins has been well studied. Rodrigo Carlessi and colleagues showed that an excess of free GTP could competitively deplete the pulled-down DAPK1 from GTP-agarose and that the artiﬁcial mutation T701N in the P-loop motif of Roc domain, which would disrupt GTP binding, signiﬁcantly increasing the kinase activity of DAPK1 compared to that of the WT [28]. Sybille Dihanich and coworkers measured GTP binding for MASL1 using GTP-agarose pull-down assay in mammalian cells and showed that GTP binding was necessary for the complex formation of MASL1 in cells [56]. There is also an abundance of evidence showing human LRRK1 and LRRK2 binding with GTP and GDP [54,57]. Both LRRK2 full-length and its isolated Roc domain or Roc-COR tandem domain have been reported to selectively bind to GDP and GTP with similar afﬁnity [12,54,55], and that mutations in the nucleotide-binding P-loop, such as K1347A or T1348N, resulted in a reduction of GTP/GDP binding [58]. p g Similarly, the GTP hydrolysis activity of the Roc domains of the Roco proteins has been well characterized, including DAPK1 [28], LRRK2 [12], and the Chlorobium tepidum Roco [55]. Among them, LRRK2 has garnered the most attention. Several groups have demonstrated that LRRK2 is able to hydrolyze GTP using radioactively labeled guanine nucleotide to assess its intrinsic GTPase activity in vitro [54], and this activity is disrupted when the key residues in the P-loop required for nucleotide described above are mutated. 3. Biochemical Activity of Roc and Its Regulatory Mechanisms The typical small G-proteins function as molecular switches by cycling between the GDP-bound inactive and GTP-bound active conformations. This biochemical cycle is composed of a binary process; 6 of 15 Int. J. Mol. Sci. 2018, 19, 4074 the GDP-bound inactive state is switched to the activated state upon GTP binding, and reciprocally, the GTP-bound active state is switched off by the intrinsic GTP hydrolysis activity of G-protein. These changes are intricately controlled by GEFs, which promote the exchange of GDP with GTP, and by GAPs, which accelerate the biochemical hydrolysis of GTP. Utilizing this highly-conserved functional G-protein fold, their different subcellular localization and speciﬁc downstream effectors enabled the small G-proteins to regulate a diverse set of biological processes in the cell. The small G-proteins in the Roco family of proteins are unique, in that they are intramolecularly-linked with a COR domain, and their nucleotide exchange process might not require GEFs or GAPs. However, the molecular details of their mechanism of action remain to be elucidated. 3.2. Regulatory Mechanisms of Roc Activity 3.2. Regulatory Mechanisms of Roc Activity As mentioned above, the Roc domain of GbpC from D. Discoideum is uniquely regulated by its RasGEF domain in the same polypeptide chain. A number of studies have set out to identify the GEFs and GAPs for LRRK2. One of the potential GEFs identified for LRRK2 is ArhGEF, which is a GEF of CDC42 and Rac1, and its interaction with LRRK2 appears to increase GTPase activity in cells and mouse brain [61,62]. ArhGEF binding to LRRK2 might be dependent on the phosphorylation state of LRRK2, which is stimulated by CK1α [63]. Interestingly, ArhGEF can also be phosphorylated by LRRK2 in vitro [62]; however, it is unclear how ArhGEF affects the kinase activity of LRRK2 and how its phosphorylation of ArhGEF affect the activity of the GEF. It has been shown that the kinase activity of LRRK2 is toxic in yeast and a genetic screen for suppressors of this toxicity has found a GAP, GCS1, as a suppressor of LRRK2-induced toxicity [60]. The mammalian homologue of GCS1, ArfGAP1, has been tested for its interaction with LRRK2 in human cells and in rodent brains, and in vitro assays showed that ArfGAP1 causes an approximately 2–3 fold acceleration of the GTPase activity of LRRK2 [64,65]. Surprisingly, ArfGAP1 is found to interact with the N-terminus and C-terminus portions of LRRK2 rather than at the expected catalytic core region where the Roc domain resides [64,65]. If ArfGAP1 is indeed a GAP for Roc, then it would indicate that the GAP activating mechanism of Roc is more complex and different than that of the canonical small GTPases. Another potential GAP for LRRK2 is RGS2, which was originally identified in C. elegans as a modulator of LRRK2 [66]. RGS2 is subsequently shown to interact with LRRK2, and in so doing, increases its GTPase activity leading to a reduction in kinase activity in vitro [66]. It is unclear how ArfGAP1 and RGS2 both activate GTP hydrolysis of LRRK2 while showing the opposite effect on its kinase activity and neuronal toxicity. It remains to be determined whether or not ArfGAP1 or RGS2 are authentic physiological GAPs of LRRK2 As mentioned above, the Roc domain of GbpC from D. Discoideum is uniquely regulated by its RasGEF domain in the same polypeptide chain. A number of studies have set out to identify the GEFs and GAPs for LRRK2. 3.2. Regulatory Mechanisms of Roc Activity 3.2. Regulatory Mechanisms of Roc Activity One of the potential GEFs identiﬁed for LRRK2 is ArhGEF, which is a GEF of CDC42 and Rac1, and its interaction with LRRK2 appears to increase GTPase activity in cells and mouse brain [61,62]. ArhGEF binding to LRRK2 might be dependent on the phosphorylation state of LRRK2, which is stimulated by CK1α [63]. Interestingly, ArhGEF can also be phosphorylated by LRRK2 in vitro [62]; however, it is unclear how ArhGEF affects the kinase activity of LRRK2 and how its phosphorylation of ArhGEF affect the activity of the GEF. It has been shown that the kinase activity of LRRK2 is toxic in yeast and a genetic screen for suppressors of this toxicity has found a GAP, GCS1, as a suppressor of LRRK2-induced toxicity [60]. The mammalian homologue of GCS1, ArfGAP1, has been tested for its interaction with LRRK2 in human cells and in rodent brains, and in vitro assays showed that ArfGAP1 causes an approximately 2–3 fold acceleration of the GTPase activity of LRRK2 [64,65]. Surprisingly, ArfGAP1 is found to interact with the N-terminus and C-terminus portions of LRRK2 rather than at the expected catalytic core region where the Roc domain resides [64,65]. If ArfGAP1 is indeed a GAP for Roc, then it would indicate that the GAP activating mechanism of Roc is more complex and different than that of the canonical small GTPases. Another potential GAP for LRRK2 is RGS2, which was originally identiﬁed in C. elegans as a modulator of LRRK2 [66]. RGS2 is subsequently shown to interact with LRRK2, and in so doing, increases its GTPase activity leading to a reduction in kinase activity in vitro [66]. It is unclear how ArfGAP1 and RGS2 both activate GTP hydrolysis of LRRK2 while showing the opposite effect on its kinase activity and neuronal toxicity. It remains to be determined whether or not ArfGAP1 or RGS2 are authentic physiological GAPs of LRRK2. LRRK2. Interestingly, biochemical characterization of the LRRK2 protein reveals that the guanosine nucleotide affinity is much weaker than that of the canonical small GTPases [12,55]. Generally, the nucleotide affinity of Ras family of proteins is in the range from picomolar to nanomolar, whereas the nucleotide affinity of LRRK2 is in the micromolar range. The low affinity or nucleotides would enable nucleotide exchanges to occur in LRRK2 in the absence of GEFs. There is a precedent for a GEF- and GAP-independent G-protein activation mechanism. 3.1. Roco Proteins are Functional GTPases The Parkinson’s disease-associated mutation G2019S aberrantly over-activate the kinase domain. A set of Parkinson’s disease-associated mutations in the Roc domain (R1441G/H/C) also activate LRRK2 kinase activity. 3.1. Roco Proteins are Functional GTPases Truncation constructs consisting of just the Roc domain or the Roc-COR tandem domain retains LRRK2 GTPase activity; however, the activity is signiﬁcantly lower than that of the full-length protein, indicating that the GTPase activity may be intramolecularly modulated via interaction with other domains (Figure 3) [12,55,59]. Although the mechanism of action of the Roc G-protein remains unclear, it might be different from that of the typical G-proteins. For example, LRRK2 carrying the mutation R1398L resulted in a protein that possesses higher GTPase activity, while the equivalent mutation in Ras would abolish GTPase activity and trap it in a persistently active form [58,60]. A dual substitution mutant, combining the R1398L with a T1343V, in P-loop has created an active form of LRRK2 with impaired GTPase activity [58]. These artiﬁcial mutations conﬁrm the GTPase activity of the Roc domain of LRRK2, and suggest that it might function a little differently than the typical G-proteins. 7 of 15 7 of 15 Int. J. Mol. Sci. 2018, 19, 4074 J. Mol. Sci. 2018, 19, 4074 7 o J. Mol. Sci. 2018, 19, x 7 of Figure 3. Schematic depiction of the catalytic activity of LRRK2. GTP binding to Roc activates the kinase domain, which in turn phosphorylates downstream substrates and potentially feedback to the Roc domain via phosphorylation. The Parkinson’s disease-associated mutation G2019S aberrantly over-activate the kinase domain. A set of Parkinson’s disease-associated mutations in the Roc domain (R1441G/H/C) also activate LRRK2 kinase activity. Figure 3. Schematic depiction of the catalytic activity of LRRK2. GTP binding to Roc activates the kinase domain, which in turn phosphorylates downstream substrates and potentially feedback to the Roc domain via phosphorylation. The Parkinson’s disease-associated mutation G2019S aberrantly over-activate the kinase domain. A set of Parkinson’s disease-associated mutations in the Roc domain (R1441G/H/C) also activate LRRK2 kinase activity. Figure 3. Schematic depiction of the catalytic activity of LRRK2. GTP binding to Roc activates the kinase domain, which in turn phosphorylates downstream substrates and potentially feedback to the Roc domain via phosphorylation. The Parkinson’s disease-associated mutation G2019S aberrantly over-activate the kinase domain. A set of Parkinson’s disease-associated mutations in the Roc domain (R1441G/H/C) also activate LRRK2 kinase activity. Figure 3. Schematic depiction of the catalytic activity of LRRK2. GTP binding to Roc activates the kinase domain, which in turn phosphorylates downstream substrates and potentially feedback to the Roc domain via phosphorylation. 3.2. Regulatory Mechanisms of Roc Activity 3.2. Regulatory Mechanisms of Roc Activity Even so, there is evidence consistent with LRRK2 being a GAD G-protein. For example, LRRK2 has been reported to predominantly exist as a homodimer in cells [68,69], its COR domain dimeric interface is the most highly-conserved region among Roco proteins [70], the isolated COR domains of LRRK2 interact with each other [55,59], and the familial mutations (R1441C/G/H) disrupt dimerization and reduce GTPase activity [71,72]. However, several lines of evidence have shown that the isolated Roc domain of LRRK2 is catalytically active in its monomeric state in solution [12,73], demonstrating that dimerization is not essential for its GTP hydrolysis activity (which is inconsistent with the GADs model). Of note is that the catalytic arginine residue R543 of the ortholog C. tepidum Roc-COR does not exist in human LRRK2; therefore, it is still unclear whether the mechanism of action of CtRoco is the same as that of the human LRRK2. Another observation of LRRK2 that is inconsistent with the GAD model is that the binding of nucleotides does not regulate LRRK2 dimerization in cells. On the other hand, the human DAPK1 forms a homodimer in cells through interactions in the Roc and kinase domain instead of the COR domain. More recently, Egon Deyaert and colleagues proposed that the GTPase catalytic activity of the C. tepidum Roco protein is regulated by the conformational changes between dimeric and monomeric states during the process of GTP turnover [74]. In contrast to the GAD model mentioned above, the current dimer-monomer cycle model posits that GTP binding induces monomerization, and that GTP hydrolysis occurs in the monomeric form of the enzyme. LRRK2 has been observed to cycle between a dimeric kinase-active form and a monomeric kinase-inactive form in vivo [69,75]. The studies demonstrated that stabilization of either the dimeric or monomeric state of LRRK2 led to decreased GTPase activity [74]. Additionally, emerging evidence has indicated that the intramolecular kinase activity might reciprocally regulate the GTPase activity [73,76]. For example, autophosphorylation sites mapping of LRRK2 using mass spectrometry has found that many of these sites are clustered in the Roc domain, such as T1343, T1348, S1403, T1404, T1410, and T1503 [77–79]. Interestingly, these residues map primarily to the P-loop motif (which involves in nucleotide binding) and the switch regions (which mediate GTP binding or GTP hydrolysis) of Roc, suggesting that autophosphorylation at the Roc domain could potentially affect nucleotide binding and/or GTP hydrolysis. 3.2. Regulatory Mechanisms of Roc Activity 3.2. Regulatory Mechanisms of Roc Activity Raphael Gasper and colleagues classified a group of G proteins that are regulated by dimerization as GADs (G-proteins activated by nucleotide-dependent dimerization), including the SRP (signal recognition particle), the dynamins, and the septins [67]. Indeed, LRRK2 has been proposed as a GAD G-protein based on the structural study of prokaryotic C. tepidum Roco protein model [55]. The basic mechanistic feature that distinguishes GADs from the typical small G-proteins is that GADs do not require GEFs and GAPs for nucleotide exchange and GTPase activation, but instead, nucleotide exchange is dependent on the Interestingly, biochemical characterization of the LRRK2 protein reveals that the guanosine nucleotide afﬁnity is much weaker than that of the canonical small GTPases [12,55]. Generally, the nucleotide afﬁnity of Ras family of proteins is in the range from picomolar to nanomolar, whereas the nucleotide afﬁnity of LRRK2 is in the micromolar range. The low afﬁnity or nucleotides would enable nucleotide exchanges to occur in LRRK2 in the absence of GEFs. There is a precedent for a GEF- and GAP-independent G-protein activation mechanism. Raphael Gasper and colleagues classiﬁed a group of G proteins that are regulated by dimerization as GADs (G-proteins activated by nucleotide-dependent dimerization), including the SRP (signal recognition particle), the dynamins, and the septins [67]. Indeed, LRRK2 has been proposed as a GAD G-protein based on the structural study of prokaryotic C. tepidum Roco protein model [55]. The basic mechanistic feature that distinguishes GADs from the typical small G-proteins is that GADs do not require GEFs and GAPs for nucleotide exchange and GTPase activation, but instead, nucleotide exchange is dependent on 8 of 15 Int. J. Mol. Sci. 2018, 19, 4074 the local concentration of the nucleotides, and GTPase activation is conferred upon dimerization [67]. The notion that LRRK2 is a GAD G-protein has been inferred from the studies of the C. tepidum Roco. A Roc-COR contiguous construct of the CtRoco has been crystallized as constitutive homodimer stabilized by the interactions between the two COR domains. Based on the structure, it was proposed that the dimerization mediated by the two COR domains would position the Roc domains in close proximity to each other, thereby exchanging an Arginine residue that would complement and constitute a productive catalytic site [55]. Katja Gotthardt and colleagues suggested that the structure of the prokaryotic CtRoc-COR construct has implications for LRRK2, although the key active-site residues are not conserved [55]. 4. The Mechanism of Roc Transmitting Its Signal Downstream The typical Ras-family GTPases undergo guanine nucleotide-dependent conformational changes that alter their interactions with various effectors in the cellular signaling cascades leading to changes in subcellular localization and/or oligomeric states. However, the Roco-family G-proteins are contiguously fused with a COR domain, and most of them comprise of an intramolecular kinase domain, among others. Given this modular arrangement that consists of a G-protein and a potential effector kinase in the same polypeptide chain, it is tempting to imagine that the Roc domain might interact with its intramolecular kinase domain in a fashion analogous to that of Ras with the MAP kinases. Indeed, in slime mold, the activation of the Roc domain of GbpC with GTP stimulates the activity of its kinase domain, which then goes on phosphorylates its downstream substrates [17,18]. Similar evidence also exists for the human Roco proteins. Daniel Korr and colleagues observed that the kinase activity of LRRK1 could be stimulated by GTP binding [57], and that this kinase activity, which modulates osteoclast function, is regulated by cycling between GTP and GDP-bound Roc domain, which is reminiscent of the canonical Ras-family small GTPases [32,33]. More recently, several lines of evidence demonstrate that the kinase activity of LRRK2 is also regulated in a GTP-dependent manner [86–88]. The Roco proteins, lacking an intramolecular kinase domain, appear to transmit their signals extramolecularly, which is similar to the canonical G-proteins. For example, the Roc domain of MASL1, which lacks an intrinsic kinase domain, is also activated by GTP-binding; however, once activated, it regulates the extrinsic kinases in the Raf/MEK/ERK signaling cascade that is involved in the erythropoiesis. Interestingly, in contrast to LRRK1/2 and MASL1, GTP binding to the Roc domain of DAPK1 results in the down-regulation of its kinase activity [17,18]. All this suggests that the mechanisms of action of the Roc G-proteins are more complex than those of typical small G-proteins; however, the mechanism of nucleotide binding-induced conformational changes leading to the toggling of the molecular switch observed in the typical G-protein appear to be conserved in the Roc proteins. It is still unclear how the nucleotide-dependent conformational changes in the Roc domain of the Roco proteins result in the activation of their kinase domain. However, several lines of evidence suggest the involvement of dimerization and/or overall conformational changes leading to rearrangement of the domains. 3.2. Regulatory Mechanisms of Roc Activity 3.2. Regulatory Mechanisms of Roc Activity It has been reported that the phosphorylation of residue T1503 may regulate GTP binding and kinase activity of LRRK2 [11]. Another study showed that the phosphorylation of residues in the Roc domain increases the rate of GTP hydrolysis [73]. In addition to the intrinsic kinase activity, an extrinsic kinase PKA has also been shown to phosphorylate residue S1444 of LRRK2, where the phospho-binding protein 14-3-3 has been shown to dock, and in turn, 14-3-3 binding causes a decrease in the kinase activity of LRRK2 [80]. In addition to the cluster of sites located in the Roc domain mentioned above, autophosphorylation sites are also found in the region between the ANK domain and LRR domain, including S910, S935, S955, and S973 [81]. Since the phosphorylation of LRRK2 is a potential regulation mechanism of LRRK2, then its dephosphorylation might also a play a role. Indeed, several pathogenic mutants in LRRK2 have been shown to have a reduced phosphorylation level at the above ANK-LRR inter-domain phosphosites [82]. Moreover, inhibition of LRRK2 also resulted in a reduction of phosphorylation at these sites, thus lending support to the notion that the phosphorylation and dephosphorylation of LRRK2 are potentially a regulatory mechanism of LRRK2, as thoroughly reviewed by Jean-Marc Taymans [83]. Two potential LRRK2 phosphatases have been identiﬁed 9 of 15 Int. J. Mol. Sci. 2018, 19, 4074 in recent years. In 2013, Evy Lobbestael and colleagues showed that the protein phosphatase 1 (PP1) can effectively dephosphorylate LRRK2 at S910, S935, S955, and S973 [82]. More recently, in 2017, Panagiotis Athanasopoulos and colleagues identiﬁed protein phosphatase 2A (PP2A) as another potential LRRK2 phosphatase [84]. This study showed that PP2A interacts with the Roc domain of LRRK2, and that knocking down the activity of PP2A aggravated cellular degeneration induced by a pathogenic LRRK2 mutant. The mechanisms of how each phosphosite affect the activity of LRRK2 is unknown, but because LRRK2 is a large mutli-domain and dual enzymatic protein, the different phosphosites might regulate the distinct characteristics. In additional to the conventional effects of phosphorylation on protein-protein interactions and conformational changes, Jing Zhao and colleagues reported recently that LRRK2 dephosphorylation could lead to ubiquitination, thus suggesting that the dephosphorylation of LRRK2 might also regulate its levels in the cell [85]. 4. The Mechanism of Roc Transmitting Its Signal Downstream For example, Rodrigo Carlessi and colleagues revealed that the Roc domain of DAPK1 mediates its homodimerization as well as its heterodimerization with other kinases, including LRRK2 and ZIPK (ZIP-kinase); however, the mechanistic signiﬁcance of the dimerization event was unclear at the time [28]. They also showed that deletion of the Roc domain resulted in a more-active DAPK1, indicating that dimerization of the Roc domain is not required for DAPK1 enzyme activity, yet dimerization might be crucial for GTPase activity which directly regulates kinase activity [28]. In contrast, a number of studies have shown that puriﬁed full-length LRRK2 forms dimers in solution in vitro and cells [68,89,90]. Saurabh Sen and colleagues observed that LRRK2 isolated from different species were only catalytically active in their dimeric form [68]. Nicholas G. James and colleagues found that the majority of cytoplasmic LRRK2 is monomeric, and that the LRRK2 Int. J. Mol. Sci. 2018, 19, 4074 10 of 15 10 of 15 oligomers, including dimers, tetramers, or higher-order oligomers, are associated with the plasma membrane or the intracellular membranous structures [49,91]. Other studies have shown that the membrane-associated LRRK2 is dimeric and is more catalytically active than the cytosolic monomeric conformation [69]. As such, it appears that guanine nucleotide-binding drives Roc conformational changes that, in turn, alter the conformation and/or oligomeric states of the Roco proteins, ultimately leading to altered protein-protein interactions and/or subcellular localization. However, the deﬁning feature that distinguishes Roc from all other G-proteins is the juxtaposition of Roc and COR. Roc always co-exists contiguously with a COR domain, suggesting that they likely function as a unit; however, how the two domains work together is still a mystery. Understanding the interplay between Roc and COR is essential for understanding the mechanism of action of this unique class of G-proteins. 5. Conclusions The Roco family of G-proteins is the most recently characterized and least understood of the Ras-like small G-protein family. It is a class of G-proteins in which the G-domain functions within a multi-domain protein and is characteristically conjoined with a COR domain. Understanding the details of the functioning of the Roco proteins will have important applications. For example, LRRK2 is currently the most promising drug target for Parkinson’s disease, and it is being intensively investigated for that purpose. Because the aberrant kinase activity of LRRK2 is associated with disease pathogenesis, most efforts in drug discovery have been focused on inhibiting its kinase domain. This has yielded a number of highly speciﬁc and potential inhibitors of LRRK2 kinase activity [92–94]. However, inhibition of LRRK2 kinase activity has led to detrimental side-effects in animal models [37,38,95,96]. Still, researchers in the ﬁeld are working to modulate LRRK2 kinase activity to achieve therapeutic doses and minimize side-effects. The unique structure comprising of the Roc-COR domains might provide an alternative venue for therapeutic development. This is an attractive strategy because, in addition to the unique structure that might offer specific drug binding sites, it is an intrinsic modulator of the kinase domain; thus, modulating the modulator might offer a tunable regulation of LRRK2 kinase activity. To achieve this, we will need to understand the biochemical and structural details of the Roc-COR domains. The bottleneck in these endeavors has been obtaining sufficient amounts of correctly folded proteins suitable for the biochemical and biophysical studies. Even so, there are ongoing efforts to achieve precisely that. Author Contributions: J.L. and Q.Q.H. wrote and revised the manuscript. Author Contributions: J.L. and Q.Q.H. wrote and revised the manuscript. Funding: J.L. acknowledges funding from the National Nature Science Foundation of China (No.315005 Q.Q.H. acknowledges funding from the NIH (1R01GM111639, R01GM115844). Conﬂicts of Interest: The authors declare no conﬂict of interest. Conﬂicts of Interest: The authors declare no conﬂict of interest. References 1. Bosgraaf, L.; Van Haastert, P.J. Roc, a Ras/GTPase domain in complex proteins. Biochim. Biophys. Acta 2003, 1643, 5–10. [CrossRef] 1. Bosgraaf, L.; Van Haastert, P.J. Roc, a Ras/GTPase domain in complex proteins. Biochim. Biophys. Acta 2003, 1643, 5–10. [CrossRef] 2. Goldberg, J.M.; Bosgraaf, L.; Van Haastert, P.J.; Smith, J.L. Identiﬁcation of four candidate cGMP targets in Dictyostelium. Proc. Natl. Acad. Sci. USA 2002, 99, 6749–6754. [CrossRef] 3. Kobe, B.; Kajava, A.V. The leucine-rich repeat as a protein recognition motif. Curr. Opin. Struct. Biol. 2001, 11, 725–732. [CrossRef] 4. Cohen, O.; Feinstein, E.; Kimchi, A. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J. 1997, 16, 998–1008. [CrossRef] 4. Cohen, O.; Feinstein, E.; Kimchi, A. DAP-kinase is a Ca2+/calmodulin-dependent, cytoskeletal-associated protein kinase, with cell death-inducing functions that depend on its catalytic activity. EMBO J. 1997, 16, 998–1008. [CrossRef] 5. Itoh, N.; Nagata, S. A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J. Biol. Chem. 1993, 268, 10932–10937. [PubMed] 5. Itoh, N.; Nagata, S. A novel protein domain required for apoptosis. Mutational analysis of human Fas antigen. J. Biol. Chem. 1993, 268, 10932–10937. [PubMed] 6. Aravind, L.; Dixit, V.M.; Koonin, E.V. Apoptotic molecular machinery: Vastly increased complexity in vertebrates revealed by genome comparisons. Science 2001, 291, 1279–1284. [CrossRef] 7. Deiss, L.P.; Feinstein, E.; Berissi, H.; Cohen, O.; Kimchi, A. Identiﬁcation of a novel serine/threonine kinase and a novel 15-kD protein as potential mediators of the gamma interferon-induced cell death. Genes Dev. 1995, 9, 15–30. [CrossRef] [PubMed] 8. Marin, I.; van Egmond, W.N.; van Haastert, P.J. The Roco protein family: A functional perspective. FASEB J. 2008, 22, 3103–3110. [CrossRef] [PubMed] 9. Paisan-Ruiz, C.; Jain, S.; Evans, E.W.; Gilks, W.P.; Simon, J.; van der Brug, M.; Lopez de Munain, A.; Aparicio, S.; Gil, A.M.; Khan, N.; et al. Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron 2004, 44, 595–600. [CrossRef] 10. Zimprich, A.; Biskup, S.; Leitner, P.; Lichtner, P.; Farrer, M.; Lincoln, S.; Kachergus, J.; Hulihan, M.; Uitti, R.J.; Calne, D.B.; et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron 2004, 44, 601–607. [CrossRef] 11. Greggio, E.; Jain, S.; Kingsbury, A.; Bandopadhyay, R.; Lewis, P.; Kaganovich, A.; van der Brug, M.P.; Beilina, A.; Blackinton, J.; Thomas, K.J.; et al. Kinase activity is required for the toxic effects of mutant LRRK2/dardarin. Neurobiol. Abbreviations Abbreviations GEFs Guanine nucleotide exchange factors GAPs GTPase-activating proteins Roc Ras of complex proteins GbpC cGMP binding protein C LRR Leucine-rich repeat COR C-terminal of Roc LRRK1/2 Leucine-rich repeat kinase 1/2 MASL1 Malignant ﬁbrous histiocytoma ampliﬁed sequence 1 (MFHAS1) DAPK1 Death-associated protein kinase 1 GRAM Glucosyltransferases Rab-like GTPase activators and Myotubularins PD Parkinson’s disease AD Alzheimer’s disease OSMD Osteosclerotic metaphyseal dysplasia CMA Chaperone-mediated autophagy 11 of 15 Int. J. Mol. Sci. 2018, 19, 4074 GADs G-proteins activated by nucleotide-dependent dimerization CK1 Casein kinase 1 alpha PP1 Protein phosphatase 1 PP2A Protein phosphatase 2A References Dis. 2006, 23, 329–341. [CrossRef] [PubMed] 12. Liao, J.; Wu, C.X.; Burlak, C.; Zhang, S.; Sahm, H.; Wang, M.; Zhang, Z.Y.; Vogel, K.W.; Federici, M.; Riddle, S.M.; et al. Parkinson disease-associated mutation R1441H in LRRK2 prolongs the “active state” of its GTPase domain. Proc. Natl. Acad. Sci. USA 2014, 111, 4055–4060. [CrossRef] [PubMed] 13. Takai, Y.; Sasaki, T.; Matozaki, T. Small GTP-binding proteins. Physiol. Rev. 2001, 81, 153–208. [CrossRef] [PubMed] 14. Lewis, P.A. The function of ROCO proteins in health and disease. Biol. Cell 2009, 101, 183–191. [CrossRef] 15. Bosgraaf, L.; Van Haastert, P.J. A model for cGMP signal transduction in Dictyostelium in perspective of 14. Lewis, P.A. The function of ROCO proteins in health and disease. Biol. Cell 2009, 101, 183 191. [CrossRef] 15. Bosgraaf, L.; Van Haastert, P.J. A model for cGMP signal transduction in Dictyostelium in perspective of 25 f GMP h J M l R C ll M til 2002 23 781 791 [C R f] [P bM d] 15. Bosgraaf, L.; Van Haastert, P.J. A model for cGMP signal transduction in Dictyostelium in perspective of 25 years of cGMP research. J. Muscle Res. Cell Motil. 2002, 23, 781–791. [CrossRef] [PubMed] 16. Bosgraaf, L.; Russcher, H.; Smith, J.L.; Wessels, D.; Soll, D.R.; Van Haastert, P.J. A novel cGMP signalling pathway mediating myosin phosphorylation and chemotaxis in Dictyostelium. EMBO J. 2002, 21, 4560–4570. [CrossRef] 17. Van Egmond, W.N.; Kortholt, A.; Plak, K.; Bosgraaf, L.; Bosgraaf, S.; Keizer-Gunnink, I.; van Haastert, P.J. Intramolecular activation mechanism of the Dictyostelium LRRK2 homolog Roco protein GbpC. J. Biol. Chem. 2008, 283, 30412–30420. [CrossRef] 18. Kortholt, A.; van Egmond, W.N.; Plak, K.; Bosgraaf, L.; Keizer-Gunnink, I.; van Haastert, P.J. Multiple regulatory mechanisms for the Dictyostelium Roco protein GbpC. J. Biol. Chem. 2012, 287, 2749–2758. [CrossRef] 19. Abysalh, J.C.; Kuchnicki, L.L.; Larochelle, D.A. The identiﬁcation of pats1, a novel gene locus required for cytokinesis in Dictyostelium discoideum. Mol. Biol. Cell 2003, 14, 14–25. [CrossRef] Int. J. Mol. Sci. 2018, 19, 4074 12 of 15 12 of 15 20. Inbal, B.; Cohen, O.; Polak-Charcon, S.; Kopolovic, J.; Vadai, E.; Eisenbach, L.; Kimchi, A. DAP kinase links the control of apoptosis to metastasis. Nature 1997, 390, 180–184. [CrossRef] 21. Greggio, E.; Lewis, P.A.; van der Brug, M.P.; Ahmad, R.; Kaganovich, A.; Ding, J.; Beilina, A.; Baker, A.K.; Cookson, M.R. References Mutations in LRRK2/dardarin associated with Parkinson disease are more toxic than equivalent mutations in the homologous kinase LRRK1. J. Neurochem. 2007, 102, 93–102. [CrossRef] [PubMed] 22. Guo, L.; Girisha, K.M.; Iida, A.; Hebbar, M.; Shukla, A.; Shah, H.; Nishimura, G.; Matsumoto, N.; Nismath, S.; Miyake, N.; et al. Identiﬁcation of a novel LRRK1 mutation in a family with osteosclerotic metaphyseal dysplasia. J. Hum. Genet. 2017, 62, 437–441. [CrossRef] [PubMed] 23. Gasser, T. Molecular pathogenesis of Parkinson disease: Insights from genetic studies. Expert Rev. Mol. Med. 2009, 11, e22. [CrossRef] [PubMed] 24. Kumari, U.; Tan, E.K. LRRK2 in Parkinson’s disease: Genetic and clinical studies from patients. FEBS J. 2009, 276, 6455–6463. [CrossRef] [PubMed] 25. Bialik, S.; Kimchi, A. The death-associated protein kinases: Structure, function, and beyond. Annu. Rev. Biochem. 2006, 75, 189–210. [CrossRef] [PubMed] 26. Raval, A.; Tanner, S.M.; Byrd, J.C.; Angerman, E.B.; Perko, J.D.; Chen, S.S.; Hackanson, B.; Grever, M.R.; Lucas, D.M.; Matkovic, J.J.; et al. Downregulation of death-associated protein kinase 1 (DAPK1) in chronic lymphocytic leukemia. Cell 2007, 129, 879–890. [CrossRef] [PubMed] 27. Henshall, D.C.; Schindler, C.K.; So, N.K.; Lan, J.Q.; Meller, R.; Simon, R.P. Death-associated protein kinase expression in human temporal lobe epilepsy. Ann. Neurol. 2004, 55, 485–494. [CrossRef] 28. Carlessi, R.; Levin-Salomon, V.; Ciprut, S.; Bialik, S.; Berissi, H.; Albeck, S.; Peleg, Y.; Kimchi, A. GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling. EMBO Rep. 2011, 12, 917–923. [CrossRef] 29. Jebelli, J.D.; Dihanich, S.; Civiero, L.; Manzoni, C.; Greggio, E.; Lewis, P.A. GTP binding and intramolecular regulation by the ROC domain of Death Associated Protein Kinase 1. Sci. Rep. 2012, 2, 695. [CrossRef] 30. Sakabe, T.; Shinomiya, T.; Mori, T.; Ariyama, Y.; Fukuda, Y.; Fujiwara, T.; Nakamura, Y.; Inazawa, J. Identiﬁcation of a novel gene, MASL1, within an amplicon at 8p23.1 detected in malignant ﬁbrous histiocytomas by comparative genomic hybridization. Cancer Res. 1999, 59, 511–515. 31. Kumkhaek, C.; Aerbajinai, W.; Liu, W.; Zhu, J.; Uchida, N.; Kurlander, R.; Hsieh, M.M.; Tisdale, J.F.; Rodgers, G.P. MASL1 induces erythroid differentiation in human erythropoietin-dependent CD34+ cells through the Raf/MEK/ERK pathway. Blood 2013, 121, 3216–3227. [CrossRef] [PubMed] 32. Xing, W.; Goodluck, H.; Zeng, C.; Mohan, S. Erratum: Role and mechanism of action of leucine-rich repeat kinase 1 in bone. Bone Res. 2017, 5, 17029. [CrossRef] [PubMed] 33. Zeng, C.; Goodluck, H.; Qin, X.; Liu, B.; Mohan, S.; Xing, W. References [CrossRef] [PubMed] 44. Parisiadou, L.; Xie, C.; Cho, H.J.; Lin, X.; Gu, X.L.; Long, C.X.; Lobbestael, E.; Baekelandt, V.; Taymans, J.M.; Sun, L.; et al. Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis. J. Neurosci. 2009, 29, 13971–13980. [CrossRef] [PubMed] 45. Civiero, L.; Cogo, S.; Biosa, A.; Greggio, E. The role of LRRK2 in cytoskeletal dynamics. Biochem. Soc Trans. 2018. [CrossRef] 46. Biskup, S.; Moore, D.J.; Celsi, F.; Higashi, S.; West, A.B.; Andrabi, S.A.; Kurkinen, K.; Yu, S.W.; Savitt, J.M.; Waldvogel, H.J.; et al. Localization of LRRK2 to membranous and vesicular structures in mammalian brain. Ann. Neurol. 2006, 60, 557–569. [CrossRef] 47. Alegre-Abarrategui, J.; Christian, H.; Luﬁno, M.M.; Mutihac, R.; Venda, L.L.; Ansorge, O.; Wade-Martins, R. LRRK2 regulates autophagic activity and localizes to speciﬁc membrane microdomains in a novel human genomic reporter cellular model. Hum. Mol. Genet. 2009, 18, 4022–4034. [CrossRef] 48. MacLeod, D.A.; Rhinn, H.; Kuwahara, T.; Zolin, A.; Di Paolo, G.; McCabe, B.D.; Marder, K.S.; Honig, L.S.; Clark, L.N.; Small, S.A.; et al. RAB7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson’s disease risk. Neuron 2013, 77, 425–439. [CrossRef] 49. Beilina, A.; Rudenko, I.N.; Kaganovich, A.; Civiero, L.; Chau, H.; Kalia, S.K.; Kalia, L.V.; Lobbestael, E.; Chia, R.; Ndukwe, K.; et al. Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. Proc. Natl. Acad. Sci. USA 2014, 111, 2626–2631. [CrossRef] 50. Purlyte, E.; Dhekne, H.S.; Sarhan, A.R.; Gomez, R.; Lis, P.; Wightman, M.; Martinez, T.N.; Tonelli, F.; Pfeffer, S.R.; Alessi, D.R. Rab29 activation of the Parkinson’s disease-associated LRRK2 kinase. EMBO J. 2018, 37, 1–18. [CrossRef] 51. Steger, M.; Tonelli, F.; Ito, G.; Davies, P.; Trost, M.; Vetter, M.; Wachter, S.; Lorentzen, E.; Duddy, G.; Wilson, S.; et al. Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. eLife 2016, 5, e12813. [CrossRef] 52. Steger, M.; Diez, F.; Dhekne, H.S.; Lis, P.; Nirujogi, R.S.; Karayel, O.; Tonelli, F.; Martinez, T.N.; Lorentzen, E.; Pfeffer, S.R.; et al. Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis. eLife 2017, 6, e31012. [CrossRef] [PubMed] 53. Dhekne, H.S.; Yanatori, I.; Gomez, R.C.; Tonelli, F.; Diez, F.; Schule, B.; Steger, M.; Alessi, D.R.; Pfeffer, S.R. A pathway for Parkinson’s Disease LRRK2 kinase to block primary cilia and Sonic hedgehog signaling in the brain. eLife 2018, 7, e40202. References Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation. Am. J. Physiol. Endocrinol. Metab. 2016, 311, E772–E780. [CrossRef] [PubMed] 34. Lewis, P.A.; Manzoni, C. LRRK2 and human disease: A complicated question or a question of complexes? Sci. Signal. 2012, 5, pe2. [CrossRef] [PubMed] 35. Cookson, M.R. The role of leucine-rich repeat kinase 2 (LRRK2) in Parkinson’s disease. Nat. Rev. Neurosci. 2010, 11, 791–797. [CrossRef] [PubMed] 36. Kalogeropulou, A.F.; Zhao, J.; Bolliger, M.F.; Memou, A.; Narasimha, S.; Molitor, T.P.; Wilson, W.H.; Rideout, H.J.; Nichols, R.J. P62/SQSTM1 is a novel leucine-rich repeat kinase 2 (LRRK2) substrate that enhances neuronal toxicity. Biochem. J. 2018, 475, 1271–1293. [CrossRef] [PubMed] 37. Tong, Y.; Yamaguchi, H.; Giaime, E.; Boyle, S.; Kopan, R.; Kelleher, R.J., 3rd; Shen, J. Loss of leucine-rich repeat kinase 2 causes impairment of protein degradation pathways, accumulation of alpha-synuclein, and apoptotic cell death in aged mice. Proc. Natl. Acad. Sci. USA 2010, 107, 9879–9884. [CrossRef] [PubMed] 38. Herzig, M.C.; Kolly, C.; Persohn, E.; Theil, D.; Schweizer, T.; Hafner, T.; Stemmelen, C.; Troxler, T.J.; Schmid, P.; Danner, S.; et al. LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice. Hum. Mol. Genet. 2011, 20, 4209–4223. [CrossRef] 39. Orenstein, S.J.; Kuo, S.H.; Tasset, I.; Arias, E.; Koga, H.; Fernandez-Carasa, I.; Cortes, E.; Honig, L.S.; Dauer, W.; Consiglio, A.; et al. Interplay of LRRK2 with chaperone-mediated autophagy. Nat. Neurosci. 2013, 16, 394–406. [CrossRef] [PubMed] 40. Gandhi, P.N.; Wang, X.; Zhu, X.; Chen, S.G.; Wilson-Delfosse, A.L. The Roc domain of leucine-rich repeat kinase 2 is sufﬁcient for interaction with microtubules. J. Neurosci. Res. 2008, 86, 1711–1720. [CrossRef] 13 of 15 13 of 15 Int. J. Mol. Sci. 2018, 19, 4074 41. Meixner, A.; Boldt, K.; Van Troys, M.; Askenazi, M.; Gloeckner, C.J.; Bauer, M.; Marto, J.A.; Ampe, C.; Kinkl, N.; Uefﬁng, M. A QUICK screen for Lrrk2 interaction partners—leucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics. Mol. Cell Proteom. 2011, 10, M110-001172. [CrossRef] 42. Jaleel, M.; Nichols, R.J.; Deak, M.; Campbell, D.G.; Gillardon, F.; Knebel, A.; Alessi, D.R. LRRK2 phosphorylates moesin at threonine-558: Characterization of how Parkinson’s disease mutants affect kinase activity. Biochem. J. 2007, 405, 307–317. [CrossRef] [PubMed] 43. Chan, D.; Citro, A.; Cordy, J.M.; Shen, G.C.; Wolozin, B. Rac1 protein rescues neurite retraction caused by G2019S leucine-rich repeat kinase 2 (LRRK2). J. Biol. Chem. 2011, 286, 16140–16149. References [CrossRef] [PubMed] 54. Ito, G.; Okai, T.; Fujino, G.; Takeda, K.; Ichijo, H.; Katada, T.; Iwatsubo, T. GTP binding is essential to the protein kinase activity of LRRK2, a causative gene product for familial Parkinson’s disease. Biochemistry 2007, 46, 1380–1388. [CrossRef] 55. Gotthardt, K.; Weyand, M.; Kortholt, A.; Van Haastert, P.J.; Wittinghofer, A. Structure of the Roc-COR domain tandem of C. tepidum, a prokaryotic homologue of the human LRRK2 Parkinson kinase. EMBO J. 2008, 27, 2239–2249. [CrossRef] [PubMed] 56. Dihanich, S.; Civiero, L.; Manzoni, C.; Mamais, A.; Bandopadhyay, R.; Greggio, E.; Lewis, P.A. GTP binding controls complex formation by the human ROCO protein MASL1. FEBS J. 2014, 281, 261–274. [CrossRef] controls complex formation by the human ROCO protein MASL1. FEBS J. 2014, 281, 261–274. [CrossRef] 57. Korr, D.; Toschi, L.; Donner, P.; Pohlenz, H.D.; Kreft, B.; Weiss, B. LRRK1 protein kinase activity is stimulated upon binding of GTP to its Roc domain. Cell Signal. 2006, 18, 910–920. [CrossRef] [PubMed] 57. Korr, D.; Toschi, L.; Donner, P.; Pohlenz, H.D.; Kreft, B.; Weiss, B. LRRK1 protein kinase activity is stimulated upon binding of GTP to its Roc domain. Cell Signal. 2006, 18, 910–920. [CrossRef] [PubMed] 58. Biosa, A.; Trancikova, A.; Civiero, L.; Glauser, L.; Bubacco, L.; Greggio, E.; Moore, D.J. GTPase activity regulates kinase activity and cellular phenotypes of Parkinson’s disease-associated LRRK2. Hum. Mol. Genet. 2013, 22, 1140–1156. [CrossRef] [PubMed] 59. Terheyden, S.; Ho, F.Y.; Gilsbach, B.K.; Wittinghofer, A.; Kortholt, A. Revisiting the Roco G-protein cycle. Biochem. J. 2015, 465, 139–147. [CrossRef] 14 of 15 Int. J. Mol. Sci. 2018, 19, 4074 60. Xiong, Y.; Coombes, C.E.; Kilaru, A.; Li, X.; Gitler, A.D.; Bowers, W.J.; Dawson, V.L.; Dawson, T.M.; Moore, D.J. GTPase activity plays a key role in the pathobiology of LRRK2. PLoS Genet. 2010, 6, e1000902. [CrossRef] 61. Habig, K.; Walter, M.; Poths, S.; Riess, O.; Bonin, M. RNA interference of LRRK2-microarray expression l i f P ki ’ di k l Neurogenetics 2008 9 83 94 [C R f] [P bM d] 61. Habig, K.; Walter, M.; Poths, S.; Riess, O.; Bonin, M. RNA interference of LRRK2-microarray expression analysis of a Parkinson’s disease key player. Neurogenetics 2008, 9, 83–94. [CrossRef] [PubMed] 62. Haebig, K.; Gloeckner, C.J.; Miralles, M.G.; Gillardon, F.; Schulte, C.; Riess, O.; Uefﬁng, M.; Biskup, S.; Bonin, M. ARHGEF7 (Beta-PIX) acts as guanine nucleotide exchange factor for leucine-rich repeat kinase 2. PLoS ONE 2010, 5, e13762. References [CrossRef] [PubMed] 63. Chia, R.; Haddock, S.; Beilina, A.; Rudenko, I.N.; Mamais, A.; Kaganovich, A.; Li, Y.; Kumaran, R.; Nalls, M.A.; Cookson, M.R. Phosphorylation of LRRK2 by casein kinase 1alpha regulates trans-Golgi clustering via differential interaction with ARHGEF7. Nat. Commun. 2014, 5, 5827. [CrossRef] [PubMed] 64. Stafa, K.; Trancikova, A.; Webber, P.J.; Glauser, L.; West, A.B.; Moore, D.J. GTPase activity and neuronal toxicity of Parkinson’s disease-associated LRRK2 is regulated by ArfGAP1. PLoS Genet. 2012, 8, e1002526. [CrossRef] 65. Xiong, Y.; Yuan, C.; Chen, R.; Dawson, T.M.; Dawson, V.L. ArfGAP1 is a GTPase activating protein for LRRK2: Reciprocal regulation of ArfGAP1 by LRRK2. J. Neurosci. 2012, 32, 3877–3886. [CrossRef] [PubMed] 65. Xiong, Y.; Yuan, C.; Chen, R.; Dawson, T.M.; Dawson, V.L. ArfGAP1 is a GTPase activating protein for LRRK2: Reciprocal regulation of ArfGAP1 by LRRK2. J. Neurosci. 2012, 32, 3877–3886. [CrossRef] [PubMed] 66. Dusonchet, J.; Li, H.; Guillily, M.; Liu, M.; Stafa, K.; Derada Troletti, C.; Boon, J.Y.; Saha, S.; Glauser, L.; 66. Dusonchet, J.; Li, H.; Guillily, M.; Liu, M.; Stafa, K.; Derada Troletti, C.; Boon, J.Y.; Saha, S.; Glauser, L.; Mamais, A.; et al. A Parkinson’s disease gene regulatory network identiﬁes the signaling protein RGS2 as a modulator of LRRK2 activity and neuronal toxicity. Hum. Mol. Genet. 2014, 23, 4887–4905. [CrossRef] [PubMed] 67. Gasper, R.; Meyer, S.; Gotthardt, K.; Sirajuddin, M.; Wittinghofer, A. It takes two to tango: Regulation of G proteins by dimerization. Nat. Rev. Mol. Cell Biol. 2009, 10, 423–429. [CrossRef] 68. Sen, S.; Webber, P.J.; West, A.B. Dependence of leucine-rich repeat kinase 2 (LRRK2) kinase activity on dimerization. J. Biol. Chem. 2009, 284, 36346–36356. [CrossRef] [PubMed] 69. Berger, Z.; Smith, K.A.; Lavoie, M.J. Membrane localization of LRRK2 is associated with increased formation of the highly active LRRK2 dimer and changes in its phosphorylation. Biochemistry 2010, 49, 5511–5523. [CrossRef] 70. Civiero, L.; Russo, I.; Bubacco, L.; Greggio, E. Molecular Insights and Functional Implication of LRRK2 Dimerization. Adv. Neurobiol. 2017, 14, 107–121. 71. Greggio, E.; Cookson, M.R. Leucine-rich repeat kinase 2 mutations and Parkinson’s disease: Three questions. ASN Neuro 2009, 1, AN20090007. [CrossRef] [PubMed] 72. Sheng, Z.; Zhang, S.; Bustos, D.; Kleinheinz, T.; Le Pichon, C.E.; Dominguez, S.L.; Solanoy, H.O.; Drummond, J.; Zhang, X.; Ding, X.; et al. Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations. Sci. Transl. Med. 2012, 4, 164ra161. References Lobbestael, E.; Zhao, J.; Rudenko, I.N.; Beylina, A.; Gao, F.; Wetter, J.; Beullens, M.; Bollen, M.; Cookson, M.R.; Baekelandt, V.; et al. Identiﬁcation of protein phosphatase 1 as a regulator of the LRRK2 phosphorylation cycle. Biochem. J. 2013, 456, 119–128. [CrossRef] [PubMed] y 83. Taymans, J.M. Regulation of LRRK2 by Phosphatases. Adv. Neurobiol. 2017, 14, 145–160. [PubMed] 84. Athanasopoulos, P.S.; Jacob, W.; Neumann, S.; Kutsch, M.; Wolters, D.; Tan, E.K.; Bichler, Z.; Herrmann, C.; Heumann, R. Identiﬁcation of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2. Biol. Chem. 2016, 397, 541–554. [CrossRef] [PubMed] Heumann, R. Identiﬁcation of protein phosphatase 2A as an interacting protein of leucine-rich repeat kinase 2. Biol. Chem. 2016, 397, 541–554. [CrossRef] [PubMed] 85. Zhao, J.; Molitor, T.P.; Langston, J.W.; Nichols, R.J. LRRK2 dephosphorylation increases its ubiquitination. Biochem. J. 2015, 469, 107–120. [CrossRef] [PubMed] 86. Li, X.; Tan, Y.C.; Poulose, S.; Olanow, C.W.; Huang, X.Y.; Yue, Z. Leucine-rich repeat kinase 2 (LRRK2)/PARK8 possesses GTPase activity that is altered in familial Parkinson’s disease R1441C/G mutants. J. NeuroChem. 2007, 103, 238–247. [PubMed] 87. Smith, W.W.; Pei, Z.; Jiang, H.; Dawson, V.L.; Dawson, T.M.; Ross, C.A. Kinase activity of mutant LRRK2 mediates neuronal toxicity. Nat. Neurosci. 2006, 9, 1231–1233. [CrossRef] [PubMed] 88. West, A.B.; Moore, D.J.; Choi, C.; Andrabi, S.A.; Li, X.; Dikeman, D.; Biskup, S.; Zhang, Z.; Lim, K.L.; Dawson, V.L.; et al. Parkinson’s disease-associated mutations in LRRK2 link enhanced GTP-binding and kinase activities to neuronal toxicity. Hum. Mol. Genet. 2007, 16, 223–232. [CrossRef] 89. Greggio, E.; Zambrano, I.; Kaganovich, A.; Beilina, A.; Taymans, J.M.; Daniels, V.; Lewis, P.; Jain, S.; Ding, J.; Syed, A.; et al. The Parkinson disease-associated leucine-rich repeat kinase 2 (LRRK2) is a dimer that undergoes intramolecular autophosphorylation. J. Biol. Chem. 2008, 283, 16906–16914. [CrossRef] 90. Klein, C.L.; Rovelli, G.; Springer, W.; Schall, C.; Gasser, T.; Kahle, P.J. Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment. J. Neurochem. 2009, 111, 703–715. [CrossRef] 91. James, N.G.; Digman, M.A.; Gratton, E.; Barylko, B.; Ding, X.; Albanesi, J.P.; Goldberg, M.S.; Jameson, D.M. Number and brightness analysis of LRRK2 oligomerization in live cells. Biophys. J. 2012, 102, L41-3. [CrossRef] [PubMed] 92. Ding, X.; Stasi, L.P.; Ho, M.H.; Zhao, B.; Wang, H.; Long, K.; Xu, Q.; Sang, Y.; Sun, C.; Hu, H.; et al. Discovery of 4-ethoxy-7H-pyrrolo[2,3-d]pyrimidin-2-amines as potent, selective and orally bioavailable LRRK2 inhibitors. Bioorg. Med. Chem. Lett. References [CrossRef] Drummond, J.; Zhang, X.; Ding, X.; et al. Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations. Sci. Transl. Med. 2012, 4, 164ra161. [CrossRef] 73. Liu, Z.; Mobley, J.A.; DeLucas, L.J.; Kahn, R.A.; West, A.B. LRRK2 autophosphorylation enhances its GTPase activity. FASEB J. 2016, 30, 336–347. [CrossRef] [PubMed] y 73. Liu, Z.; Mobley, J.A.; DeLucas, L.J.; Kahn, R.A.; West, A.B. LRRK2 autophosphorylation enhances its GTPase activity. FASEB J. 2016, 30, 336–347. [CrossRef] [PubMed] 74. Deyaert, E.; Wauters, L.; Guaitoli, G.; Konijnenberg, A.; Leemans, M.; Terheyden, S.; Petrovic, A.; Gallardo, R.; Nederveen-Schippers, L.M.; Athanasopoulos, P.S.; et al. A homologue of the Parkinson’s disease-associated protein LRRK2 undergoes a monomer-dimer transition during GTP turnover. Nat. Commun. 2017, 8, 1008. [CrossRef] 75. Plotegher, N.; Gratton, E.; Bubacco, L. Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells. Biochim. Biophys. Acta 2014, 1840, 2014–2024. [CrossRef] [PubMed] 76. Kamikawaji, S.; Ito, G.; Iwatsubo, T. Identiﬁcation of the autophosphorylation sites of LRRK2. Biochemistry 2009, 48, 10963–10975. [CrossRef] [PubMed] 77. Greggio, E.; Taymans, J.M.; Zhen, E.Y.; Ryder, J.; Vancraenenbroeck, R.; Beilina, A.; Sun, P.; Deng, J.; Jaffe, H.; Baekelandt, V.; et al. The Parkinson’s disease kinase LRRK2 autophosphorylates its GTPase domain at multiple sites. Biochem. Biophys. Res. Commun 2009, 389, 449–454. [CrossRef] 78. Gloeckner, C.J.; Boldt, K.; von Zweydorf, F.; Helm, S.; Wiesent, L.; Sarioglu, H.; Uefﬁng, M. Phosphopeptide analysis reveals two discrete clusters of phosphorylation in the N-terminus and the Roc domain of the Parkinson-disease associated protein kinase LRRK2. J. Proteome Res. 2010, 9, 1738–1745. [CrossRef] 79. Webber, P.J.; Smith, A.D.; Sen, S.; Renfrow, M.B.; Mobley, J.A.; West, A.B. Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modiﬁes kinase and GTP-binding activities. J. Mol. Biol. 2011, 412, 94–110. [CrossRef] 15 of 15 15 of 15 Int. J. Mol. Sci. 2018, 19, 4074 80. Muda, K.; Bertinetti, D.; Gesellchen, F.; Hermann, J.S.; von Zweydorf, F.; Geerlof, A.; Jacob, A.; Uefﬁng, M.; Gloeckner, C.J.; Herberg, F.W. Parkinson-related LRRK2 mutation R1441C/G/H impairs PKA phosphorylation of LRRK2 and disrupts its interaction with 14-3-3. Proc. Natl. Acad. Sci. USA 2014, 111, E34–43. [CrossRef] 81. Lobbestael, E.; Baekelandt, V.; Taymans, J.M. Phosphorylation of LRRK2: From kinase to substrate. Biochem. Soc. Trans. 2012, 40, 1102–1110. [CrossRef] [PubMed] 82. References 2018, 28, 1615–1620. [CrossRef] [PubMed] 93. Li, W.; Zhou, Y.; Tang, G.; Wong, N.K.; Yang, M.; Tan, D.; Xiao, Y. Chemoproteomics Reveals the Antiproliferative Potential of Parkinson’s Disease Kinase Inhibitor LRRK2-IN-1 by Targeting PCNA Protein. Mol. Pharm. 2018, 15, 3252–3259. [CrossRef] [PubMed] 94. Reith, A.D.; Bamborough, P.; Jandu, K.; Andreotti, D.; Mensah, L.; Dossang, P.; Choi, H.G.; Deng, X.; Zhang, J.; Alessi, D.R.; et al. GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor. Bioorg. Med. Chem. Lett. 2012, 22, 5625–5629. [CrossRef] [PubMed] 95. Fuji, R.N.; Flagella, M.; Baca, M.; Baptista, M.A.; Brodbeck, J.; Chan, B.K.; Fiske, B.K.; Honigberg, L.; Jubb, A.M.; Katavolos, P.; et al. Effect of selective LRRK2 kinase inhibition on nonhuman primate lung. Sci. Transl. Med. 2015, 7, 273ra15. [CrossRef] [PubMed] 96. Rudenko, I.N.; Chia, R.; Cookson, M.R. Is inhibition of kinase activity the only therapeutic strategy for LRRK2-associated Parkinson’s disease? BMC Med. 2012, 10, 20. [CrossRef] [PubMed] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
https://openalex.org/W4220960517	https://seer.ufs.br/index.php/revtee/article/download/16900/12601	Portuguese	null	School daily life in times of pandemic: perceptions of education professionals in the public network of Rio de Janeiro	Revista Tempos e Espaços em Educação	2,022	cc-by	11,886	La vida cotidiana escolar en tiempos de pandemia: percepciones de profesionales de la educación de la red pública de Rio de Janeiro Renata Cristina Barreto Pinto1 , Patricia Lorena Quiterio1 1 Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brasil. Autor correspondente: Patricia Lorena Quiterio Email: patricialorenauerj@gmail.com Como citar: Pinto, R. C. B., & Quiterio, P. L. (2022). School daily life in times of pandemic: perceptions of professionals of the public education system of Rio de Janeiro. Revista Tempos e Espaços em Educação, 15(34), e16900. http://dx.doi.org/10.20952/revtee.v15i34.16900 ABSTRACT The Covid-19 pandemic brought changes and need for adaptation in the educational sector, especially in public schools. The study aimed to analyze the statements of teachers/professionals in the public network about the access by both students and education professionals to the World Wide Web and digital teaching platforms. The data was collected from March to August 2020, in the Facebook Social Network, in posts and comments from groups open and intended for education professionals from the Public Network of the State and Municipalities of Rio de Janeiro. The data were analyzed using the Iramuteq software, constituting a corpus with 1,086 text segments and a 91.07% utilization rate. The results were presented in two thematic areas (a) remote teaching with three classes: remote teaching modality, conditions of access to technology and union organization and (b) pandemic in the school context with three classes: safety measures for the return and school structure, concern with the preservation of life and school as a place of risk and propagation of the virus. Education professionals showed concern about the possibility of contamination in face of the physical conditions of school units and the exclusion of access to remote activities for most students. Keywords: Digital exclusion. Public network. Social isolation. Teaching. RESUMEN La pandemia de Covid-19 trajo consigo cambios y la necesidad de adaptación en el sector educativo, especialmente en las escuelas públicas. El estudio pretendía analizar las declaraciones de los profesores/profesionales de la red pública sobre el acceso tanto de los estudiantes como de los profesionales de la educación a la World Wide Web y a las plataformas digitales para la enseñanza. La recolección de datos se realizó en el período de marzo a agosto de 2020, en la Red Social Facebook, en posts y comentarios de grupos abiertos y destinados a profesionales de la educación de la Red Pública del Estado y Municipios de Río de Janeiro. Los datos se analizaron con el software Iramuteq, constituyendo un corpus con 1.086 segmentos de texto y un 91,07% de utilización. Los resultados fueron presentados en dos áreas temáticas (a) enseñanza a distancia con tres clases: modalidad de enseñanza a distancia, condiciones de acceso a la tecnología y organización sindical y (b) pandemia en el contexto escolar con tres clases: medidas de seguridad para el regreso y estructura escolar, preocupación con la preservación de la vida y la escuela como lugar de riesgo y propagación del virus. Los profesionales de la educación mostraron su preocupación por la posibilidad de contaminación frente a las condiciones físicas de las unidades escolares y por la exclusión del acceso a las actividades a distancia para la mayoría de los alumnos. Palabras clave: Aislamiento social. Enseñanza. Exclusión digital. Red pública. RESUMO A pandemia do Covid-19 trouxe mudanças e necessidade de adaptação no setor educacional, principalmente nas escolas públicas. O estudo teve como objetivo analisar os depoimentos dos professores/ profissionais da rede pública acerca do acesso, tanto pelos alunos, quanto pelos profissionais de educação à rede mundial de computadores e às plataformas digitais de ensino. A Rev. Tempos Espaços Educ. \| 2022 \| v.15, n. 34, e16900, \| e-ISSN 2358-1425 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro coleta de dados foi feita no período de março a agosto de 2020, na Rede Social Facebook, em postagens e comentários de grupos abertos e destinados aos profissionais de educação da Rede Pública do Estado e Municípios do Rio de Janeiro. Os dados foram analisados por meio do software Iramuteq constituindo um corpus com 1.086 segmentos de texto e aproveitamento de 91,07%. Os resultados apresentaram-se em duas áreas temáticas (a) ensino remoto com três classes: modalidade de ensino remoto, condições de acesso à tecnologia e organização sindical e (b) pandemia no contexto escolar com três classes: medidas de segurança para o retorno e estrutura escolar, preocupação com a preservação da vida e escola como lugar de risco e propagação do vírus. Os profissionais de educação mostraram-se preocupados com a possibilidade de contaminação diante as condições físicas das unidades escolares e com a exclusão de acesso às atividades remotas da maior parte dos estudantes. Palavras-chave: Ensino. Exclusão digital. Isolamento social. Rede pública. INTRODUÇÃO A pesquisa surgiu do cenário de mudanças trazidas pela pandemia de Covid-19 - doença respiratória causada pelo agente SARS-CoV-2 (síndrome respiratória aguda grave coronavírus 2) (Franco Adriano et al., 2020) e a necessidade do isolamento social no fazer pedagógico. Em fevereiro de 2020, confirmou-se o primeiro episódio no Brasil e desde então, convivemos com casos de infecção e reinfecção pelo coronavírus, e o surgimento de novas variantes do vírus (Rodrigues, 2020). Como medida de redução do contágio, todos os setores considerados não essenciais foram fechados, incluindo as escolas, até que as curvas de contágio e mortes reduzissem e se fez necessário buscar um modelo de ensino que permitisse a manutenção do distanciamento social. A opção, para dar continuidade às aulas, foi utilizar as tecnologias digitais, por meio do ensino remoto, para intermediar o aprendizado, inclusive na Educação Básica (Brasil, 2020). Assim, a pandemia proporcionou mudança na forma de transmissão da educação, principalmente com o uso de tecnologias, com uma interrupção de grande impacto na interação professor-aluno. Gomes et al. (2020) destacam que nessa nova forma de mediação do ensino, não houve mudança ou adaptação das aulas e atividades, somente a distância é o elemento novo, visto que a maior parte das escolas públicas mantiveram as aulas expositivas, com leitura dos livros didáticos e pedidos de exercícios, agora enviados pelas redes sociais. Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro Alguns pontos a observar, nesse período em que o ensino na Educação Básica ocorre unicamente na modalidade remota, são: o panorama da educação pública em sua estrutura física, logística, a formação de profissionais e a (im)possibilidade em garantir o acesso de todos alunos à educação conforme Art. 206. Inciso I da Constituição Federal “igualdade de condições para o acesso e permanência na escola” (Brasil, 1988); e, a diferença existente entre a Educação à Distância (EaD) e o ensino remoto nesse período emergencial. Em relação ao retorno às aulas presenciais, os representantes dos poderes públicos instituíram um comitê para desenvolvimento e acompanhamento de estratégias para a retomada dos serviços (Diário Oficial do Município do Rio de Janeiro, 2020). INTRODUÇÃO Neste sentido, antes da pandemia, o Tribunal de Contas do Município do Rio de Janeiro (TCMRJ) (2019) ressalta a importância de o poder público sanar aspectos relacionados à falta de professores e a estrutura física das escolas municipais. De Oliveira Brito e Siveres (2015), em um estudo in loco, destacam a importância da participação da comunidade escolar em um modelo de gestão compartilhada, incluindo a tomada de decisão. O documento elaborado pela Organização Mundial de Saúde (OMS), destaca três pontos principais para retorno das aulas presenciais: “(i) o conhecimento sobre transmissão e gravidade da Covid-19 em crianças e adolescentes, (ii) a situação epidemiologia em cada local específico, e (iii) a capacidade das escolas de manter medidas de prevenção e controle da doença” (OMS citado por Soares & Schoen, 2020, p.5). Soares e Schoen (2020) analisaram protocolos para reabertura de instituições educacionais em 13 países e identificaram medidas quanto ao alunado que pode frequentar a escola, higiene pessoal e limpeza escolar, uso de máscara e distanciamento social. O ensino remoto e as estratégias de atendimento escolar na pandemia Precisam de motivação com algo que traga significado para o aprender, além de instalações adequadas em casa para o estudo. Hodges et al. (2020) indicam a comparação da experiência de aulas on-line com o EaD e o questionamento da qualidade do que se oferece nesse período emergencial. Pode-se observar, como exemplo, o tempo disponibilizado para o planejamento das atividades e conteúdos. A mudança para ensino on-line trouxe a possibilidade de manter o aprendizado, mas exige que professores e alunos aprendam e improvisem diante das circunstâncias não ideais. Dentro desse contexto, discute-se as diferenças entre os dois ensinos. No chamado EaD são considerados diversos tipos de interação do estudante, seja com o conteúdo, com o professor ou com os demais estudantes. Existe um planejamento do aprendizado e de qual forma será possível dar suporte aos diversos tipos de interação reconhecendo o processo da aprendizagem como social e cognitivo, muito além do que transmitir informações. Para que isso ocorra, faz-se necessário pensar e desenvolver diversas formas de transformar o ambiente virtual em uma comunidade de aprendizagem, em que todos possam contribuir na construção do conhecimento (Hodges et al., 2020). Em contrapartida, o ensino remoto emergencial trata de algo temporário e alternativo devido ao contexto de saúde pública. Deve ser feito exclusivamente à distância e tão somente com a estrutura existente na moradia de cada aluno. Assim, o principal objetivo dessa modalidade de ensino é dar acesso aos conteúdos, que deveriam ser estudados, de forma rápida e o mais universal possível e, paralelamente, fazer um levantamento das necessidades e limitações de alunos e professores. Diante disso, é fácil deduzir que a qualidade será amplamente afetada, e devem ser vistas como algo temporário que precisará de soluções a médio e longo prazo para resgatar o que foi perdido nesse período. Ressalta-se o seu caráter emergencial e a impossibilidade de comparar o ensino oferecido nesse período com o EaD (Hodges et al., 2020). Segundo Cardoso et al. (2020), com o atendimento escolar exclusivo em meio digital pode- se observar que além das políticas públicas serem aquém do necessário para implementar uma educação que utilize os meios tecnológicos no ambiente escolar, também não contemplam a universalização do acesso às tecnologias fora do ambiente escolar. A pandemia mostrou que a exclusão digital se estendeu para a exclusão do ensino, levando em conta que o único meio de acesso à educação é o virtual. O ensino remoto e as estratégias de atendimento escolar na pandemia Segundo T. Cunha et al. (2020) a educação pública enfrenta problemas anteriores a pandemia, como falta de manutenção das instalações físicas, ausência de acesso à tecnologia no ambiente escolar, classes numerosas, dificuldades na formação docente e falta de profissionais. Neste sentido, o profissional da educação pública, como mediador do poder público nesse ambiente, é capaz de observar a (in)eficiência das medidas e alternativas ofertadas pelos representantes públicos em garantir a universalização do ensino. A pandemia tem revelado muitas desigualdades e, no campo educacional, as diferenças na forma de acesso à educação ultrapassam questões geográficas (área rural ou urbana; bairros seguros ou violentos), questões de gênero (homem, mulher), questões socioculturais (maior ou menor renda; ambiente e materiais adequados para estudo, seja em casa ou no ambiente escolar) (Nascimento & Santos, 2020). Tais questões sobrepõem-se entre si, interagindo e interferindo no modo e no acesso à educação (Gomes et al., 2020). Ao longo do tempo, estudiosos e profissionais em educação pontuam que as escolas precisam se atualizar usando as tecnologias digitais no processo ensino-aprendizagem (T. Cunha et al, 2020). Sabe-se, que tal empreendimento requer investimentos substanciais em equipamentos, adequação da estrutura predial e formação dos profissionais e alunos para lidarem com a tecnologia enquanto ferramenta educacional (Novello et al., 2020). Com o isolamento social a única forma de manter a continuidade do sistema educacional foi por meios tecnológicos, à distância. Com isso, as desigualdades sociais foram intensificadas. Os estudantes só poderiam utilizar os recursos que dispunham em suas residências e o capital cultural familiar. Ou seja, precisam de apoio para orientar as tarefas e estudo, acesso a livros, ter hábitos e local próprio para estudar. E, o principal em tempos de isolamento social, acesso a equipamentos eletrônicos e internet (Gomes et al., 2020; Pereira & Barros, 2020). Para Pereira e Barros (2020), a qualidade da aprendizagem nesse período emergencial depende de três fatores relacionados a professores e alunos. O primeiro está relacionado aos profissionais de ensino, sua formação para uso das tecnologias, acesso e familiaridade com as mesmas. O segundo se relaciona à exclusão dos alunos sem equipamentos e acesso à internet. O Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro terceiro, também, se relaciona aos alunos e a falta de motivação em realizar as tarefas e acompanhar o ensino. O ensino remoto e as estratégias de atendimento escolar na pandemia Sentir-se-á os impactos desse período na educação por muito tempo, e as diferenças e disparidades sociais serão agravadas trazendo uma falta de perspectiva na mobilidade social, que se refletirá no mercado de trabalho e na economia. Melo (2020) destaca que a mudança repentina para o ensino não presencial traz para o corpo docente inúmeros desafios, visto que sem formação continuada voltada para recursos tecnológicos tiveram de desenvolver aulas, apresentar conteúdos e atividades por meios digitais. Para além da carência de formação dos professores, há a questão socioeconômica dos alunos e a dificuldade dos gestores públicos em promover oportunidade de acesso a todos. As medidas são implementadas pelo governo por meio da verticalização das propostas de atendimento que visam à apresentação dos conteúdos acadêmicos e o acesso ao material didático, mas torna-se fundamental a escuta dos profissionais e o entendimento das especificidades de cada unidade escolar, com vistas a promover a equidade no acesso à educação. Ressalta ainda que nos casos em que o aluno não tem acesso as tecnologias digitais, recebem o material impresso e/ou assistem programas didáticos na televisão ou rádio e tais procedimentos não fornecem a orientação do professor, cabendo a responsabilidade aos familiares. As estratégias de atendimento remoto no estado e município do Rio de Janeiro seguiram as normativas gerais, com o uso das redes sociais, aplicativos de conversas instantâneas, materiais preparados de acesso virtual, programas educativos na televisão, canal da prefeitura no YouTube e fornecimento de material didático impresso (Brasil, 2020). Tomou-se como procedimento Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro alternativo à falta de equipamentos de alunos, somente a entrega de material impresso. Contudo, essa alternativa não possibilita atendimento para explicação, execução e correção das atividades. Com o prolongamento do afastamento escolar, plataformas de ensino a distância foram implementadas. Em março de 2021, foi disponibilizado um aplicativo com a transferência de dados patrocinada pela Prefeitura da Cidade do Rio de Janeiro (Rio Prefeitura, 2021). Corroborando com as pesquisas sobre o contexto pandêmico, L. Cunha et al. (2020) ressaltam que devemos considerar as implicações de ordem didática-pedagógica e socioeconômica e as grandes limitações das estratégias disponibilizadas em substituição às aulas presenciais. O ensino remoto e as estratégias de atendimento escolar na pandemia Em primeiro lugar, pela autonomia dos estudantes na Educação Básica que, dependendo do segmento, terão maior ou menor autossuficiência para execução das atividades e necessitarão de orientação e acompanhamento de um familiar ou outra pessoa por ele delegada. Soma-se aqui a impossibilidade de suporte, por falta de tempo e/ou falta de escolaridade necessária para tal. Seguindo para os que têm acesso às tecnologias digitais, é possível destacar a quantidade insuficiente de equipamentos para todos os estudantes do domicílio, a oscilação do sinal para acesso à internet e a obsolescência dos equipamentos. Já para os que assistem pela televisão e rádio, os programas educativos nem sempre seguem uma ordem cronológica dos conteúdos programáticos. Por último, outra limitação ocorre pela falta ou pouca interação aluno-professor, tendo em vista a maioria das atividades sendo oferecidas de maneira assíncrona1. Somado a estes fatores faz-se necessário um local minimamente apropriado para o estudo, com iluminação e silêncio. Buscou-se observar o desenvolvimento e a continuidade do trabalho dos professores e funcionários da educação pública do Estado do Rio de Janeiro neste contexto e, possíveis efeitos psicológicos, para auxiliar na recuperação dos possíveis danos que a pandemia traria para o setor educacional. Assim, este artigo teve como objetivo analisar as percepções dos profissionais de educação acerca do acesso às plataformas digitais de ensino e as alterações na dinâmica escolar decorrente do contexto pandêmico. E como objetivo específico discutir as medidas preventivas para retorno à escola. O presente estudo pretende dar voz aos profissionais de educação sobre a inclusão/exclusão digital e a infraestrutura no retorno às atividades escolares. 1 Atividades assíncronas são aquelas em que não ocorre interação imediata, ou seja, o professor envia a aula e/ou atividade para o aluno acessar em outro momento. Enquanto as atividades síncronas são aquelas em que professor e aluno interagem imediatamente, similar às aulas presenciais (Joye et al. 2020). METODOLOGIA Trata-se de uma pesquisa qualitativa, exploratória e documental. Segundo Fontelles et al. (2009), a pesquisa qualitativa propicia o entendimento de fenômenos de natureza social e cultural, mediante descrições, interpretações e comparações por parte do pesquisador e a pesquisa exploratória possibilita uma aproximação inicial com o tema e os aspectos relacionados ao fenômeno. Ainda segundo os autores, a pesquisa documental utiliza a coleta de documentos como base constituindo-se uma técnica adequada de dados qualitativos. O corpus foi coletado das declarações dos profissionais de educação, principalmente professores, da Rede Pública do Estado e Municípios do Rio de Janeiro. Profissionais de educação são todos aqueles que atuam dentro da unidade escolar e participam da dinâmica da instituição, ou seja, professores, coordenadores, diretores, merendeiras, agentes educadores e funcionários administrativos. Análise de dados Os dados foram analisados por meio do software Iramuteq® (Interface de R pour les Analyses Multidimensionnelles de Textes et de Questionnaires). Trata-se de um software gratuito que permite analisar estatisticamente corpus de textos, visualizar e comparar formas linguísticas e características das palavras utilizadas. Ou seja, acessar o conteúdo simbólico dos textos, dos discursos. Desta forma, pode-se realizar um estudo qualitativo através da análise dos discursos que compõem o corpus, buscando representações ideológicas e socioculturais manifestos no léxico (Camargo & Justo, 2013; Salviati, 2017). Primeiramente, os dados foram transcritos na íntegra de maneira ordenada em uma tabela no software aplicativo de textos Microsoft® Word, separados por meses e conteúdo de postagens com seus respectivos comentários. Ao final, contabilizou-se um total de 1.157 entradas de postagens e comentários distintos retirados das páginas e divididos entre os meses: março - 227; abril - 95; maio - 104; junho - 251; julho - 345; e, agosto - 130. A partir dessa primeira organização dos dados, levantou-se a hipótese que o foco das preocupações se modificara no tempo, conforme a situação de reabertura/funcionamento das escolas se mantinha incerta e a divulgação de notícias e pesquisas sobre as formas de contágio e protocolos de enfrentamento e contenção do contágio se propagavam. Por isso, o corpus foi dividido em seis textos de acordo com os meses do período de coleta com o intuito de identificar os comentários ao longo do tempo e as possíveis mudanças. Devido ao corpus extenso, utilizou-se somente os vocábulos que se repetiam a partir de dez vezes, e para a formação da nuvem de palavras somente as formas ativas. Vale ressaltar que o número de postagens e comentários não é equivalente ao que se configura Segmentos de Texto (STs) no programa. Segundo Camargo e Justo (2013), STs são fragmentos dimensionados pelo software em acordo com o tamanho do corpus de três linhas. Destaca-se que ao inserir um corpus no software, este deve ter um aproveitamento de 75% para ser considerado apto (Camargo & Justo, 2013). Logo após, os dados foram tratados conforme os parâmetros indicados para análise no software Iramuteq®. Os textos foram revisados ortograficamente, retirados os emoticons e as pontuações excessivas, padronizou-se as siglas e escreveu-se por extenso as abreviações. As palavras que juntas trazem uma única ideia, foram ligadas por um traço baixo, como em Ministério_Público; escola_pública; escola_particular; Rio_de_Janeiro; entre outros. Procedimentos A coleta de dados foi feita na Rede Social Facebook nos grupos destinados aos participantes dessa pesquisa. Tendo por período determinado a data de fechamento das escolas no Estado do Rio 1 Atividades assíncronas são aquelas em que não ocorre interação imediata, ou seja, o professor envia a aula e/ou atividade para o aluno acessar em outro momento. Enquanto as atividades síncronas são aquelas em que professor e aluno interagem imediatamente, similar às aulas presenciais (Joye et al. 2020). Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro de Janeiro, 18 de março de 2020 até 31 de agosto do mesmo ano, completando cinco meses e 13 dias de coleta. Por questões éticas, somente os grupos públicos foram usados para a coleta de dados: “SEPE” e “SEPE RJ – Oficial”. Os dois grupos são representativos do Sindicato Estadual dos Profissionais de Educação do Estado do Rio de Janeiro (SEPE), segundo seu estatuto, Art. 2º, Inciso I, uma de suas finalidades é “reunir e congregar os professores, funcionários administrativos, orientadores e supervisores, ativos e aposentados, enfim, os profissionais de educação das redes públicas de educação Estadual e Municipais do Estado do Rio de Janeiro” (SEPE, 2014, p. 1). Como fonte de coleta de dados utilizou-se postagens e comentários resultantes da busca nas páginas com os descritores “aula” e “pandemia”. O critério de inclusão foi postagens realizadas por profissional de educação, conforme devidamente delimitado acima. Para identificação usou-se (a) o autorreconhecimento, diretamente por sua fala, ao se colocar nesse lugar e (b) por pesquisa nas informações públicas contidas em seu perfil na referida rede social. O critério de exclusão foi postagens de comentários e publicações com menções a partidos políticos, ainda que profissional da educação. Dentre as postagens resultantes da busca, quando compartilhadas pelos sujeitos do corpus, transcreveu-se suas observações sobre o compartilhamento e os comentários feitos na publicação que atendiam aos critérios de inclusão. Neste caso, o texto compartilhado também foi considerado como corpus, a maior parte trazia notas de esclarecimento, reivindicações, cartas abertas e posicionamentos do sindicato. Análise de dados A seguir, as postagens e os comentários foram analisados no software. Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 6 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro Na análise textual, considerou-se as classes de palavras: substantivos, formas não comuns, adjetivos e verbos como formas ativas; artigos e preposições foram eliminados; e, as demais classes como formas suplementares. Dentre as alternativas de análise de dados fornecidas pelo programa, escolheu-se Classificação Hierárquica Descendente (CHD), Análise Fatorial por Correspondência (AFC) e nuvem de palavras. A análise CHD, também conhecida por método Reinert, agrupa as palavras dos textos em classes de acordo com a proximidade semântica observadas nos STs. A partir disso, apresenta um dendograma que organiza as palavras por: quantidade, mostrando as com maior frequência primeiro e em tamanho maior que as demais; e, por composição das classes, mostrando a ramificação dos sentidos identificados nos STs (Tavares, 2019). A análise AFC representa em um plano fatorial cartesiano as palavras e suas classes, conforme agrupadas na análise CHD, sendo possível visualizar a proximidade e as interações entre palavras e classes dos STs, ou seja, observar graficamente os diversos contextos e sentidos do corpus (Tavares, 2019). Por último, a análise de Nuvem de Palavras agrupa e organiza as palavras do corpus de acordo com a frequência. Quanto maior a frequência, maior o tamanho da palavra, facilitando a observação dos termos que mais aparecem no conjunto de textos (Camargo & Justo, 2013; Salviati, 2017). RESULTADOS E DISCUSSÃO O corpus geral foi constituído por seis textos representativos dos dados de cada mês analisado (março, abril, maio, junho, julho e agosto), com 1.086 STs, e aproveitamento de 989 STs, 91,07% do total, conforme Figura 1. Apresentaram-se 39.103 ocorrências de palavras, com 5.520 palavras distintas e 2.946 aparecendo uma única vez. Formaram-se seis classes de categorias divididas em dois ramos temáticos: classe 1 (em vermelho) com 128 STs, 12,94% do total; classe 2 (em cinza) com 174 STs, 17,59% do total; classe 3 (em verde claro) com 208 STs, 21,03% do total; classe 4 (em azul claro) com 128 STs, 12,94% do total; classe 5 (em azul escuro) com 198 STs, 20,02% do total; e, classe 6 (em rosa) com 153 STs, 15,47% do total. Figura 1. Dendograma da Classificação Hierárquica Descendente – CHD. Figura 1. Dendograma da Classificação Hierárquica Descendente – CHD. Observa-se na Figura 1, que os textos se dividiram em duas áreas temáticas. A primeira, Ramo 1, trouxe questões relacionadas ao ensino remoto, envolvendo três classes: Classe 3 – Modalidade de ensino remoto; Classe 4 – Condições de acesso à tecnologia; e, Classe 6 – Organização sindical. Destaca-se a conexão entre as classes 3 e 4, pois levantaram discussões sobre os tipos de modalidade de ensino remoto e a disponibilidade de recursos tecnológicos para garantir Figura 1. Dendograma da Classificação Hierárquica Descendente – CHD. Observa-se na Figura 1, que os textos se dividiram em duas áreas temáticas. A primeira, Ramo 1, trouxe questões relacionadas ao ensino remoto, envolvendo três classes: Classe 3 – Modalidade de ensino remoto; Classe 4 – Condições de acesso à tecnologia; e, Classe 6 – Organização sindical. Destaca-se a conexão entre as classes 3 e 4, pois levantaram discussões sobre os tipos de modalidade de ensino remoto e a disponibilidade de recursos tecnológicos para garantir Observa-se na Figura 1, que os textos se dividiram em duas áreas temáticas. A primeira, Ramo 1, trouxe questões relacionadas ao ensino remoto, envolvendo três classes: Classe 3 – Modalidade de ensino remoto; Classe 4 – Condições de acesso à tecnologia; e, Classe 6 – Organização sindical. RESULTADOS E DISCUSSÃO Destaca-se a conexão entre as classes 3 e 4, pois levantaram discussões sobre os tipos de modalidade de ensino remoto e a disponibilidade de recursos tecnológicos para garantir tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro a universalização da educação, e a classe 6 retratou o posicionamento político enquanto servidor público. A segunda, Ramo 2, problematizou a pandemia no contexto escolar e agrupou a Classe 1 – Medidas de segurança para o retorno e estrutura escolar; Classe 2 – Preocupação com a preservação da vida; e a Classe 5 – Escola como lugar de risco e propagação do vírus. As classes 1 e 2 relacionaram a preservação da vida e a necessidade de cumprir um protocolo de segurança nas escolas com a estrutura física apresentada nas unidades escolares, enquanto a classe 5 se preocupou com a propagação do vírus para as famílias de alunos e profissionais. Figura 2. Nuvem de palavras ativas - Classes do Ramo 1. Figura 2. Nuvem de palavras ativas Classes do Ramo 1. A classe 3 (Figura 2) teve predominância nos meses de março e abril trazendo o tema da implementação do ensino remoto emergencial e do uso das plataformas de EaD, como na fala dessa professora: “Estamos falando de política educacional durante uma crise epidemiológica. E achar que dá para substituir a aula presencial por EaD, sabendo da realidade dos nossos alunos, não é uma saída. ” (ST, 20 mar. 2020). Os professores, desde o início da pandemia, ressaltaram as diferenças entre as modalidades de ensino corroborando com os estudos publicados posteriormente sobre a educação em contexto de pandemia (T. Cunha et al., 2020; Gomes et al., 2020; Hodges et al., 2020; Novello et al., 2020). De fato, existem muitas diferenças entre a modalidade de ensino EaD e a oferecida emergencialmente. A primeira é complexa e tem uma legislação própria, possui profissionais qualificados, materiais didáticos adequados, planejamento com visão multidisciplinar A classe 3 (Figura 2) teve predominância nos meses de março e abril trazendo o tema da implementação do ensino remoto emergencial e do uso das plataformas de EaD, como na fala dessa professora: “Estamos falando de política educacional durante uma crise epidemiológica. E achar que dá para substituir a aula presencial por EaD, sabendo da realidade dos nossos alunos, não é uma saída. ” (ST, 20 mar. 2020). Os professores, desde o início da pandemia, ressaltaram as diferenças entre as modalidades de ensino corroborando com os estudos publicados posteriormente sobre a educação em contexto de pandemia (T. Cunha et al., 2020; Gomes et al., 2020; Hodges et al., 2020; Novello et al., 2020). De fato, existem muitas diferenças entre a modalidade de ensino EaD e a oferecida emergencialmente. A primeira é complexa e tem uma legislação própria, possui profissionais qualificados, materiais didáticos adequados, planejamento com visão multidisciplinar A classe 3 (Figura 2) teve predominância nos meses de março e abril trazendo o tema da implementação do ensino remoto emergencial e do uso das plataformas de EaD, como na fala dessa professora: “Estamos falando de política educacional durante uma crise epidemiológica. E achar que dá para substituir a aula presencial por EaD, sabendo da realidade dos nossos alunos, não é uma saída. ” (ST, 20 mar. 2020). Área temática 1 – ensino remoto Observa-se nessa área a busca por soluções para continuação das atividades escolares. Nesse momento, o uso das tecnologias digitais se apresentou como o único modo de promover a continuidade da oferta de ensino, e os professores questionaram a igualdade de acesso, tendo em vista a desigualdade social vivenciada, principalmente nas instituições públicas de ensino. Outro ponto levantado pelos profissionais, nos meses de março e abril, foi a impossibilidade de se transformar as aulas presenciais em EaD. Até aquele momento não se falava em ensino remoto emergencial. A confusão entre EaD e aulas emergenciais estava posta. Depois de muito questionamento e constatação das diferenças entre o oferecido emergencialmente e o EaD iniciou- se o ensino remoto emergencial. Devido à falta de formação continuada e a falta de estrutura sanitária observou-se inquietações e questionamentos dos profissionais nas classes 3 e 4, dentro da área temática do ensino remoto. Já a classe 6, teve seu foco temático na luta sindical e, embora esteja no Ramo 1, é presente em todos os meses de coleta, conforme demonstra a Figura 2. Figura 2. Nuvem de palavras ativas - Classes do Ramo 1. Figura 2. Nuvem de palavras ativas - Classes do Ramo 1. Os professores, desde o início da pandemia, ressaltaram as diferenças entre as modalidades de ensino corroborando com os estudos publicados posteriormente sobre a educação em contexto de pandemia (T. Cunha et al., 2020; Gomes et al., 2020; Hodges et al., 2020; Novello et al., 2020). De fato, existem muitas diferenças entre a modalidade de ensino EaD e a oferecida emergencialmente. A primeira é complexa e tem uma legislação própria, possui profissionais qualificados, materiais didáticos adequados, planejamento com visão multidisciplinar A classe 3 (Figura 2) teve predominância nos meses de março e abril trazendo o tema da implementação do ensino remoto emergencial e do uso das plataformas de EaD, como na fala dessa professora: “Estamos falando de política educacional durante uma crise epidemiológica. E achar que dá para substituir a aula presencial por EaD, sabendo da realidade dos nossos alunos, não é uma saída. ” (ST, 20 mar. 2020). Os professores, desde o início da pandemia, ressaltaram as diferenças entre as modalidades de ensino corroborando com os estudos publicados posteriormente sobre a educação em contexto de pandemia (T. Cunha et al., 2020; Gomes et al., 2020; Hodges et al., 2020; Novello et al., 2020). De fato, existem muitas diferenças entre a modalidade de ensino EaD e a oferecida emergencialmente. A primeira é complexa e tem uma legislação própria, possui profissionais qualificados, materiais didáticos adequados, planejamento com visão multidisciplinar Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro e, é focada no aluno como protagonista do processo ensino-aprendizagem. A segunda não possui legislação, e tem por objetivo transmitir o conteúdo do programa presencial, com pouca interação com os professores e muito menor com os colegas, sendo em sua totalidade improvisada (Joye et al., 2020). A preocupação dos profissionais se estendeu para uma possível utilização precária dos recursos tecnológicos que seriam disponibilizados. Outro questionamento referiu-se a falta de formação dos professores para utilizar as tecnologias digitais como ferramentas pedagógicas, tornando o ensino mais deficitário: “Bom, também não acredito que implementar o Ead sem a devida qualificação e treinamento dos professores será efetivo... o Ead dá certo justamente porque os profissionais são qualificados para esse tipo de curso e os materiais são feitos com cuidado e técnica ...” (ST, 20 mar. 2020). Figura 2. Nuvem de palavras ativas - Classes do Ramo 1. A primeira experiência de isolamento sanitário para o Covid-19 foi a da cidade de Wuhan, na China, epicentro da disseminação do vírus. A cidade ficou em isolamento por 76 dias, findando em sete de abril de 2020 (G1, 2020). Com base nessas informações, havia a expectativa de que as mesmas medidas e prazos de isolamento seriam implementadas pelos Estados, em especial o Estado do Rio de Janeiro. Nesse contexto, os profissionais de educação questionavam a forma de reposição das aulas no retorno presencial. E, se acaso houvesse reposição, não teria sentido um atendimento remoto nesse momento em que a preocupação primeira seria a manutenção da vida: “Nesse momento precisamos nos cuidar e cuidar dos nossos. Não é hora pra pensar em reposição ou aula virtual... eu hein.” (ST, 20 mar. 2020). A classe 4 (Figura 2) apresentou ocorrências predominantemente nos meses de março, abril e maio, e corroborando com a literatura, refletiu a preocupação dos profissionais de educação com a falta de recursos tecnológicos e a dificuldade de acesso à internet por grande parte dos alunos da rede pública de ensino (Cardoso et al., 2020; L. Cunha et al., 2020; Pereira & Barros, 2020). Esta classe também revelou a preocupação com o acesso dos professores, seja por questões materiais, seja por falta de qualificação para utilizar equipamentos, programas e plataformas de ensino. Esse dado vai ao encontro dos estudos de Melo (2021) e Novello et al. (2020). Os STs com maiores escores estavam relacionados à carência de recursos próprios e a observada nos alunos para a efetivação das aulas em meio eletrônico: “Dou aula em comunidade, vários alunos não possuem acesso à internet. Como vão fazer as aulas no Teams? E os professores que não têm computador? Esse app não tem as mesmas ferramentas de PC para uso no celular...” (ST, 13 mai. 2020). Examinando as declarações coletadas, uma das preocupações dos profissionais em educação estava no uso, exclusivamente, das tecnologias mediando o processo educativo, nesse período pandêmico. Principalmente pela dificuldade no acesso às tecnologias. Tal fato, impossibilita o acesso de todos à educação possível no momento de crise. Segundo Joye et al. (2020), nos dados da última pesquisa, em 2019, sobre Tecnologias da Informação e Comunicação (TICs), somente 14% dos domicílios das classes D e E, possuem computador. Figura 2. Nuvem de palavras ativas - Classes do Ramo 1. Neste caso, no contexto atual de isolamento, as pessoas de classes econômicas mais vulneráveis têm o acesso à internet restrito, cerca de 26% da população. Outro dado relevante da mesma pesquisa é o meio de acesso, 57% dos que acessam é pelo smartfone. Um professor trouxe o seguinte depoimento: Por mais que a ideia de EaD em um primeiro momento pareça boa, na prática se mostra um caos e um desastre por todos os motivos que conhecemos, principalmente porque fere a garantia de universalidade de ensino para todos os filhos e filhas da classe trabalhadora, que é o maior propósito da educação pública, visto que o Estado não fornece os meios e os recursos para que haja igualdade de condições para todos. (ST, 11 abr. 2020). Ao pensar em inclusão digital primeiramente se apresenta os equipamentos e o acesso à rede mundial de computadores. Contudo, a inclusão digital não é somente ter meios e saber acessá- los. Como todo processo de aprendizagem, é importante que haja apropriação, que os sujeitos aprendentes possam não só consumir, mas também produzir cultura e conhecimento no meio Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro digital. Assim, mesmo que tenham acesso e façam uso das tecnologias digitais em seu cotidiano, a inclusão digital não está garantida, porque precisam deixar o lugar de meros consumidores, recebendo as informações sem pensar sobre, ou sem uma visão crítica do que acessam (Joye et al., 2020). Porém, a maior parte das unidades escolares da rede pública não possuem estrutura e/ou equipamentos tecnológicos para uso coletivo de alunos e professores (TCMRJ, 2019). Não é possível planejar e executar aulas que incluam o uso das tecnologias de maneira crítica, seja para produzir conteúdo, compartilhar saberes ou buscar direitos. Dessa forma, pôde-se deduzir que a deficiência da rede de ensino em propiciar a inclusão digital é anterior à pandemia, e afetou, inclusive, aos alunos que possuem acesso aos meios digitais, pois não estavam aptos a usar essa forma para construir seu conhecimento (Gomes et al., 2020; Joye et al., 2020). A classe 6 (Figura 2) teve predominância nos meses de julho e agosto, embora aparecessem STs com escore alto em março. Nesse grupo de STs, o foco referiu-se ao movimento sindical organizado dos profissionais da educação para garantir seus direitos e dos alunos. Figura 2. Nuvem de palavras ativas - Classes do Ramo 1. Foram chamados, apelos para a organização e luta enquanto categoria. Foi possível observar o discurso sindical em busca da garantia dos direitos dos servidores públicos e dos usuários do serviço público, coincidindo com a ação pública do sindicato (SEPE, 2021). No mês de março, o posicionamento foi contra à implementação das atividades remotas. A preocupação estava na impossibilidade dos professores em acessar: “A partir do momento em que vão votar na ALERJ o EaD como dia letivo, logo, quem não entrar vai tomar falta, 10 consecutivas é processo administrativo, abandono e tchau! A não ser que o sindicato judicialize e consiga liminar” (ST, 28 mar. 2020). E, na impossibilidade dos alunos: “Escola do interior, metade dos alunos não terão acesso. ” (ST, 24 mar. 2020). Outro ponto nesse mês foi a abertura das escolas do município do Rio de Janeiro para fornecer alimentação. Os profissionais pediam, outras formas de atendimento alimentar, tendo em vista que abrir as escolas para alimentação, também traria a aglomeração e circulação do vírus: “A diretora do Sepe comenta a decisão da SMERJ de abrir as escolas para o almoço, no recesso do combate à pandemia. O sindicato já movimentou seu departamento jurídico para resguardar a saúde das funcionárias e professoras.” (ST, 20 mar. 2020). Os STs predominantes, em julho e agosto, mostraram as convocações e movimentações para impedir legalmente o possível retorno das atividades presenciais: “A campanha, intitulada ‘A vida é mais importante: volta às aulas na pandemia é genocídio’, está sendo realizada pelos núcleos do Sepe” (ST, 24 jul. 2020). A presente área temática expressa as preocupações compartilhadas pelos profissionais de educação com a falta de infraestrutura tecnológica no ambiente escolar, falta de formação tecnológica dos professores e, principalmente, da impossibilidade de acesso dos alunos às tecnologias digitais. Tais deficiências para acessar a internet e interagir nas atividades de ensino, não poderiam ser supridas imediatamente, pois necessitam da implementação de políticas públicas nesse setor. Os relatos dos educadores contribuem com Cardoso et al. (2020) quando ressalta que para construir um cidadão crítico incluso nas tecnologias digitais, faz-se necessário uma política pública educacional voltada para propiciar o uso pedagógico das tecnologias de maneira efetiva no ambiente escolar e social, bem como é essencial uma formação efetiva dos profissionais de educação que favoreça a apropriação das tecnologias nesse novo modelo educacional. Área temática 2 – contexto escolar A área temática do contexto escolar teve predominância nos meses de junho e julho, e abrangeu a falta de elaboração de um protocolo de segurança eficaz e a impossibilidade física e organizacional das instituições de ensino públicas em executar esse protocolo. Das três classes que compõe o ramo 2 (ver Figura 3), a Classe 1 – Medidas de segurança para o retorno e estrutura escolar – e Classe 2 – Preocupação com a preservação da vida – se mostraram próximas, como duas partes de um mesmo assunto. Enquanto, a Classe 5 – Escola como local de risco e propagação do 10 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro vírus – evidenciou a situação precária em que as unidades escolares já se encontravam antes da pandemia, seja em relação ao espaço físico, seja em relação aos recursos humanos. Fato que dificulta a execução de qualquer protocolo de segurança. Figura 3. Nuvem de palavras ativas - Classes do Ramo 2. Figura 3. Nuvem de palavras ativas - Classes do Ramo 2. Figura 3. Nuvem de palavras ativas - Classes do Ramo 2. A Classe 1 (Figura 3) evidenciou a necessidade de protocolos de segurança para o retorno das atividades presenciais. Soares e Schoen (2020) destacam que a reabertura de escolas deve considerar a situação epidemiológica de cada região e as condições de infraestrutura de cada instituição. Os profissionais de educação se mostraram preocupados com um retorno presencial quando a curva de contágio subia e a ocupação dos leitos estava alta: “Com a curva só aumentando, é descaso com a vida de alunos, profissionais da educação e responsáveis” (ST, 03 jun. 2020). Principalmente, pela imensa rede de contatos que o ambiente escolar envolve, com muitas pessoas de convívio próximo e que se movimentam pela cidade. Nesse momento os representantes dos poderes públicos anunciavam o retorno das Figura 3. Nuvem de palavras ativas - Classes do Ramo 2. Figura 3. Nuv do Ramo 2. A Classe 1 (Figura 3) evidenciou a necessidade de protocolos de segurança para o retorno das atividades presenciais. Soares e Schoen (2020) destacam que a reabertura de escolas deve considerar a situação epidemiológica de cada região e as condições de infraestrutura de cada instituição. Área temática 2 – contexto escolar \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 11 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro afastamento da criança infestada, é provável que a maioria dos alunos da turma, incluindo o professor, “pegará” piolho. A Classe 2 (Figura 3) teve como assunto principal a vida e sua preservação e conforme a imprensa noticiava o aumento do número de mortes diárias e infecções (Exame, 2020), os professores/profissionais educação não apoiavam o retorno das aulas presenciais: “Não é hora de voltar às aulas, não é questão de opinião, estamos no meio de uma grave pandemia, há risco de morte e a prioridade é preservar a VIDA!” (ST, 23 jul. 2020). Na presente classe, os profissionais de educação se apegaram às questões legais e sindicais para evitar a pressão social de retorno das aulas presenciais (Diário Oficial do Município do Rio de Janeiro, 2020). Difere ao observado na Classe 6, cujo foco foi a preservação da vida. Os profissionais de educação baseavam-se na experiência de funcionamento escolar anterior à pandemia: turmas lotadas, algumas passando de 45 alunos; falta de insumos de limpeza; preservação precária de banheiros. E ressalta-se que a escola é um ambiente frequentado não só por alunos e professores, mas também por funcionários de apoio administrativo, de serviços gerais e da família dos alunos e a comunidade local (De Oliveira Brito & Siveres, 2015). A Classe 5 (Figura 3) foi predominante no mês de julho. Nesse grupo de STs, o tema estava no contágio e na exposição ao vírus dentro do ambiente escolar, caso as aulas presenciais retornassem naquele momento. Observamos o medo do contágio e/ou de contaminar os familiares: “Pior os professores que não são do grupo de risco, porém convivem com familiares que são. E aí, como fica? Vai trabalhar, pega o vírus, leva para casa e contamina familiares com comorbidades? Absurdo sem tamanho!!!” (ST, 07 jul. 2020). De alunos também: “Sem mencionar que às crianças levam para dentro de casa o vírus transmitindo assim para pessoas do grupo de risco” (ST, 04 jul. 2020). Também, preocupação com o grande número de profissionais em grupo de risco, se fossem afastados aumentaria o déficit de professores e, os que ficassem não seriam suficientes para atender todos alunos: “vão contratar novos professores para suprir a carência desses que apresentam comorbidades? Vai haver essa contratação ou chamada de concursados relâmpago?? Área temática 2 – contexto escolar Se for, ótimo!” (ST, 07 jul. 2020). O segundo eixo temático trouxe a percepção, desse grupo de profissionais da educação, a respeito das dinâmicas, precariedade das instalações físicas e falta de profissionais nas unidades escolares. O observado condiz com recomendações do relatório das atividades do TCMRJ “R7 – Que se envide esforços para solucionar a carência de professores, bem como as questões relativas à infraestrutura das escolas.” (TCMRJ, 2019). E, com dados fornecidos pela imprensa “Segundo o estudo, cerca de 73% das escolas do ensino fundamental estão em situação precária. Em 2008, quando o levantamento começou a ser produzido, eram apenas 14%.” (G1, 2019). A educação pública no estado do Rio de Janeiro já estava carente de uma efetiva modificação. E, nesse momento de crise sanitária, seriam a base para o retorno gradual das atividades presenciais. Área temática 2 – contexto escolar Os profissionais de educação se mostraram preocupados com um retorno presencial quando a curva de contágio subia e a ocupação dos leitos estava alta: “Com a curva só aumentando, é descaso com a vida de alunos, profissionais da educação e responsáveis” (ST, 03 jun. 2020). Principalmente, pela imensa rede de contatos que o ambiente escolar envolve, com muitas pessoas de convívio próximo e que se movimentam pela cidade. Nesse momento, os representantes dos poderes públicos anunciavam o retorno das atividades presenciais (Diário Oficial do Município do Rio de Janeiro, 2020), contudo não apresentavam o planejamento estratégico de acompanhamento destes protocolos ou formas que protegessem a comunidade escolar. Muitos evidenciaram a distância entre o que se propunha de medidas e o que acontecia no ambiente escolar: Na teoria, tem tapete sanitizante, álcool em gel e uso obrigatório de máscara. Na prática, tem um monte de criança com saudade dos amigos, se abraçando, incomodada com a máscara e mexendo o tempo todo, tem compartilhamento de lanche, sem dar tempo de a professora interferir, tem empréstimo de borracha, tem falta de profissional de limpeza de banheiros a cada uso e não tem sabonete pra lavar as mãos, durante todo o tempo. (ST, 24 jun. 2020). A palavra vacina apareceu, em diversos STs desta classe, como alternativa diante da impossibilidade de se resolver tantos problemas estruturais observados nas escolas: “Precisamos de vacina antes de qualquer coisa, tudo além disso é engodo.” (ST, 24 jul. 2020); “Só acredita na segurança da ‘volta às aulas com protocolo’ quem nunca mais pisou numa escola, desde que terminou os estudos. ” (ST, 24 jul. 2020). O vocábulo “piolhar”, que apareceu com destaque médio na nuvem de palavras dessa classe, trouxe curiosidade. Fez-se uma busca nos STs e a palavra “piolho” apareceu associada a “controlar” e “covid_19”: “Volta as aulas com vacina, escola não controla piolho. Vai controlar COVID?” (ST, 24 jul. 2020). A analogia entre propagação de piolhos com a propagação do vírus no ambiente escolar também apareceu em outras discussões: “Só uma coisinha pra quem não entende xxx nenhuma sobre escolas e seu funcionamento: se um aluno pegar piolho, todos, ou quase todos, vão pegar também, ok?! Entenderam agora?” (ST, 24 jun. 2020). De acordo com estes STs, mesmo com todos os cuidados da família e protocolos de Rev. Tempos Espaços Educ. Apreciando o conjunto de dados Tal evidência concorre com estudos de análise do discurso trabalhista-sindical que indicam ser este “constituído por formações discursivas provenientes de diferentes campos: do político, do jurídico- trabalhista, do patronal, do governamental, do trabalhador, que se opõem hierarquicamente de forma instável.” (Steingenberger, 2007. p. 42). Correlacionou-se a AFC (Figura 4) com o litograma (Figura 5), usando as seis primeiras palavras mais citadas em cada classe para buscar possíveis inferências. Na Classe 6 (destacada em cor rosa), temos: sepe; rio_de_janeiro; diretor; sindicato; rede_municipal; conselho. São palavras ligadas diretamente ao sindicato enquanto instituição (sepe, sindicato), ao local de atuação (rio_de_janeiro, rede_municipal), e a gestão das unidades escolares (diretor, conselho). Nesse contexto, o gestor representa o poder público, é o agente que efetiva as medidas ordenadas, enquanto é pelo sindicato e organização de seus filiados que se torna possível ir contra as determinações percebidas como “irresponsabilidade dos governantes para com os profissionais de educação e a população” (SEPE, 2021). Na Classe 3, verde-claro: ead; distância; ensino; repor; reposição; solução. E, Classe 4, (cor azul-claro): acesso; internet; computador; celular; aluno; whatsapp. Nessas classes, evidencia-se a interdependência, para que o ensino remoto, Classe A Classe 6, nos meses de julho e agosto, embora se apresente no mesmo eixo, no gráfico AFC está bem destacada das demais classes do corpus. São STs de construção semântica diversa e isolados dos demais, tão destacados que seria possível classificá-los como um ramo independente. Tal evidência concorre com estudos de análise do discurso trabalhista-sindical que indicam ser este “constituído por formações discursivas provenientes de diferentes campos: do político, do jurídico- trabalhista, do patronal, do governamental, do trabalhador, que se opõem hierarquicamente de forma instável.” (Steingenberger, 2007. p. 42). Correlacionou-se a AFC (Figura 4) com o litograma (Figura 5), usando as seis primeiras palavras mais citadas em cada classe para buscar possíveis inferências. Na Classe 6 (destacada em cor rosa), temos: sepe; rio_de_janeiro; diretor; sindicato; rede_municipal; conselho. São palavras ligadas diretamente ao sindicato enquanto instituição (sepe, sindicato), ao local de atuação (rio_de_janeiro, rede_municipal), e a gestão das unidades escolares (diretor, conselho). Nesse contexto, o gestor representa o poder público, é o agente que efetiva as medidas ordenadas, enquanto é pelo sindicato e organização de seus filiados que se torna possível ir contra as determinações percebidas como “irresponsabilidade dos governantes para com os profissionais de educação e a população” (SEPE, 2021). Na Classe 3, verde-claro: ead; distância; ensino; repor; reposição; solução. Apreciando o conjunto de dados Após a análise das classes e seus STs, comprovou-se a percepção inicial de que os temas das falas se modificaram entre os meses de coleta. O primeiro eixo temático – Ensino remoto – é discutido nos primeiros meses de fechamento das unidades escolares, nas classes 3 e 4. Quando observamos o gráfico de coordenadas cartesianas (AFC – Figura 4) estas classes estão correlacionadas. Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 12 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro Figura 4. Análise Fatorial por Correspondência – AFC. Figura 4. Análise Fatorial por Correspondência – AFC. Figura 4. Análise Fatorial por Correspondência – AFC. A Classe 6, nos meses de julho e agosto, embora se apresente no mesmo eixo, no gráfico AFC está bem destacada das demais classes do corpus. São STs de construção semântica diversa e isolados dos demais, tão destacados que seria possível classificá-los como um ramo independente. Tal evidência concorre com estudos de análise do discurso trabalhista-sindical que indicam ser este “constituído por formações discursivas provenientes de diferentes campos: do político, do jurídico- trabalhista, do patronal, do governamental, do trabalhador, que se opõem hierarquicamente de forma instável.” (Steingenberger, 2007. p. 42). Correlacionou-se a AFC (Figura 4) com o litograma (Figura 5), usando as seis primeiras palavras mais citadas em cada classe para buscar possíveis inferências. Na Classe 6 (destacada em cor rosa), temos: sepe; rio_de_janeiro; diretor; sindicato; rede_municipal; conselho. São palavras ligadas diretamente ao sindicato enquanto instituição (sepe, sindicato), ao local de atuação (rio_de_janeiro, rede_municipal), e a gestão das unidades escolares (diretor, conselho). Nesse contexto, o gestor representa o poder público, é o agente que efetiva as medidas ordenadas, enquanto é pelo sindicato e organização de seus filiados que se torna possível ir contra as determinações percebidas como “irresponsabilidade dos governantes para com os profissionais de educação e a população” (SEPE, 2021). Na Classe 3, verde-claro: ead; distância; ensino; repor; reposição; solução. E, Classe 4, (cor azul-claro): acesso; internet; computador; celular; aluno; whatsapp. Nessas classes, evidencia-se a interdependência, para que o ensino remoto, Classe A Classe 6, nos meses de julho e agosto, embora se apresente no mesmo eixo, no gráfico AFC está bem destacada das demais classes do corpus. São STs de construção semântica diversa e isolados dos demais, tão destacados que seria possível classificá-los como um ramo independente. Apreciando o conjunto de dados E, Classe 4, (cor azul-claro): acesso; internet; computador; celular; aluno; whatsapp. Nessas classes, evidencia-se a interdependência, para que o ensino remoto, Classe Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 13 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro 3 (cor verde), se efetive e concretize faz-se necessário um meio para acessar a rede mundial de computadores (internet) e equipamentos. Assim, o aluno precisa ter acesso à internet, equipamentos (celular, computador) e programas (whatsapp). Essa correlação é vista na AFC (Figura 4), na qual as palavras das classes 3 e 4 se misturam no plano cartesiano. O segundo eixo temático – Contexto escolar – foi predominante nos meses posteriores, junho e julho, justamente no momento de pressão pública para a reabertura das escolas e retorno das aulas presenciais. Neste eixo, as três classes interagem entre si, os vocábulos estão próximos e misturados no plano cartesiano (Figura 4) mostrando uma correlação semântica na construção dos STs. Na Classe 1, (cor vermelha), destacou-se: vacina; protocolo; curva; voltar; previsão; segurança. E, na Classe 2, (cor cinza): vida; filho; voltar; pai; matar. No litograma (Figura 5), apareceram como nuances do mesmo assunto. A Classe 2 mostrou a preocupação com a preservação da vida, por isso, a necessidade de se estabelecer um protocolo de segurança nas escolas, Classe 1. Conforme destacam T. Cunha et al. (2020) para a implementação do protocolo de segurança faz-se necessário avaliar as instalações físicas, insumos e recursos humanos das unidades escolares com o intuito de proteger a comunidade escolar da contaminação – Classe 5, (cor azul escuro): risco, grupo, vírus, criança, lugar, comorbidades. Profissionais (comorbidades, grupo, risco) e alunos (crianças) iriam interagir no ambiente escolar (lugar) e poderiam espalhar o vírus em suas casas. Figura 5. Filograma da Classificação Hierárquica Descendente – CHD. Os três pontos principais destacados pela OMS para retorno das aulas presenciais (OMS citado por Soares & Schoen, 2020) foram observados também, nos dados coletados no presente estudo e apareceram como preocupação nos discursos dos profissionais. Destes, o que mais apareceu foi a capacidade das escolas em manter tais medidas, principalmente pela falta de materiais e insumos constantes, depreciação das estruturas físicas das unidades escolares, superlotação das turmas e profissionais de apoio insuficientes. Apreciando o conjunto de dados Assim, os riscos de infecção e transmissão tornam-se aumentados pela deficiência em manter a higiene e dificuldade em controlar o acesso e circulação dos alunos, por falta de insumos e recursos humanos. Para uma efetiva separação da comunidade escolar em pequenos grupos permanentes e com ausência de contatos entre si, seria necessário investimento e ampliação de recursos físicos e humanos. O contexto pandêmico em seus vieses escolar e social no Estado do Rio de Janeiro, dentro do período coletado Figura 5. Filograma da Classificação Hierárquica Descendente – CHD. Os três pontos principais destacados pela OMS para retorno das aulas presenciais (OMS citado por Soares & Schoen, 2020) foram observados também, nos dados coletados no presente estudo e apareceram como preocupação nos discursos dos profissionais. Destes, o que mais apareceu foi a capacidade das escolas em manter tais medidas, principalmente pela falta de materiais e insumos constantes, depreciação das estruturas físicas das unidades escolares, superlotação das turmas e profissionais de apoio insuficientes. Assim, os riscos de infecção e transmissão tornam-se aumentados pela deficiência em manter a higiene e dificuldade em controlar o acesso e circulação dos alunos, por falta de insumos e recursos humanos. Para uma efetiva separação da comunidade escolar em pequenos grupos permanentes e com ausência de contatos entre si, seria necessário investimento e ampliação de recursos físicos e humanos. O contexto pandêmico em seus vieses escolar e social no Estado do Rio de Janeiro, dentro do período coletado Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 14 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro (18 de março a 31 de agosto de 2020), foi percebido por meio dos STs coletados nas redes sociais abertas que a suspensão das aulas presenciais seria a medida possível de evitar a propagação do vírus e preservar mais vidas. CONSIDERAÇÕES FINAIS O objetivo deste estudo foi analisar as percepções dos profissionais de educação acerca do acesso tanto pelos alunos, quanto pelos profissionais de educação às plataformas digitais de ensino e as alterações na dinâmica escolar decorrente do contexto pandêmico, bem como discutir as medidas preventivas para retorno à escola. No momento de fechamento desse artigo, observa-se um retorno escalonado do ensino presencial após um ano de escolas fechadas, no Estado do Rio de Janeiro. Passado esse tempo, continuamos enfrentando a pandemia no ambiente escolar com políticas públicas isoladas para redução da exclusão digital, e movimentos pontuais de reestruturação das unidades escolares. Foi evidenciado nos STs analisados que os profissionais de educação executaram suas atividades sem recursos, planejamento e orientação para efetivação do atendimento remoto. Com cada unidade escolar buscando soluções para manterem o vínculo dos estudantes com a escola em um momento em que não havia uniformidade no atendimento. No momento, a instabilidade se fez presente, e os profissionais de educação manifestaram ansiedade e medo quanto ao futuro retorno das atividades presenciais: “Prioridade é a vida. Educação se recupera, se houver uma política de qualidade no futuro. Até parece que teremos um ano letivo de qualidade em se voltando às aulas. Esse prefeito está louco, desrespeitando os protocolos da saúde mundial. O Rio está em plena contaminação.” (ST, Classe 1, 23 jun. 2020). O atendimento escolar ofertado, quase exclusivamente no meio digital, segrega a maior parte dos alunos. Pode-se pensar na exclusão da exclusão, caso se leve em conta a deficiência do poder público em promover uma educação universal e de qualidade. Em meio a esses processos, o professor/profissional da educação, vivencia os problemas sociais estruturais, pois se replicam no ambiente escolar: a adesão por parte dos alunos é mínima, uma mentira, uma enganação. O esperado. Com familiares desempregados, passando necessidades, irmão menores em casa sem aula, sem acesso a uma internet boa e a computadores, sem um ambiente favorável ao estudo, sem privacidade e silêncio, dividindo cômodos com muitas pessoas, somado ao total despreparo da rede para este tipo de aula online, o resultado só poderia ser esse. Uma derrota. Uma vergonha. (ST, Classe 4, 15 abr. 2020). a adesão por parte dos alunos é mínima, uma mentira, uma enganação. O esperado. CONSIDERAÇÕES FINAIS Com familiares desempregados, passando necessidades, irmão menores em casa sem aula, sem acesso a uma internet boa e a computadores, sem um ambiente favorável ao estudo, sem privacidade e silêncio, dividindo cômodos com muitas pessoas, somado ao total despreparo da rede para este tipo de aula online, o resultado só poderia ser esse. Uma derrota. Uma vergonha. (ST, Classe 4, 15 abr. 2020). Por isso, torna-se importante refletir o efeito limitado dessa estratégia e seus impactos na ampliação da desigualdade, para então, desenvolver programas educacionais que assegurem a aprendizagem de todos (Nascimento & Santos, 2020). No corpus desse estudo, os STs expressaram a preocupação com o retorno às aulas presenciais e com a disponibilidade de formas de acesso as TICs, para professores e alunos. Evidencia-se que tal posicionamento se justificava pelas condições da infraestrutura das unidades escolares em que atuavam. Para um retorno presencial, muitos fatores interconectados devem ser observados: questões sanitárias do ambiente com adequação do espaço e continuidade de procedimentos de higienização; manutenção dos protocolos de segurança por todos envolvidos; vacinação; propagação de novas variantes do vírus e letalidade. Deste modo, faz-se importante o estabelecimento de estratégias para o retorno que atentem para alternativas construídas no coletivo, como por exemplo, um retorno progressivo. Exemplificando o exposto – parte da turma frequenta as aulas em formato presencial e outra parte da turma realiza atividades assíncronas. Provavelmente, esta estratégia pode auxiliar na adesão a participação dos alunos. Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 15 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro Outro ponto a ser observado são os resultados de pesquisas. Destaca-se a divulgação recente de uma pesquisa da Fundação Oswaldo Cruz (FIOCRUZ) em conjunto com a Universidade da Califórnia (UCLA) e a London School of Hygiene and Tropical Medicine (LSHTM) (Azevedo, 2021). Os estudos indicaram que crianças e adolescentes foram mais infectados por adultos que atuaram como transmissores no período de maio a setembro de 2020 (momento em as escolas estavam fechadas no Rio de Janeiro). Concluem dizendo que mesmo com esses resultados, se faz necessário imunizar esse grupo, além dos adultos. Agradecimentos: Não aplicável. Agradecimentos: Não aplicável. Contribuições dos Autores: Pinto, R. C. B.: concepção e desenho, aquisição de dados, análise e interpretação dos dados, redação do artigo, revisão crítica relevante do conteúdo intelectual; Quiterio, P. L.: concepção e desenho, aquisição de dados, análise e interpretação dos dados, redação do artigo, revisão crítica relevante do conteúdo intelectual. As autoras leram e aprovaram a versão final do manuscrito. CONSIDERAÇÕES FINAIS Tendo em vista que 25% da população brasileira tem menos de 18 anos e o nível de proteção para que não se perpetue a epidemia de Covid-19 é uma imunização de 85% dos indivíduos suscetíveis (Azevedo, 2021), a vacinação dos professores/profissionais de educação antes de um retorno presencial auxiliaria na contenção da propagação do vírus. O que justifica sua inclusão como grupo prioritário para a vacinação. Esta pesquisa contribuiu com elementos, indicados pelos STs de profissionais de educação, para auxiliar no planejamento da recuperação das lacunas no acesso aos recursos digitais deste período pandêmico. Incita pesquisas sobre os possíveis impactos psicológicos nos profissionais de ensino, pois poderão influenciar na gestão educacional e na prática pedagógica. Pesquisas que relatem as experiências dos profissionais se fazem necessárias para sustentar um planejamento capaz de amenizar, significativamente, os danos educacionais agravados pela pandemia de Covid- 19. E, auxiliam no estudo da eficiência das medidas e alternativas ofertadas pelos representantes públicos em garantir a continuidade do ensino de forma que todos tenham acesso. Apesar disso, apresenta algumas limitações, uma vez que para uma comprovação ou melhor verificação das inferências apresentadas, precisa-se aumentar e diversificar as fontes de coleta dos dados. Neste estudo, foi possível pesquisar em dois grupos, abertos, por questões éticas. Usar somente grupos abertos não permitiu uma universalização dos resultados. Contudo, foi possível fazer um levantamento de hipóteses sobre as percepções e sentimentos no discurso dos professores e demais profissionais de educação quanto as circunstâncias da educação pública do Estado e Municípios do Rio de Janeiro. Um discurso voltado para seus pares, aqueles com quem divide as angústias, limitações, experiências e dificuldades enfrentadas no ambiente escolar; cheio de esperança, ideias e consciência do importante papel social de uma educação pública universal e de qualidade. Contribuições dos Autores: Pinto, R. C. B.: concepção e desenho, aquisição de dados, análise e interpretação dos dados, redação do artigo, revisão crítica relevante do conteúdo intelectual; Quiterio, P. L.: concepção e desenho, aquisição de dados, análise e interpretação dos dados, redação do artigo, revisão crítica relevante do conteúdo intelectual. As autoras leram e aprovaram a versão final do manuscrito. Aprovação Ética: Não aplicável. REFERÊNCIAS Azevedo, C. (2021). Covid-19: Adultos são fonte de infecção mais comum que crianças. Agência Fiocruz de Notícias. Recuperado de: https://portal.fiocruz.br/noticia/covid-19-adultos-sao-fonte-de-infeccao-mais-comum-que-criancas Brasil. Conselho Nacional de Educação. (2020). Parecer n 5, 28 de abril de 2020. Recuperado de: http://portal.mec.gov.br/index.php?option=com_docman&view=download&alias=14511-pcp005- 20&category_slud=marco-2020-pdf&Itemid=30192 Brasil. Casa Civil. (1988). Constituição da república federativa do Brasil de 1988. Recuperado de: http://www.planalto.gov.br/ccivil_03/constituicao/constituicao.htm Camargo, B. V., & Justo, A. M. (2013). Tutorial para uso do software de análise textual IRAMUTEQ. Santa Catarina: Editora da Universidade Federal de Santa Catarina. Azevedo, C. (2021). Covid-19: Adultos são fonte de infecção mais comum que crianças. Agência Fiocruz de Notícias. Recuperado de: https://portal.fiocruz.br/noticia/covid-19-adultos-sao-fonte-de-infeccao-mais-comum-que-criancas Brasil. Conselho Nacional de Educação. (2020). Parecer n 5, 28 de abril de 2020. Recuperado de: http://portal.mec.gov.br/index.php?option=com_docman&view=download&alias=14511-pcp005- 20&category_slud=marco-2020-pdf&Itemid=30192 asil. Casa Civil. (1988). Constituição da república federativa do Brasil de 1988. Recuperado de: tp://www.planalto.gov.br/ccivil_03/constituicao/constituicao.htm Camargo, B. V., & Justo, A. M. (2013). Tutorial para uso do software de análise textual IRAMUTEQ. Santa Catarina: Editora da Universidade Federal de Santa Catarina. Camargo, B. V., & Justo, A. M. (2013). Tutorial para uso do software de análise textual IRAMUTEQ. Santa Catarina: Editora da Universidade Federal de Santa Catarina. Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 16 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro Cardoso, C. A., Ferreira, V. A., Barbosa, & F. C. G. (2020). (Des)igualdade de acesso à educação em tempos de pandemia: Uma análise do acesso às tecnologias e das alternativas de ensino remoto. Revista Com Censo: Estudos Educacionais do Distrito Federal, 7(3), 38-46. Cardoso, C. A., Ferreira, V. A., Barbosa, & F. C. G. (2020). (Des)igualdade de acesso à educação em tempos de pandemia: Uma análise do acesso às tecnologias e das alternativas de ensino remoto. Revista Com Censo: Estudos Educacionais do Distrito Federal, 7(3), 38-46. Cunha, L. F. F., Silva, A. S., & Silva, A. P. (2020). O ensino remoto no Brasil em tempos de pandemia: Diálogos acerca da qualidade e do direito e acesso à educação. Revista Com Censo: Estudos Educacionais do Distrito Federal, 7(3), 27-37. Cunha, T. C., Scriviano, I., & Vieira, E. S. (2020). Educação Básica em tempos de pandemia: Padronizada, remota, domiciliar e desigual. Revista Interinstitucional Artes de Educar, 6, 118-139. http://dx.doi.org/10.12957/riae.2020.51907 De Oliveira Brito, R., & Siveres, L. (2015). As características da participação da comunidade escolar em um modelo de gestão compartilhada. Sophia Educación, 11(1), 9-20. REFERÊNCIAS Recuperado de: https://dianet.uniroja.es/servlet/articlo?codigo=5163700 Diário Oficial do Município do Rio de Janeiro. (2020). Decreto Rio n 47.488, 02 de junho de 2020. Recuperado de: https://doweb.rio.rj.gov.br/apifront/portal/edicoes/imprimir_materia/659805/4588 Exame. (2020, julho 01). Com recorde de casos em junho, Brasil chega a 60 mil mortes por covid-19 [web log post]. Recuperado de: https://exame.com/brasil/com-recorde-de-casos-em-junho-brasil-chega-a-60-mil-mortes-por-covid- 19/ 19/ Franco Adriano, M. S. P. (2020). Síndrome respiratória aguda grave e a COVID-19 (SARS-Cov-2): Uma revisão narrativa. Enfermagem em Foco, 11(2e), 66-76. Fontelles, M. J., Simões, M. G., Farias, S. H., & Fontelles, R. G. S. (2009). Metodologia da pesquisa científica: Diretrizes para a elaboração de um protocolo de pesquisa. Revista paraense de medicina, 23(3), 1-8. G1. (2019, abril 16). Mais de 70% das escolas municipais do Rio estão em condição precária, aponta lev G1. (2019, abril 16). Mais de 70% das escolas municipais do Rio estão em condição precária, aponta levantamento do TCM [web log post] Recuperado de: https://g1 globo com/rj/rio de janeiro/noticia/2019/04/16/mais de 70percent G1. (2019, abril 16). Mais de 70% das escolas municipais do Rio estão em condição precária, aponta levantamento do TCM [web log post]. Recuperado de: https://g1.globo.com/rj/rio-de-janeiro/noticia/2019/04/16/mais-de-70percent- das-escolas-municipais-do-rio-estao-em-condicao-precaria-aponta-levantamento-do-tcm.ghtml TCM [web log post]. Recuperado de: https://g1.globo.com/rj/rio-de-janeiro/noticia/2019/04/16/mais-d TCM [web log post]. Recuperado de: https://g1.globo.com/rj/rio-de-janeiro/noticia/2019/04/16/mais-de-70percent- das-escolas-municipais-do-rio-estao-em-condicao-precaria-aponta-levantamento-do-tcm.ghtml das-escolas-municipais-do-rio-estao-em-condicao-precaria-aponta-levantamento-do-tcm.ghtml G1. (2020, abril 07). Acaba o confinamento em Wuhan, primeiro epicentro da Covid-19 [web log post]. Recuperado de: https://g1.globo.com/bemestar/coronavirus/noticia/2020/04/07/acaba-o-confinamento-em-wuhan-primeiro- epicentro-da-covid-19.ghtml Gomes, C. A., Sá, S. O., Vásquez-Justo, E., & Costa-Lobo, C. (2020). A Covid-19 e o direito à educação. Revista Internacional de Educación para la Justicia Social, 9(3e), 1-14. Hodges, C., Trust, T., Moore, S., Bond, A., & Lockee, B. (2020). The difference between emergency remote teaching and online learning. Educause Review, 2, 1-12. Joye, C., Moreira, M., & Rocha, S. (2020). Educação a distância ou atividade educacional remota emergencial: Em busca do elo perdido da educação escolar em tempos de COVID-19. Research, Society and Development, 9(7), 1-29. http://dx.doi.org/10.33448/rsd-v9i7.4299 Melo, I. V. (2020). As consequências da pandemia (COVID-19) na rede municipal de ensino: Impactos e desafios [Monografia]. Recuperado de: https://repositorio.ifgoiano.edu.br/bitstream/prefix/1377/1/Artigo%20Cient%C3%ADfico%20Corrigido%20- %20Italo%20Vaz%20de%20Melo%20-%20versao%20final%20corrigida%2014-09.pdf Nascimento, I. S., & Santos, P. C. (2020). A normalidade da desigualdade social e da exclusão educacional no Brasil. Caderno de Administração, 28, 122-130. https://doi.org/10.4025/cadadm.v28iEdição E.53834 Novello, T. P., Junior, E. F. Z. P., & Ribeiro, N. F. (2020). Ambientes virtuais de aprendizagem: Limitações digitais dos professores em época de pandemia do Covid-19. In Simpósio nacional de estratégias e multidebates da educação. SEMEDUC, Online, 481-487. Recuperado de: http://anais.uesb.br/index.php/semeduc/article/viewFile/9494/9302 Pereira, M. REFERÊNCIAS D., & Barros, E. A. (2020). A educação e a escola em tempos de Corona Vírus. Scientia Vitae Rio Prefeitura. (2021). Aplicativo de estudos da SME, o Rioeduca em Casa, está disponível para IOS e Android [web log post]. Recuperado de: http://www.rio.rj.gov.br/web/sme/exibeconteudo?id=12489394 Rio Prefeitura. (2021). Aplicativo de estudos da SME, o Rioeduca em Casa, está disponível para IOS e Android [web log post]. Recuperado de: http://www.rio.rj.gov.br/web/sme/exibeconteudo?id=12489394 Rodrigues, A. (2020). Ministério da Saúde confirma primeiro caso de coronavírus no Brasil: Ministro concede entrevista coletiva sobre o assunto. Agência Brasil. Recuperado de: Rodrigues, A. (2020). Ministério da Saúde confirma primeiro caso de coronavírus no Brasil: Ministro concede entrevista coletiva sobre o assunto. Agência Brasil. Recuperado de: Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 17 tidiano escolar em tempos de pandemia: percepções dos profissionais de educação da rede pública do Rio de Janeiro Salviati, M. E. (2017). Manual do Aplicativo Iramuteq: Compilação, organização e notas. Recuperado de: http://www.iramuteq.org/documentation/fichiers/manual-do-aplicativo-iramuteq-par-maria-elisabeth-salviati SEPE. (2021). SEPE entra com ação na justiça contra aulas presenciais na Rede Municipal RJ [web log post]. Recuperado de: https://www.seperj.org.br/sepe-entra-com-acao-na-justica-contra-aulas-presenciais-na-rede- municipal-rj/ Sindicato Estadual dos Profissionais de Educação do Rio de Janeiro (SEPE) (2014). Estatuto do Sindicato. Recuperado de: http://www.seperj.org.br/admin/fotos/estatuto/estatuto1.pdf Soares, L., & Schoen, T. H. (2020). Covid-19 preventive measures for the return to school: Documents from 13 Countries. Scielo Preprints. 1-20. https://doi.org/10.1590/SciELOPreprints.1082 Soares, L., & Schoen, T. H. (2020). Covid-19 preventive measures for the return to school: Documents from 13 Countries. Scielo Preprints. 1-20. https://doi.org/10.1590/SciELOPreprints.1082 Soares, L., & Schoen, T. H. (2020). Covid-19 preventive measures for the return to school: Documents from 13 Countries. Scielo Preprints. 1-20. https://doi.org/10.1590/SciELOPreprints.1082 Steingenberger, F. F. (2007). A subjetividade dispersa no discurso sindical da CUT. [Monografia]. Recuperado de: http://www.bibliotecadigital.uel.br/document/?view=vtls000123607 Steingenberger, F. F. (2007). A subjetividade dispersa no discurso sindical da CUT. [Monografia]. Recuperado de: http://www.bibliotecadigital.uel.br/document/?view=vtls000123607 Tavares, Í. (2019). Iramuteq: Um software para análises estatísticas qualitativas em corpus textuais. [Monografia]. Recuperado de: https://docplayer.com.br/180088996-Iramuteq-um-software-para-analises-estatisticas-qualitativas- em-corpus-textuais.html Tavares, Í. (2019). Iramuteq: Um software para análises estatísticas qualitativas em corpus textuais. [Monografia]. Recuperado de: https://docplayer.com.br/180088996-Iramuteq-um-software-para-analises-estatisticas-qualitativas- em-corpus-textuais.html Tavares, Í. (2019). Iramuteq: Um software para análises estatísticas qualitativas em corpus textuais. [Monografia]. Recuperado de: https://docplayer.com.br/180088996-Iramuteq-um-software-para-analises-estatisticas-qualitativas- em-corpus-textuais.html Tribunal de Contas do Município do Rio de Janeiro (TCMRJ) (2019). Relatório das atividades do TCMRJ: 3º trimestre 2019. Recuperado de: http://www.tcm.rj.gov.br/Noticias/14011/REL3TRI2019.pdf Tribunal de Contas do Município do Rio de Janeiro (TCMRJ) (2019). Relatório das atividades do TCMRJ: 3º trimestre 2019. Recuperado de: http://www.tcm.rj.gov.br/Noticias/14011/REL3TRI2019.pdf Tribunal de Contas do Município do Rio de Janeiro (TCMRJ) (2019). Relatório das atividades do TCMRJ: 3º trimestre 2019. Recuperado de: http://www.tcm.rj.gov.br/Noticias/14011/REL3TRI2019.pdf Recebido: 17 de dezembro de 2021 \| Aceito: 12 de fevereiro de 2022 \| Publicado: 15 de março de 2022 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. Rev. Tempos Espaços Educ. \| 2022 \| http://dx.doi.org/10.20952/revtee.v15i34.16900 18
https://openalex.org/W4206798056	https://revistaseletronicas.pucrs.br/ojs/index.php/oficinadohistoriador/article/download/40969/27251	Portuguese	null	O canto e a vida das quebradeiras de coco do Maranhão	Oficina do Historiador	2,021	cc-by	7,737	http://dx.doi.org/10.15448/2178-3748.2021.1.40969 http://dx.doi.org/10.15448/2178-3748.2021.1.40969 Recebido em: 21 mai. 2021. Aprovado em: 08 set. 2021. Publicado em: 14 dez. 2021. O canto e a vida das quebradeiras de coco do Maranhão The singing and the life of the coconut breakers of Maranhão Resumo: Este trabalho analisa o universo sociocultural das “quebradeiras” de coco babaçu do Maranhão, através de uma coletânea de cantos, para tentar entender por outro prisma alguns elementos que compõem suas visões de mundo. Para tanto, faz uma leitura histórica sobre a organização sociopolítica dos grupos de mulheres no território maranhense, nas últimas décadas. Em seguida, o texto se debruça sobre as músicas, compiladas em um livro, intitulado Canto e encanto nos babaçuais. Músicas sob domínio popular selecionadas por ‘As Encantadeiras’.... Um dos principais objetivos é buscar nos cantos as expressões de sua vida e de seu cotidiano, assim como entender a forma como elas leem o mundo e sua própria realidade. Nesse sentido, as canções, não são apenas manifestações artísticas, separadas da própria vida. Assim, faz-se uma leitura dessas composi- ções, na tentativa de revelar mais sobre o mundo dessas mulheres, alcançar o que se pode chamar de “pontos cegos”, os elementos não intencionais de suas expressões. Dessa forma, tentar compreender melhor o cotidiano, as crenças, os medos e as perspectivas de vida desse grupo social. A arte, no que tange a literatura, é mais que um meio para se distrair, pois ela pode revelar mundos ocultos e os reconstruir. OFICINA DO HISTORIADOR Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 e-ISSN: 2178-3748 Raimundo Lima dos Santos1 orcid.org/0000-0001-5242-580X raimundosantos81@gmail.com Raimundo Lima dos Santos1 orcid.org/0000-0001-5242-580X raimundosantos81@gmail.com Raimundo Lima dos Santos1 a tos orcid.org/0000-0001-5242-580X raimundosantos81@gmail.com Recebido em: 21 mai. 2021. Aprovado em: 08 set. 2021. Publicado em: 14 dez. 2021. Palavras-chave: Cantos. Quebradeiras de coco. Maranhão. Palavras-chave: Cantos. Quebradeiras de coco. Maranhão. Abstract: This work analyzes the sociocultural universe of the babassu coco- nut “quebradeiras” in Maranhão, through a collection of songs, in an attempt to understand from another perspective some elements that make up their views of the world. Therefore, it makes a historical reading of the sociopolitical orga- nization of women’s groups in Maranhão territory in recent decades. Then, the text focuses on the songs, compiled in a book, entitled “Canto e enchantment in babaçuais. Popular songs selected by ‘As Encantadeiras’...”. One of the main goals is to seek expressions of their life and daily life in the corners, as well as understanding how they read the world and their own reality. In this sense, songs are not just artistic manifestations, separate from life itself. Thus, there is a reading of these compositions, in an attempt to reveal more about the world of these women, reaching what might be called “blind spots”, the unintentional elements of their expressions. Thus, trying to better understand the daily life, beliefs, fears and life perspectives of this social group. Art, literature is more than a means of distraction, as it can reveal hidden worlds and reconstruct them. Keywords: Corners. Coconut breakers. Maranhão. Artigo está licenciado sob forma de uma licença Creative Commons Atribuição 4.0 Internacional. Universidade Estadual da Região Tocantina do Maranhão (UEMASUL), Imperatriz, MA, Brasil. 1 Universidade Estadual da Região Tocantina do Maranhão (UEMASUL), Imperatriz, MA, Brasil. 2/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 2/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 pois parte de zonas tangenciais do texto para alcançar um tipo de compreensão que não é secundária, como na psicanálise, ao buscar em “atos falhos” a compreensão de certos aspectos da personalidade. Essa maneira de entender autores e textos investiga atos involuntários para compreender percepções densas sobre de- terminados indivíduos ou grupos. “Escavando os meandros dos textos, contra as intenções de quem os produziu, podemos fazer emergir vozes incontroladas” (GINZBURG, 2007, p. 11). Introdução No século XX, a história se consolidou no diálo- go com outras disciplinas, isso provocou mudan- ças filosóficas e metodológicas. Elas se alinharam na diversidade de temas e de abordagens e trou- xeram novas questões, que não poderiam existir sem esse movimento. Talvez uma das principais mudanças na historiografia atual envolva a noção de verdade, cada vez mais plástica e menos am- biciosa, embora não menos importante. Na medida em que a história dialoga com a arte, com a música, com a física, com a literatura ou qualquer outro ramo do conhecimento, ela se volta para diferentes indagações e compreende verdades distintas das formas convencionais. Ao dialogar com a literatura, há a possibilidade de explorar novos caminhos e novas descobertas e, assim, oferecer outras perspectivas ao conhe- cimento. Em um cenário de múltiplas verdades, não há a ambição de um fato universal, apenas o anseio de uma verdade possível. Ler os testemunhos históricos contra as inten- ções dos próprios autores pode ser um exercício fecundo para alcançar outros graus de com- preensão, não necessariamente melhores, mas indispensáveis para se perceber outros pontos de vistas. Assim como as pessoas, os textos re- velam mais do que aquilo que está na superfície das intenções. Os fatos são também construções sociais e a relação das fontes com as explicações é elabo- rada dentro de amarras na visão de mundo do historiador. O que se escolhe e o que se descarta depende também da relação com o lugar social, condicionantes institucionais às quais todos es- tão, em alguma medida, sujeitos. Nesse sentido, afirma Michel De Certeau (2002, p. 66), Ao percorrer esse caminho, procura-se, neste trabalho, ver as “quebradeiras” de coco por meio de seus cantos, abarcados aqui no rol de uma produção literária. Aqui entende-se literatura no seu conceito mais amplo, da produção de texto, ficcionais ou não, ajustados pela criatividade. Por meio de versos, escritos ou cantados, é possível encontrar pistas de um grupo de pessoas, de uma comunidade ou de um indivíduo. Busca-se aqui por percepções econômicas, políticas e culturais, assim como por fatores aparentemente secundá- rios, mas que podem ser de grande importância para o entendimento desses grupos de mulheres. [...] Encarar a história como uma operação será tentar, de maneira necessariamente limitada, compreendê-la como a relação entre um lugar (profissão), procedimentos de análise (uma disciplina) e a construção de um texto (uma literatura). Raimundo Lima dos Santos1 Essa busca, no entanto, não precisa descartar os elementos explícitos e intencionais do texto, pois elas podem se complementar. Introdução É admitir que ela faz parte da “reali- dade” da qual trata, e que essa realidade pode ser apropriada “enquanto atividade humana”, “enquanto prática” [...]. A vida Para Carlo Ginzburg (1989, p. 177) todo texto possui elementos incontrolados, pois, muitas vezes, as ideias secundarizadas pelos autores são as mais reveladoras de suas intenções ocultas, enquanto as centrais são escolhidas para veicu- lar a mensagem desejada. Nesse sentido, “Se a realidade é opaca, existem zonas privilegiadas – sinais, indícios, que permitem decifrá-la”. Desde o período colonial, a palmeira do baba- çu, Orrbignya speciosa, beneficia as populações rurais do Maranhão e, no século XX, o fruto ga- nhou destaque na indústria do estado. O coco babaçu pode ser encontrado em alguns países da América, a exemplo do México, da Bolívia e Colômbia. No Brasil, há incidência nos estados de Minas Gerais, Mato Grosso, Pará, Tocantins e Piauí, mas é no Maranhão que se encontra a Esse autor busca uma “realidade às avessas”, Raimundo Lima dos Santos O canto e a vida das quebradeiras de coco do Maranhão 3/11 Raimundo Lima dos Santos O canto e a vida das quebradeiras de coco do Maranhão 3/11 tas do estado. A entidade é constituída por várias associações, sindicatos e cooperativas (BARBIERI, 2004, p. 60). Soma-se a ela, no grupo das entida- des mais importantes, a Cooperativa de Pequenos Agricultores de Lago do Junco (COOPALJ). Essa se especializou na extração do óleo de babaçu, para fins alimentícios. Assim como a MMTR, ela alcançou o mercado externo. Outras organizações de menor porte, em diferentes regiões do estado, se dedicaram a diversos subprodutos do coco babaçu, a exemplo do artesanato e da farinha de mesocarpo, extraída da casca. maior cobertura vegetal, pois cerca de 25% do seu território é coberto por florestas de babaçu (SANTOS, 2010, p. 98). As primeiras organizações femininas no Ma- ranhão começaram na região do Médio Mearim, na parte central do estado. Essa região contém a maior floresta de babaçuais do estado e por muitos anos liderou as estatísticas dos confli- tos sociais vinculados à concentração fundiária (SANTOS, 2010, p. 122). Essas tensões sempre estiveram relacionadas à falta do acesso à terra para a pequena lavoura e, depois, a impossibi- lidade de alcance ao coco babaçu, importante componente na renda e na vida de muitas famílias maranhenses. No início da década de 1990, surgiu a mais importante organização política das quebradeiras, o Movimento Interestadual das Quebradeiras de Coco Babaçu (MIQCB). Além da atuação propria- mente política, o movimento tem importante papel econômico, ao dar suporte na venda dos produtos das mulheres. A vida A entidade é compos- ta por trabalhadoras do Maranhão, Piauí, Pará e Tocantins e contou, inicialmente, com apoio financeiro da União Europeia (ALMEIDA, 2000). Na década de 1970 começou uma significativa transformação nas relações sociais e econômicas na região do Mearim. Esse divisor de águas se impôs com as restrições impostas por fazendei- ros às quebradeiras de coco aos babaçuais. De acordo com Barbosa (2008, p. 5), o capim para a pecuária tomou o lugar de florestas de babaçu, com isso, o acesso ao fruto teria se tornado mais difícil. Foi nesse momento que os grupos femini- nos começaram a se mobilizar para impor uma resistência aos fazendeiros. Na década seguinte, começaram a surgir clubes de mães, associações e outras agremiações em defesa dos babaçuais e seu aproveitamento sustentável (AYRES JÚNIOR, 2007, p. 118). Por meio dessas organizações, as quebradeiras de coco conquistaram um importante espaço político na região. Com a conquista desse lugar de fala, sua voz em defesa do ambiente, dos babaçuais, do seu aproveitamento integral e de melhores condições de vida, passou a ecoar com mais intensidade. Essa voz ressoou para todas as direções, graças ao apoio de organizações interestaduais e internacionais, bem como a adesão de políticos, além da conquista de espa- ço na mídia. Em consequência disso, boa parte da sociedade brasileira conhece os problemas dessas mulheres, suas lutas e aspirações. As mulheres criaram um grupo de estudos do babaçu com a proposição de medidas alternativas para o aproveitamento do fruto. A Associação Maranhense dos Trabalhadores Rurais (AMTR) desenvolveu a produção de sabonetes do óleo do coco. A produção, ainda que em pequena es- cala, ganhou o mercado europeu (AYRES JÚNIOR, 2007, p. 118). Desde então, além do sabonete, a entidade produz o óleo corporal e tem a parceria de dezenas de outras associações em diferentes municípios da região. As quebradeiras de coco do Maranhão, além de buscarem melhores condições de vida com o fruto, querem mostrar que os valores tradicionais comunitários, em harmonia com o ambiente, po- dem sobreviver, ao menos em parte, atualmente. Por isso, a luta desses grupos é mais ampla do que supõe um olhar apressado. É uma batalha de valores, de visões de mundo, em que elas es- peram influenciar a sociedade de alguma forma. Outra importante organização fundada no fim da década de 1980 foi a Associação em Áreas de Assentamentos do Maranhão (ASSEMA). O canto O canto, música ou verso, faz parte da vida das quebradeiras de coco, ele as acompanha logo ao nascer do dia, quando muitas vão aos cocais, antes que o incômodo do sol arrebate as terras mara- nhenses. Ao começar cedo, podem estar em casa antes do meio-dia para o almoço com a família. A depender do lugar, o cocal pode estar no “quintal” de casa, mas o comum é uma caminhada de alguns ou vários quilômetros para a coleta do fruto. Durante esse percurso aos babaçuais ou no trabalho, elas cantam as músicas ensinadas pelas mães e avós ou fazem suas próprias, também passadas às filhas e netas. Essas canções ex- pressam muitos sentimentos, medos, insatisfa- ções ou podem celebrar a alegria de ter saúde e alimento. Conforme a região e suas variantes culturais, os cantos pedem chuva, nos tempos de seca, sol em tempo de chuva, amores perdidos ou, dentre outros desejos, abrigo em outra vida para as almas que deixam esse mundo. Com músicas que traduzem suas vidas e tra- balhos como mulheres, as quebradeiras de coco utilizam o seu canto e seus encantos para expressar o valor do seu trabalho na agricultura e extrativismo do babaçu e na luta pela terra e pelo livre acesso aos babaçuais. Assim, esse livro surge do desejo das mulheres quebra- deiras de coco compartilharem suas músicas com crianças, jovens e adultos de suas co- munidades tradicionais (AS ENCANTADEIRAS, 2014, p. 5). Influenciado por seus organizadores, o livro expressa uma mensagem polissêmica com al- cance na política, no ambiente e na cultura. Além disso, chama atenção para os problemas vividos por comunidades extrativistas, suas dificuldades e suas aspirações. Contudo, quem canta os sen- timentos, as dores e as alegrias são as mulheres camponesas que vivem em busca de sonhos singelos, voltados ao sossego do lar, à família e aos amigos, em simbiose também com seu meio natural. Cada lugar tem suas próprias canções, que se somam às de outras localidades. Para reunir todos esses cantos seria necessária uma pesquisa extensa em vários municípios maranhenses e em outros estados. Para uma amostra significativa, este traba- lho se utiliza de um livro intitulado Canto e encanto nos babaçuais: músicas sob domínio popular sele- cionados por “As encantadeiras”, lançado em 2014. A obra contém 44 músicas, de diferentes regiões. A vida Essa é uma das principais organizações em defesa de trabalhadores e trabalhadoras rurais e extrativis- Para entender melhor a luta dessas mulheres do coco, é preciso conhecer quem são elas e uma 4/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 4/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 visto que os cantos, em sua maioria, são com- postos por elas. forma de fazê-lo é observar suas manifestações culturais. Elas têm o costume de cantar enquanto se destinam ao trabalho cotidiano nos cocais e, as canções, expressam seu cotidiano, suas lutas e, dentre outros aspectos, sua visão de mundo. Dessa forma, é possível entender melhor suas vidas no sentido coletivo e privado. O repertório não tem precisão geográfica, essas músicas podem ser conhecidas no Mara- nhão, Pará e Tocantins ou apenas em um desses estados. O mais importante para este trabalho é saber que elas pertencem ao universo das que- bradeiras de coco, em especial as maranhenses. O MIQCB, composto por trabalhadoras extrati- vistas de quatro estados, contribuiu para formar o grupo “As encantadeiras”, com a participação de mulheres oriundas de várias regiões. Esse coletivo feminino já se apresentou em desfile de carnaval na capital maranhense, no Palácio do Planalto, no Encontro Mundial de Comunidade do Alimento, na Itália, e em dezenas de outros eventos regionais, nacionais e até fora do País. O livro pode ser resumido da seguinte maneira: Com músicas que traduzem suas vidas e tra- balhos como mulheres, as quebradeiras de coco utilizam o seu canto e seus encantos para expressar o valor do seu trabalho na agricultura e extrativismo do babaçu e na luta pela terra e pelo livre acesso aos babaçuais. Assim, esse livro surge do desejo das mulheres quebra- deiras de coco compartilharem suas músicas com crianças, jovens e adultos de suas co- munidades tradicionais (AS ENCANTADEIRAS, 2014, p. 5). Aspectos econômicos Na parte econômica, pode-se ver como a vida dessas mulheres está estruturada em uma realidade de pobreza e de dificuldades diversas. Na falta de dinheiro, as dezenas de subprodutos do babaçu são indispensáveis para a vida nessas comunidades. “O coco é para nós grande riqueza,/ é obra da nature- za,/ ninguém vai dizer que não./ Porque da palha se faz casa pra morar,/já é um meio de ajudar a maior população”. (AS ENCANTADEIRAS, 2014, p. 8-9). Eu vim depressa, eu não vim de caminhão, eu vim a jato, neste asfalto, neste chão. Achei difícil a viagem até aqui, mas eu cheguei, mas eu cheguei. (2x) Eu vim por causa daquilo que não se vê, Vim nu, descalço, sem dinheiro e na pior. Achei difícil a viagem até aqui, mas eu cheguei, mas eu cheguei. (2x) O óleo do coco é muito utilizado no preparo de alimentos, na produção de sabão e na venda in natura. “Se faz o óleo pra temperar comida,/ é um dos meios de vida pra os fracos de condição./ Re- conhecemos o valor que o coco tem,/ a casca serve também para fazer o carvão” (AS ENCANTADEIRAS, 2014, p. 8-9). Além disso, da palha, o artesanato, do caule da palmeira, o adubo. O próprio coco pode ser vendido com as amêndoas extraídas, o que também contribui para a renda. Todos esses produtos têm a importância de substituir ou, com a venda, comprar produtos do supermercado. Eu tive ajuda de quem você não acredita, tive esperança de chegar até aqui. Vim caminhando, aqui estou. Me decidi: eu vou ficar, eu vou ficar. (2x). Eu tive ajuda de quem você não acredita, tive esperança de chegar até aqui. Vim caminhando, aqui estou. Me decidi: eu vou ficar, eu vou ficar. (2x). Muitas famílias continuaram a migração para fugir dos conflitos e conquistar um pedaço de terra para cultivar. Em muitos casos, essas constantes migrações não surtiram o efeito esperado, pois em todos os lugares havia conflitos agrários. As terras cobertas por babaçuais, lagos e rios nem sempre foram “terras prometidas”. Os conflitos com fazendeiros e grileiros impedia essa paz. “Do vale do Mearim, eu corri pro Pindaré./ Quando eu olhei pra trás,/ O bicho vinha no meu pé./ Da região do Tocantins, fui parar no Araguaia./ Quando eu chegava lá, O bicho tava na tocaia” (AS ENCANTADEIRAS, 2014, p. 24). O canto Por ter a colaboração da Assema, Capes, Mu- seu Emílio Goedi, UFPA, Universidade do Estado do Amazonas, dentre outros colaboradores, é natural que o repertório do livro tenha a necessi- dade de expressar elementos políticos e culturais de interesse dessas entidades. Mesmo assim, o livro não perde a feição das próprias mulheres, Esses cantos expressam diferentes valores, uma parte deles coletivo e outra individual, uma parte consciente, outra inconsciente. À medida que se percorre os cantos, encontra-se uma interação, um diálogo do texto com a vida, de modo a entender que os dois são inseparáveis e podem ser compreendidos um através do outro. Eu vim de longe, pra encontrar o meu caminho, tinha um sorriso e o sorriso ainda valia. Achei difícil a viagem até aqui, mas eu cheguei, mas eu cheguei. (2x) Aspectos econômicos Esse bicho metafórico, o fazendeiro, marca a vida de muitas famílias no Maranhão. Elas compreendem, ainda que algumas vezes de forma intuitiva, que a diferença de valor entre seus produtos e os do mercado se organiza em uma relação de genuína desigualdade de classe ou de grupo. Diz um canto (AS ENCANTADEIRAS, 2014, p. 20-21): “Eu sou roceiro, vivo de cavar o chão,/ Tenho as mãos calejadas, sim senhor,/ Me falta terra, falta casa, falta pão”. Quando dizem “só tenho a enxada e um título de eleitor”, é porque compreendem que essa desigualdade não é espontânea, pelo contrário, se organiza em uma relação estruturada para esse fim. Esse é um dos motivos que levam populações inteiras a se deslocar em busca de um pedaço de terra para viver. Para muitos moradores de zonas rurais, em especial do Maranhão, a falta de instrução formal e qualificação técnica fez da terra a única forma de viver. Tais conflitos são causa e efeito das difíceis condições de vida de muitas famílias desampara- das. As roças, os rios para a pesca e os babaçuais são de grande importância, para atenuar a falta de dinheiro. Boa parte da população maranhense é reflexo de migrações, de pessoas que abandonaram seus lares, em diferentes partes do Nordeste, devido à seca e por causa da fome. Vieram para cá na intenção de realizar o sonho de uma vida melhor, conforme poema a seguir (AS ENCANTADEIRAS, 2014, p. 23): Nessas condições, ainda que o babaçu tenha grande importância para as famílias nos cocais, por conta das dificuldades de acesso ao fruto, pelas distâncias e pelas restrições de donos de terra, é preciso buscar outras formas de ganho. A roça de arroz, feijão, milho, dentre outros produ- 6/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 6/11 3): “Mulher frágil era um ditado, pra menos te tornar./ Mas quem viu a revolução, sem a mulher funcionar?”. Em uma compreensão clássica, toda grande transformação começa com uma mudan- ça no pensamento, na mentalidade coletiva. A estrofe seguinte do verso afirma que as mulhe- res precisam se libertar de concepções que as aprisionam em uma percepção de inferioridade e diz que o jeito feminino de criar é necessário para mudar o mundo. tos, garante o alimento básico de muitas pessoas. Aspectos econômicos Trabalhos braçais no campo e na cidade são uma alternativa aos homens e mulheres. Mesmo que o ganho seja uma “mixaria”, as mulheres colocam a trouxa de roupas na cabeça e descem para o rio. “[...] O sol quente na cabeça, ô ô ô!/ E os pés na água fria ô ô ô!/ É a mulher lavadeira, trabalha a semana inteira,/para poder ganhar a vida [...]” (AS ENCANTADEIRAS, 2014, p. 11). Apesar das difíceis condições, quando elas ganham algum dinheiro com o coco, vem a alegria e o otimismo. O verso continua e conclama as mulheres a se expressarem, abrirem a boca, na linguagem coloquial, para equilibrar o diálogo. Ao afirmarem que “é preciso temperar”, estão reivindicando esse espaço de fala das mulheres, que antes de serem dominadas por um sistema econômico, estão subjugadas por um sistema cultural, onde o machismo aprisiona as mulheres na coadjuvação do lar. Esse rompimento só ocorrerá quando elas deixarem de esperar e fizerem sua parte, lembra o verso. Esse equilíbrio ou tempero é necessário, pois “[...] O prato da vida é insosso, sem o sal do teu falar!” (AS ENCANTADEIRAS, 2014, p. 11). Na década de 1970 essas mulheres e seus es- posos começaram a se organizar em sindicatos e associações, pois entendiam que a realidade de miséria e exclusão passava por decisões políticas. Esses conflitos, ocasionados pela resistência aos fazendeiros resultaram em mortes e persegui- ções. Com o tempo muitos líderes sindicais foram mortos, presos ou foragidos. A mulher, em casa com os filhos, recebia as intimações, as cestas básicas da Igreja e os convi- tes para participar de reuniões no sindicato. Com o tempo elas deixaram de ser apenas represen- tantes dos homens e assumiram o protagonismo de uma luta política. Com essa mudança de pro- tagonismo, a luta mudou seu formato. Ao invés da reivindicação da terra, apenas o acesso aos babaçuais, cercados pelos fazendeiros; em lugar do conflito armado, o convencimento; ao contrá- rio do fogo na roça, a preservação das florestas. É preciso lutar para que o processo de mu- dança social ocorra mais rápido. Não será fácil, a luta exige sacrifícios e perdas. “O sangue será semente”, as vidas perdidas, pensam, serão lem- bradas e darão lugar a outras na luta. A canção lembra que o povo sabe o que quer, a resistência nos conflitos agrários é resultado da consciência de uma classe explorada. Aspectos políticos É preciso combater, con- clama a canção, pois foi assim que conseguiram ampliar suas forças enquanto movimento social. A descrença se revela em todas as instâncias da política, local ou nacional. Em suas casas de taipa, elas escutam o rádio e as notícias falam de reforma agrária, uma espera longa, paleada por “Falsos projetos pra poder me tapear”. Para elas, a cidadania passa por uma vida digna, com trabalho, lazer, saúde, educação, dentre outros aspectos. Muitas famílias dos cocais passam longe disso, por isso, diz o verso, “sou brasileiro só na hora de votar” (AS ENCANTADEIRAS, 2014, p. 20-21). Uma canção (AS ENCANTADEIRAS, 2014, p. 18) fala dos efeitos de escolhas políticas. O dinheiro destinado à saúde não se converte em benefícios às populações extrativistas. “Nosso povo tá do- ente”, diz o verso, mas nada acontece no poder público, apesar dos altos impostos. Elas sabem que só a pressão social pode impedir que o povo não “morra à míngua”. É preciso combater, con- clama a canção, pois foi assim que conseguiram ampliar suas forças enquanto movimento social. O canto “Samba das quebradeiras” (AS ENCAN- TADEIRAS, 2014, p. 27) diz que nasceu um projeto e um sonho se concretizou. Certamente foi somente após as mulheres terem se organizado que sua voz, seus ideais e seus produtos ganharam um es- paço fora de seus próprios povoados. Alcançaram diferentes localidades do Brasil e até da Europa. Mesmo com o aprendizado político nas associa- ções e sindicatos sobre uma revolução, liderada pelos excluídos, a vida dessas mulheres está permeada pela realidade imediata da política local. É essa que define cotidianamente seu tra- balho, seu sustento e sua vida. Pode não haver dúvidas que uma profunda transformação social ocorrerá algum dia, mas é inevitável pensar e lutar hoje. “Mulher parada, deixa de ser tão medrosa!/ Seja um pouco corajosa, segura na minha mão./ Lutemos juntas com coragem e com amor,/ pra o governo dar valor a esta nossa profissão” (AS ENCANTADEIRAS, 2014, p. 8-9). Se elas não con- quistarem seus direitos, pensam, haverá uma perda não apenas para as quebradeiras, mas para toda a sociedade. A descrença se revela em todas as instâncias da política, local ou nacional. Em suas casas de taipa, elas escutam o rádio e as notícias falam de reforma agrária, uma espera longa, paleada por “Falsos projetos pra poder me tapear”. Aspectos políticos Contudo, outra canção (AS ENCANTADEIRAS, 2014, p. 13) lembra que além do sangue derramado, é preciso derramar o amor. A Igreja Católica teve destacada importância na organização de movi- mentos sociais no Brasil e no Maranhão. Uma das influências desse legado é o ensinamento do amor cristão, mesmo com os inimigos. É importante ter “o coração cheio de amor”, ainda que o processo de disputa por terra e o acesso aos cocais seja permeado por violência física e simbólica. Os cantos expressam perspectivas políticas em um horizonte de longo prazo, a qual anseia uma transformação mais profunda da sociedade. Além disso, tratam dos problemas mais imediatos da política local ou regional. A primeira vertente expressa os ensinamentos nas lutas sindicais e demais associações de classe, focada em uma grande transformação social, com base em princí- pios de igualdade. A segunda está mais associada a desejos e necessidades da vida cotidiana. Essa tem inspiração empírica e é de mais fácil com- preensão pela maioria das pessoas, quebradeiras de coco, lavradores e demais seguimentos afins. A luta vem do desejo de mudança, ou da inevitabilidade do conflito. “Não tenho mais para onde ir, todo lugar o bicho tá./ Não vou mais sair daqui,/ Não vou caçar outro lugar!” (AS ENCAN- TADEIRAS, 2014, p. 24). As organizações sindicais Diz um canto (AS ENCANTADEIRAS, 2014, p. Raimundo Lima dos Santos O canto e a vida das quebradeiras de coco do Maranhão 7/11 Trabalhei muito para fazer um prefeito, para ver se havia um jeito da minha vida me- lhorar. Mas o indivíduo quando está no palacete, no pobre mete o cacete, com força para matar. [...]. no Maranhão surgiram com essa inevitabilida- de. A AMTR é uma das principais entidades do estado em favor de agricultores e extrativistas, graças a ela, as quebradeiras tiveram mais força para defender os babaçuais. Uma canção (AS ENCANTADEIRAS, 2014, p. 41) é dedicada a esta organização, ao dizer como ela ajuda as mulhe- res a desenvolverem produtos para o mercado. Uma canção (AS ENCANTADEIRAS, 2014, p. 18) fala dos efeitos de escolhas políticas. O dinheiro destinado à saúde não se converte em benefícios às populações extrativistas. “Nosso povo tá do- ente”, diz o verso, mas nada acontece no poder público, apesar dos altos impostos. Elas sabem que só a pressão social pode impedir que o povo não “morra à míngua”. Aspectos políticos Um caráter mais pacífico prevaleceu, ainda que o embate direto ocorra constantemente. Uma identidade feminina se impôs no modo de fazer em todas as esferas. Na política, o mesmo ocor- rerá, novas práticas surgiram. Quando as quebradeiras de coco começaram a ter protagonismo político há algumas décadas, as lutas sociais ganharam tonalidades próprias. Um caráter mais pacífico prevaleceu, ainda que o embate direto ocorra constantemente. Uma identidade feminina se impôs no modo de fazer em todas as esferas. Na política, o mesmo ocor- rerá, novas práticas surgiram. As mulheres são seres de capacidade, diz uma estrofe, é por isso que elas são necessárias nos espaços públicos e reivindicam um lugar mere- cido “nesta pátria varonil” (AS ENCANTADEIRAS, 2014, p. 15). Não tem sido fácil protagonizar nos espaços dominados por homens, sejam eles filhos, esposos, pais, líderes de associações e de sindicatos ou políticos. Para elas, é preciso romper com a segregação feminina para se conquistar a igualdade. Na música “Viva mulher brasileira” (AS ENCANTADEIRAS, 2014, p. 19) se perguntam quem traz a vida ao mundo, quem luta a vida in- teira, quem cuida da família e quem se organiza na comunidade. Além do engajamento em associações, clubes de mães e sindicatos, as quebradeiras de coco entenderam a importância de sua mobilização na política partidária. Elas levaram a proposta de lei do “Babaçu Livre” ao Congresso Nacional. Lá não teve apoio suficiente para ser aprovada. A garantia da propriedade privada se sobrepôs à liberdade dos babaçuais para as quebradeiras. Pela proposta elas poderiam entrar nas fazendas para colher o coco. No estado do Maranhão, essa lei foi aprovada no município de Lago do Junco em 1997, cinco anos depois em Imperatriz. Ao todo, seis municípios no Maranhão aprovaram a lei, dois no Pará, dois no Tocantins e um no Piauí (ANTUNES, 2006, p. 124). Elas tiveram que enfrentar um grande obs- táculo cultural: o medo. O medo do marido, do preconceito da comunidade, o medo da violência física dos fazendeiros, o medo de fracassar. O canto “Sem medo de ser mulher” (AS ENCAN- TADEIRAS, 2014, p. 30), diz que a luta não é só dos homens, mas é preciso deixar de ter medo de ser mulher. Para a canção, se as mulheres não se libertarem desse sentimento negativo, desse senso de inferioridade, o movimento não terá a mesma força. Aspectos políticos Ainda que haja problemas na efetivação da Lei, isso mostra que essas mulheres organizadas foram mais longe do que muitos esperavam. “Essa luta não é fácil,/ Mas vai ter que acontecer!/ As mulheres organizadas,/ tem que chegar ao poder!” (AS ENCANTADEIRAS, 2014, p. 13). No aspecto político-partidário, elas procuram se aliar com vereadores, prefeitos, deputados e senadores que apoiam sua causa. No Mearim, elegeram a primeira vereadora quebradeira de coco do Maranhão e do Brasil. Antes de assumirem papel importante nos movimentos sociais no Maranhão, eram ape- nas agentes sem rosto e sem razão, conforme mostra um verso (AS ENCANTADEIRAS, 2014, p. 31): “Ninguém escuta meu grito,/ Desconhecem meu sufoco./ Escondida lá na mata,/ Com fome, quebrando o coco”. Outra estrofe lembra das infâncias perdidas nos babaçuais, “o machado é meu brinquedo/ cortando minha esperança”. Muitas quebradeiras estão presas aos donos de terra, aos compradores de coco e a uma vida de pobreza e de sofrimento. Aspectos políticos Para elas, a cidadania passa por uma vida digna, com trabalho, lazer, saúde, educação, dentre outros aspectos. Muitas famílias dos cocais passam longe disso, por isso, diz o verso, “sou brasileiro só na hora de votar” (AS ENCANTADEIRAS, 2014, p. 20-21). O mesmo canto expressa a descrença em determinados políticos. “Sou sismado com um grão de traição”, isso pode expressar uma traição a cada dois ou quatro anos. Alguns fogem dos “rostos lisos”, mas outros são “comprados por cem gramas de sorriso” (AS ENCANTADEIRAS, 2014, p. 20-21). Nesse caso, há uma fraqueza no povo, o coração bom, que perdoa e esquece as mentiras. Esse problema, pensam, contribui para perpetuar as desigualdades e as injustiças. Em termos locais, as músicas expressam a velha relação entre políticos e eleitores. Nela há um interesse momentâneo em tempos de elei- ção, após isso, a parte mais pobre da sociedade não vê os benefícios da política. As quebradeiras entram nesse grupo, pois “Só tem um título e uma machada”. Dessa forma, assim descrevem alguns políticos locais (AS ENCANTADEIRAS, 2014, p. 17): Nas organizações, as mulheres se tornam mais fortes e mais esclarecidas. Quanto mais atomizadas, menos valor e menos força política, em consequência menos mudanças. “Nós desco- brimos o valor da união,/ que é arma poderosa e derruba até dragão!/ E já sabemos que a riqueza do patrão/ E o poder dos governantes passa pela nossa mão!” (AS ENCANTADEIRAS, 2014, p. 39). Deixa esse bicho, não traz o bicho pra cá. Larga esse bicho, deixa o bicho se virá. Mata esse bicho, que o bicho quer te matar. Pelo que o bicho me disse, tá querendo te enrolar. 8/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 8/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 8/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 Tudo passa pela política, enquanto uns ganham milhões, o povo vive de tostões, diz o verso. intitulado “Oito de março” (AS ENCANTADEIRAS, 2014, p. 14) a primeira estrofe diz: “Tenho orgulho em ser mulher!/ Enfrento coco, enfrento a roça, enfrento a casa”. Tem que ser “brasa viva” para abarcar tantas responsabilidades. Quando as quebradeiras de coco começaram a ter protagonismo político há algumas décadas, as lutas sociais ganharam tonalidades próprias. Aspectos culturais Quando dizem “vamos quebrar a corrente do machismo e do poder” (AS ENCANTADEIRAS, 2014, p. 13), mostram a ampla dimensão de suas aspirações. À medida que suas organizações se fortalecem, igualmente se fortifica sua identidade, pois os conflitos que estão inseridas envolvem também uma disputa de narrativas, expressas em projetos sociais, culturais, econômicos. No canto Apesar de a quebra do coco ser um trabalho árduo, por causar dores nas costas, devido ao longo tempo sentadas; causar cortes nas mãos e dedos, às vezes amputação; o ofício em si não Raimundo Lima dos Santos O canto e a vida das quebradeiras de coco do Maranhão 9/11 Raimundo Lima dos Santos O canto e a vida das quebradeiras de coco do Maranhão 9/11 9/11 é o pior, aos olhos de muitas dessas mulheres. O que torna a situação pior são as duras condições de trabalho em um contexto de falta de acesso aos babaçuais, baixo preço da amêndoa e de outros subprodutos por causa de atravessadores. Além disso, a falta de organização dessa cadeia produtiva em favor das próprias quebradeiras. Em algumas regiões, elas conseguiram se estabele- cer em associações e cooperativas para organizar a produção, mas a maior parte das quebradeiras vive dispersa e sem grandes oportunidades. os palmeirais resulta em menos renda, menos tempero, alimento, remédios, artesanato e muitos outros subprodutos indispensáveis para vida de milhares de famílias pobres do Maranhão. Mesmo sem as terras, mesmo com as cercas e o gado, o acesso aos cocais já é uma vitória para elas. Elas têm outra concepção de posse, diferente dos fazendeiros. “Você é dono do gado, do açude e do curral,/ Mas não é dono do coco,/ nem também do coqueiral,/ Você corta boi de corte,/ mas não corte o palmeiral” (AS ENCANTA- DEIRAS, 2014, p. 21). Nessa concepção, os recur- sos naturais são de todos, não podem pertencer a alguns. Ao justificar a existência econômica, social e cultural do extrativismo do babaçu, elas pressionam donos de terras a preservar a floresta. Quando elas estão organizadas ou com essa perspectiva, sua concepção do trabalho ganha outro sentido. Na canção “Eu sou feliz quebrando coco” (AS ENCANTADEIRAS, 2014, p. 38), está expresso o sentimento positivo com a profissão. Mas alerta: “se ficar aqui parada, nada vamos con- seguir”. Nesse sentido, o ofício de quebrar coco ganha valor na esfera coletiva, protagonizada por mulheres, de acordo com sua identidade. Aspectos culturais Essas mulheres perceberam ser mais fácil con- tornar as dificuldades da vida no trabalho conjunto. Os clubes de mães ou as câmaras municipais são resultados da necessidade de uma vida melhor para elas e suas comunidades. Elas aprenderam a criar Associações e fazer parcerias com outras organizações. Compreenderam a importância do convencimento, da narrativa vitoriosa, por isso deixaram de ser meras quebradeiras anônimas para serem agentes sociais, transformadoras da sociedade. Para isso, se reinventaram, se adap- taram, aprenderam novas linguagens políticas e apreenderam novas concepções de vida. Umas das características das lutas femininas nos cocais, além de um perfil mais pacífico, é a proposta de preservação do ambiente. As que- bradeiras aceitaram a pauta do acesso à terra no lugar de sua aquisição, como reivindicam os trabalhadores rurais, isso modifica a dinâmica dos conflitos e das reivindicações. Ao colocarem o debate ambiental em pauta, surgiram mudanças no debate político. Contudo, as mulheres dos babaçuais vêm do campo, portanto, sua visão de mundo, sua cultura, seus valores, expressam essa atmosfera socio- cultural. A árdua tarefa cotidiana de quebrar o coco é vista como algo ora negativo, ora positivo, a depender da mensagem que querem passar. Mas geralmente elas encaram seu “destino” com orgulho, seguem a tradição das mães e avós e repassam as suas descendentes. Preservar as florestas implica menos capim, menos pecuária extensiva, menos fogo nos co- cais. Essas premissas levam ao fortalecimento do trabalho extrativista e sua organização sociopo- lítica. A narrativa da preservação ganhou força e trouxe consigo mudanças de valores no âmbito coletivo. Sem a participação das quebradeiras de coco do Maranhão e de outros estados, isso não teria ocorrido da mesma maneira. A primeira canção do livro O xote das quebra- deiras de coco (AS ENCANTADEIRAS, 2014, p. 8-9), traz a mensagem para não derrubar a palmeira. Para elas, a pauta ambiental surgiu da necessi- dade de sobrevivência. A canção “Ó liberdade” (AS ENCANTADEIRAS, 2014, p. 16) conclama as mulheres a lutarem juntas pelo ambiente, pois é do coco que elas levam o pão à mesa. Derrubar O trabalho nos cocais pode ser mais árduo que o trabalho na roça. Às vezes, elas se levantam com o sol no horizonte, já levam seu almoço e voltam no final do dia. É comum passarem o dia inteiro na quebra do coco. Considerações finais Esse é parte do universo econômico, social, política e cultural dessas trabalhadoras extrati- vistas. Por meio de cantos como estes aqui des- tacados, elas promulgam suas percepções sobre o mundo nas esferas coletiva e individual. A arte literária é capaz de tornar lúdica uma realidade dura, marcada pela pobreza e pela violência em distintas esferas, da mesma forma, ela também pode expressar, de maneira contundente, a saga de lutas antigas, que persistem à medida que as injustiças sociais permanecem. Apesar dessas mulheres terem convivido com os movimentos sociais, terem se apropriado de narrativas políticas que defendem igualdade social, suas visões de mundo são pautadas, prin- cipalmente, pela própria vida comunitária e por valores religiosos. A maior parte dessas mulheres é católica, mesclada com crenças africanas e outros segmentos religiosos. Por meio dessas canções, foi possível ter uma ideia dos aspectos visíveis das lutas dessas mu- lheres por uma vida melhor. Mais que isso, foi possível percorrer nas entrelinhas e encontrar anseios que vão além de seus combates políticos, ao mostrar aspectos peculiares, únicos, dessas mulheres sobre a vida pessoal e em comunida- de. A maneira como elas enxergam a política e a própria vida, está diretamente ligada à relação com a natureza e à religiosidade e isso leva a um modo singular de viver e de reagir diante das tensões sociais. Tanto as que são participantes ativas nos mo- vimentos sociais, quanto aquelas mais distantes, têm a crença política em um mundo melhor, mas pode-se afirmar que essa certeza é menos pela leitura política que religiosa, pois creem em um deus bondoso, preocupado com os oprimidos. Diz uma canção (AS ENCANTADEIRAS, 2014, p. 10): “Confiando em Cristo Rei, que nasceu lá em Belém/ E morreu crucificado, porque nos queria bem/ Confiando em seu amor, se reclama até a doutor,/ Mas nossos direitos vêm!”. Ainda que os fazendeiros tenham a terra, a canção alerta os opressores e lembra que essa vida é curta e ao final dela não há diferença entre ricos e pobres. “Cuidado com teu mistério!/ Um dia no cemitério, nossa carne se mistura!” (AS ENCANTADEIRAS, 2014, p. 10). Lembra, ainda, que na morte, a mo- rada é a cova e o verme, a única companhia. Aspectos culturais Também o é carregar cofos ou cestos de palha na cabeça e andar longas distâncias sob sol e chuva. A canção “Pobre Rita” (AS ENCANTADEIRAS, 2014, p. 26), relata: 10/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 10/11 Oficina do historiador, Porto Alegre, v. 14, n. 1, p. 1-11, jan.-dez. 2021 \| e-40969 Cabocla faceira É aquela Rita Muito dengosa, muito bonita Quando ela passa de macete na mão Machadinho amolado Vestidinho de algodão Vai quebrar o coco Pra ganhar dinheiro Pra compra arroz Pra comprar feijão. Pra comprar farinha, pra comprar o pão (2x). [...]. de ruim aos outros, receberemos em dobro, diz um verso, assim como a morte é certa, a justiça divina também não falha. Referências ALMEIDA, Alfredo Wagner Berno de. (org.). Economia do Babaçu: Levantamento preliminar de dados. São Luís: MIQCB, 2000. ALMEIDA, Alfredo Wagner Berno de. (org.). Economia do Babaçu: Levantamento preliminar de dados. São Luís: MIQCB, 2000. Os textos deste artigo foram revisados pela Poá Comunicação e submetidos para validação do autor antes da publicação. Os textos deste artigo foram revisados pela Poá Comunicação e submetidos para validação do autor antes da publicação. ANTUNES, Marta. As guardiãs da floresta do babaçu e o tortuoso caminho do empoderamento. In: WOORT- MANN, Ellen F.; MENACHE, Renata; HEREDIA, Beatriz (org.). NEAD Especial. Brasília: MDA, IICA, 2006. AYRES JÚNIOR, José Costa. A organização das que- bradeiras de coco babaçu e a refuncionalização de um espaço regional na Microrregião do Médio Mearim Maranhense. 2007. 186 f. Dissertação (Mestrado em Geografia) – Pós-Graduação em Geografia, Universi- dade Federal de Santa Catarina, Florianópolis, 2007. AS ENCANTADEIRAS. Canto e encanto nos babaçuais. Músicas sob domínio popular selecionadas por “As En- cantadeiras”. [S. I.]: AS ENCANTADEIRAS; AMTR; MIQCB; ASSEMA; NCADR-UFPA, 2014. Disponível em: http:// www.asmubip.org.br/wp-content/uploads/2016/04/ livro-quebradeiras-de-coco-babacu.pdf. Acesso em: 1 ago. 8 2017. BARBIERI, Ana Linhares Cavalcante. As tendências ao desenvolvimento sustentável no manejo do babaçu pelas comunidades rurais do estado do Maranhão. 2004. 114 f. Dissertação (Mestrado em Economia) – Pós-Graduação em Economia, Universidade Federal de Pernambuco, Recife, 2004. BARBOSA, Viviane de Oliveira. Histórias de trabalha- dores rurais no Maranhão: Gênero, Identidades e Mo- bilização. In: XIX ENCONTRO REGIONAL DE HISTÓRIA: PODER, VIOLÊNCIA E EXCLUSÃO, São Paulo. Anais [...]. São Paulo: ANPUH/USP, 2008. p. 1-13. DE CERTEAU, Michel. A escrita da História. 2. ed. Rio de Janeiro: Forense Universitária, 2002. GINZBURG, Carlo. Mitos, emblemas e sinais: morfologia e história. São Paulo: Companhia das Letras, 1989. GINZBURG, Carlo. Mitos, emblemas e sinais: morfologia e história. São Paulo: Companhia das Letras, 1989. GINZBURG, Carlo. O fio e os rastros: Verdadeiro, falso, fictício. São Paulo: Companhia das Letras, 2007. GINZBURG, Carlo. O fio e os rastros: Verdadeiro, falso, fictício. São Paulo: Companhia das Letras, 2007. GINZBURG, Carlo. O fio e os rastros: Verdadeiro, falso, fictício. São Paulo: Companhia das Letras, 2007. Endereço para correspondência Raimundo Lima dos Santos Universidade Estadual da Região Tocantina do Ma- ranhão Rua Godofredo Viana, 1300 Centro, 659000-000 Imperatriz, MA, Brasil Raimundo Lima dos Santos Universidade Estadual da Região Tocantina do Ma- ranhão Rua Godofredo Viana, 1300 Centro, 659000-000 Imperatriz, MA, Brasil Raimundo Lima dos Santos Universidade Estadual da Região Tocantina do Ma- ranhão Rua Godofredo Viana, 1300 Centro, 659000-000 Imperatriz, MA, Brasil Raimundo Lima dos Santos Universidade Estadual da Região Tocantina do Ma- ranhão Universidade Estadual da Região Tocantina do Ma ranhão Rua Godofredo Viana, 1300 Centro, 659000-000 Imperatriz, MA, Brasil Rua Godofredo Viana, 1300 Centro, 659000-000 Imperatriz, MA, Brasil Rua Godofredo Viana, 1300 Centro, 659000-000 Imperatriz, MA, Brasil Considerações finais O fato de essas mulheres serem fiéis as suas crenças e superstições, muitas vezes acima de concepções políticas formais, não diminui a im- portância de seu aprendizado nos movimentos sociais, pelo contrário, aumenta seu valor. Elas apenas ressignificam novos aprendizados de acordo com suas concepções culturais. Esse saber único atente a necessidades também úni- cas e mostra que o mundo poder ser construído à imagem e à semelhança de cada sociedade. Os versos compilados nesse livro de cantos são só uma amostra de uma realidade muito mais profunda e rica. Mesmo assim, é possível extrair algumas reflexões sobre esses grupos de mulheres que têm ganhado notoriedade em diversas regiões. É possível colher fragmentos que revelam suas relações sociais não apenas no âmbito coletivo. Ainda que muito mais poderia A riqueza não servirá na outra vida e todos aqueles que oprimiram os pobres prestarão contas de sua avareza. “Quero ver tua defesa, onde está tua riqueza/ Que comprava o mundo inteiro?”. Essa é uma concepção corrente em um catolicismo mais tradicional, especialmente liga- do aos movimentos sociais. Tudo o que fazemos Raimundo Lima dos Santos O canto e a vida das quebradeiras de coco do Maranhão 11/11 Raimundo Lima dos Santos O canto e a vida das quebradeiras de coco do Maranhão 11/11 ter sido revelado, o que se viu aqui mostra quão complexo e dinâmico poder ser um conjunto de pessoas que sonham em remodelar o mundo em seu favor, especialmente quando esse grupo é composto por quebradeiras de coco babaçu. Raimundo Lima dos Santos Doutor em História pela Universidade Federal de Minas Gerais (UFMG), em Belo Horizonte, MG, Brasil; mestre pela Universidade Fede-ral de Goiás (UFG), em Goiânia, GO, Brasil; graduado pela Universidade Estadual do Maranhão (UEMA), em Imperatriz, MA, Brasil. Professor de História da América da Universidade Estadual da Região Tocantina do Ma- ranhão (UEMASUL), em Imperatriz, MA, Brasil.
https://openalex.org/W2080723158	https://europepmc.org/articles/pmc4205119?pdf=render	English	null	A Synaptic Mechanism for Temporal Filtering of Visual Signals	PLoS biology	2,014	cc-by	15,108	Introduction transmission of the visual signal might also reflect intrinsic variations between the various synaptic compartments of a BC. The property we concentrate on is the volume of the terminal, which is expected to influence the amplitude and kinetics of the presynaptic calcium signal controlling neurotransmission [12]. transmission of the visual signal might also reflect intrinsic variations between the various synaptic compartments of a BC. The property we concentrate on is the volume of the terminal, which is expected to influence the amplitude and kinetics of the presynaptic calcium signal controlling neurotransmission [12]. To observe the activity of multiple BC synapses we used zebrafish expressing fluorescent proteins reporting the fusion of synaptic vesicles or the presynaptic calcium signal driving fusion [13,14]. Here we demonstrate that BC terminals of different sizes tend to transform the visual signal in different ways. On average, smaller terminals generate calcium transients that are larger and faster, resulting in a higher initial gain of responses to an increase in temporal contrast followed by more profound adaptation. Small terminals also transmit high frequencies more effectively. Such differences in the outputs of small and large terminals are also observed in individual cells, which therefore have an intrinsic ability to filter visual information through channels with different gains, temporal filters and adaptive properties. The retina analyzes the visual world through a series of spatio- temporal filters that establish parallel representations for trans- mission to the brain [1–4]. The anatomical organization of these channels is established in the inner plexiform layer (IPL), which is organized into five to six distinct strata containing the dendrites of as many as 20 different types of retinal ganglion cell (RGC) [2]. In each stratum, bipolar cells (BCs) with distinct filtering properties make excitatory synaptic connections with defined subsets of RGCs [5]. For instance, ‘‘transient’’ RGCs are thought to receive excitatory inputs from BCs with bandpass characteristics, while ‘‘sustained’’ RGCs receive inputs from BCs acting as low-pass filters [6]. p y p g g [ ] To observe the activity of multiple BC synapses we used zebrafish expressing fluorescent proteins reporting the fusion of synaptic vesicles or the presynaptic calcium signal driving fusion [13,14]. Here we demonstrate that BC terminals of different sizes tend to transform the visual signal in different ways. Abstract Abbreviations: AI, adaptation index; BC, bipolar cell; DC, direct current; dpf, days post fertilization; EGFP, enhanced green fluorescent protein; IP, intermediate pool; IPL, inner plexiform layer; OGB, Oregon green BAPTA; RGC, retinal ganglion cell; ROI, region of interest; RP, reserve pool; RRP, readily releasable pool. * Email: L Lagnado@sussex ac uk * Email: L.Lagnado@sussex.ac.uk . These authors contributed equally to this work. . These authors contributed equally to this work. Introduction On average, smaller terminals generate calcium transients that are larger and faster, resulting in a higher initial gain of responses to an increase in temporal contrast followed by more profound adaptation. Small terminals also transmit high frequencies more effectively. Such differences in the outputs of small and large terminals are also observed in individual cells, which therefore have an intrinsic ability to filter visual information through channels with different gains, temporal filters and adaptive properties. A long-standing question is how multiple spatio-temporal filters are built using the limited numbers of neurons responding to a particular region of the visual field [7–9]. An answer might lie in reconsidering the fundamental neural element through which the visual signal is transmitted to the inner retina: although individual BCs have diverse properties, their output is transmitted through a much more numerous and heterogeneous component of neural circuits–synapses [10]. Indeed, recent evidence indicates that different synapses of the same BC transmit the visual signal with varying kinetics because of local interactions with different types of inhibitory amacrine cells [11]. Here we ask whether heterogeneous Tom Baden1., Anton Nikolaev1., Federico Esposti1, Elena Dreosti1, Benjamin Odermatt1, Leon Lagnado1,2* Tom Baden1., Anton Nikolaev1., Federico Esposti1, Elena Dreosti1, Benjamin Odermatt1, Leon Lagnado1,2* 1 MRC Laboratory of Molecular Biology, Neurobiology Division, Cambridge, United Kingdom, 2 School of Life Sciences, University of Sussex, Brighton, United Kingdom 1 MRC Laboratory of Molecular Biology, Neurobiology Division, Cambridge, United Kingdom, 2 School of Life Sc Abstract The visual system transmits information about fast and slow changes in light intensity through separate neural pathways. We used in vivo imaging to investigate how bipolar cells transmit these signals to the inner retina. We found that the volume of the synaptic terminal is an intrinsic property that contributes to different temporal filters. Individual cells transmit through multiple terminals varying in size, but smaller terminals generate faster and larger calcium transients to trigger vesicle release with higher initial gain, followed by more profound adaptation. Smaller terminals transmitted higher stimulus frequencies more effectively. Modeling global calcium dynamics triggering vesicle release indicated that variations in the volume of presynaptic compartments contribute directly to all these differences in response dynamics. These results indicate how one neuron can transmit different temporal components in the visual signal through synaptic terminals of varying geometries with different adaptational properties. Citation: Baden T, Nikolaev A, Esposti F, Dreosti E, Odermatt B, et al. (2014) A Synaptic Mechanism for Temporal Filtering of Visual Signals. PLoS Biol 12(10): e1001972. doi:10.1371/journal.pbio.1001972 Citation: Baden T, Nikolaev A, Esposti F, Dreosti E, Odermatt B, et al. (2014) A Synaptic Mechanism for Temporal Filtering of Visua e1001972. doi:10.1371/journal.pbio.1001972 Academic Editor: Stephen A. Baccus, Stanford University School of Medicine, United States of America Received May 2, 2014; Accepted September 10, 2014; Published October 21, 2014 Received May 2, 2014; Accepted September 10, 2014; Published October 21, 2014 den et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2014 Baden et al. This is an open-access article distributed under the terms of the Creative Commons Attributi unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: Wellcome Trust (083220). Medical Research Council, UK (MRC-LMB; no grant number applicable). 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. PLOS Biology \| www.plosbiology.org A Synaptic Mechanism for Temporal Filtering of Visual Signals 1., Anton Nikolaev1., Federico Esposti1, Elena Dreosti1, Benjamin Odermatt1, d 1 2* Author Summary The process of neurotransmission involves the conversion of electrical signals into the release of a chemical neurotransmitter from the neurons synaptic terminal, and the key trigger for this release is a rise in calcium concentration. Accordingly, the amplitude and speed of this calcium signal controls the amplitude and time-course of synaptic communication. Working on the synaptic terminals of fish retinal bipolar cells, we show that the presynaptic calcium signal and the subsequent neuro- transmitter release are shaped by the basic property of synapse volume. Using a combination of experimental approaches and computational models, we found that large synapses are slow and adapt little during ongoing stimulation, while small synapses are fast and show more profound adaptation. This observation leads to a second key concept: since neurons usually have several presyn- aptic terminals that may vary in volume, a single neuron can, in principle, forward different synaptic signals to different postsynaptic partners. We provide direct evi- dence that this is the case for bipolar cells of the fish retina. Does the correlation between the size of the terminal compartment and the release rate reflect the network in which the terminal is embedded? For instance, one scenario might be that large terminals respond with lower gain because they receive stronger inhibitory feedback from amacrine cells. To test this possibility, we blocked GABAergic inhibition through ionotropic receptors by injecting 100 mM picrotoxin into the eye (Figure 2). This manipulation increased the amplitude of the exocytic response across terminals of all sizes, as would be expected when feedback inhibition is reduced [18,19], but larger terminals continued to respond with lower gain. This observation suggests that the correlation between terminal size and release rate reflects intrinsic properties of the terminal rather than the influence of the network it is embedded in. Might the correlation between stimulus-dependent changes in the brightness of terminals and estimates of their size be caused by a bias in the detection algorithm, perhaps causing smaller terminals to be detected only when their fluorescence signal was strong enough? Two lines of evidence indicated that this was not the case. First, the distribution of terminal sizes was very similar when measured in transgenic fish expressing synaptophysin- enhanced green fluorescent protein (EGFP), a fluorescent marker of synapses that is not affected by neural activity (Figure 1C, red bars). Different Calcium Dynamics in Small and Large Terminals of Individual Bipolar Cells Are differences in gain maintained in small and large terminals of the same cell? We investigated this question both in vivo and in vitro. In vivo experiments used zebrafish transiently expressing Ribeye::GCaMP5 in sparse populations of BCs. Square-wave stimuli (100% contrast) were presented at various frequencies, and Figure 3A–3C shows results from one BC responding to a 1 Hz stimulus. The calcium signal in the smaller of the two terminals clearly exhibited the larger degree of modulation (Figure 3B; cf. Figure 1G1), and the power of the response peaked at a higher frequency (Figure 3C). Qualitatively similar results were obtained from four BCs. How do these variations in the volume of the presynaptic compartment affect the calcium signal that is generated when a stimulus alters the membrane potential and the rate of calcium influx? To investigate this question we imaged SyGCaMP2 and measured the average response over the whole region of interest (ROI) defining each terminal. Although this procedure will neglect calcium gradients within the terminal, these gradients will not be maintained when calcium influx is modulated at frequencies relevant to vision (see below). In a sample of 932 ON terminals, the initial rate of rise of the calcium signal elicited by a full field step of light was approximately twice as fast in the smallest terminals compared to the largest, and the calcium concentration reached a higher steady-state level (Figure 1E1,2). Variations in terminal size were also correlated with differences in the kinetics of exocytosis, as measured in fish expressing sypHy. In a sample of 438 ON terminals, the relative rate of vesicle release in response to the same stimulus (quantified as a percentage of vesicles released per second) varied by a factor of at least two between the smallest and largest terminals (Figure 1F1,2) [14,18]. A similar relationship between release amplitude and terminal size was observed in the OFF channel (Figure S1G). We next used slices of goldfish retina to make whole-cell recordings directly from the large terminal of ‘‘mixed’’ BCs, which is connected to smaller presynaptic compartments by narrow processes. Introduction of the Ca2+ indicator Oregon green BAPTA (OGB-5N) allowed us to compare presynaptic calcium signals in the large and small compartments while achieving direct electrical control of the terminal system (Figure 3D; cf. Figure 1A) [20]. A BC generating a graded voltage response is shown in Figure 3E. Author Summary Second, estimates of terminal size did not differ significantly when measurements were compared under conditions of high and low activity, as described in Figure S1 (See Methods). The results in Figure 1 therefore indicate that the volume of the presynaptic compartment is closely linked to the gain with which BCs transmit the visual signal to the inner retina. terminals of various sizes (Figure 1A). To survey the activity of many BC synapses in a live animal, we imaged eight to ten days post fertilisation (dpf) zebrafish larvae expressing sypHy, a reporter of vesicle fusion (Figure 1B) [14,15], or SyGCaMP2, which signals the presynaptic calcium transient [13,16]. Regions of interest defining individual terminals in images of the IPL were generated using an algorithm based on a Laplace operator (Figure 1B) [17], from which we estimated the ‘‘effective terminal radius’’ (Materials and Methods). A survey of 5,061 terminals revealed a wide variation in radius with a mean of 1.1360.40 mm (Figure 1C, black bars). Terminals of different sizes were not distributed randomly through the IPL (Figures 1D and S1I): terminals in layers 2 and 6 were significantly larger than the average across the whole population of BCs, while terminals in layers 1, 3, 4, and 5 were significantly smaller. The Volume of Bipolar Cell Terminals Determines the Gain and Kinetics of Signal Transmission In the vertebrate retina, signals from rod and cone photore- ceptors travel through BCs to be transmitted through synaptic October 2014 \| Volume 12 \| Issue 10 \| e1001972 October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 1 Synaptic Filtering of Visual Signals of the calcium signal at the fundamental frequency was greater in small terminals (r = 0.6–1.3 mm) than in large (r = 2.1–4.1 mm), varying by a factor of at least three across the population (Figure 1G2). October 2014 \| Volume 12 \| Issue 10 \| e1001972 The Relation between the Decay of the Calcium Transient and the Radius of the Terminal but the signal in the large terminal (black) was smaller and slower, and the response to a train of spikes was dominated by the gradual accumulation of Ca2+. Figures 3G–3I summarize these properties in 16 pairs of terminals (eight graded and eight spiking), each pair from a different BC. In all cases, global Ca2+ signals were faster and larger in the smaller of two connected terminals. The same observation was made when the patch-pipette was placed on the soma (Figure S3). The results in Figure 3 demonstrate that variations in terminal volume profoundly affected the activity of different synapses providing the output from a single BC. The time-constant of decay of a calcium transient (tdecay) was directly proportional to the radius of the terminal (r), as shown Figure 3J. This observation was made both when r was estimated from images of terminals filled with dye, and when r was calculated from the capacitance of terminals detached from the cell body. This simple linear relation is notable because it is predicted by single compartment models of calcium dynamics in which there are no significant calcium gradients [24,25]. The appropriateness of a single compartment model for BC terminals can be understood in terms of the characteristic time, teq, with which a calcium gradient collapses in a volume of radius r after calcium influx ceases: Figure 2. The relation between terminal volume and release rate persists when GABAergic feedback inhibition is blocked. The initial rate of release in response to a step of light in control terminals (black, from Figure 1F2) is compared with measurements in which 100 M picrotoxin was injected in the front of the eye (77 ON terminals from three fish). The line fitted to the control has a slope of 20.09660.023, and the line fitted to the picrotoxin measurements has a slope of 20.13860.01 dependence peak release size (Figure 1F2). Note that picrotoxin also increased the amplitude of the exocytic response, as would be expected when feedback inhibition is reduced. doi:10.1371/journal.pbio.1001972.g002 teq& r2 6DCa where DCa is the diffusion coefficient of calcium [26]. Assuming DCa = 220 mm2/s [27], teq, is only ,0.75 ms in a terminal of r = 1 mm, indicating that a calcium gradient will collapse on a much shorter time-scale than stimuli relevant to vision. Different Calcium Dynamics in Small and Large Terminals of Individual Bipolar Cells Distribution of sizes calculated with SyGCaMP2 is shown in Figure S1H. (D) Variations in average radius of terminals in each stratum. The average radius over the whole IPL was 1.1360.40 mm (dashed line). Stars mark strata in which the average radius was significantly greater or smaller (p,0.001, Wilcoxon rank-sum test, n varies between 560 and 930 terminals). Similar distributions calculated for individual layers are shown in Figure S1I. (E) Relation between terminal size and calcium signals. (E1) SyGCaMP2 signals in response to a step of light (l = 590 nm; for details see methods) in ON terminals, averaged over groups of the effective radius shown (n = 143, 347, 286, 114, and 36, respectively). Straight lines fitted over the initial phase. Significant difference between groups 1, 3, and 5 (Student’s t test: p(1–3) = 0.004; p(3–5) = 0.005; p(1–5),0.001). (E2) The rate of rise of the SyGCaMP2 signal varies as the inverse of the radius, as shown by the fitted curve (n = 926 from five fish). Spearman correlation coefficient = 21, critical value (p = 0.05) = 0.85. (F) Relation between terminal size and peak rate of vesicle release. (F1) SypHy signals in response to a step of light in ON terminals, averaged over groups of the effective radius shown (n = 117, 176, 113, and 33 terminals from five fish). The lower panel shows the conversion into relative release rates, as described [14]. (F2) The initial rate of vesicle release as a function of radius (total n = 438 terminals from five fish). The points fall on a line. Spearman correlation = 21, critical value (p = 0.05) = 0.9. (G) terminal size and modulation of presynaptic calcium. (G1) SyGCaMP2 signal driven by modulation of light intensity at 1 Hz (100% contrast, square wave). Red trace averaged over OFF contrast responding terminals with r = 0.6– 1.3 mm; black trace averaged over OFF contrast responding terminals with r = 2.1–4.1 mm. (G2) relative power of the signal at 1 Hz as a function of radius (n = 98, 89, 68, 37, and 9 terminals from four fish). Points were fitted with a line. Responses to light decrements are shown in Figure S1G. doi:10.1371/journal.pbio.1001972.g001 The Relation between the Decay of the Calcium Transient and the Radius of the Terminal Even in the largest terminals with r = 5 mm, teq is ,19 ms, which is less than half the period of a stimulus fluctuating at 20 Hz. The lack of any appreciable calcium gradients on these time-scales was confirmed in a 3-D model in which calcium influx occurred through clusters of calcium channels (Figure S3). Calcium signals on these spatial scales have been demonstrated to trigger neurotransmitter release from ribbon synapses of fish bipolar cells, although calcium nanodomains very close to calcium channels also play a role in ribbon-type synapses in other neurons and other species [28]. These considerations indicate that there is a direct and causal link between the volume of the presynaptic compartment and the amplitude and kinetics of the calcium transient caused by a stimulus. But why is the volume of the presynaptic compartment also correlated with its output measured as vesicle release (Figure 1F)? Such a link is expected, because it is the calcium signal in the terminal that drives the output. Figure 2. The relation between terminal volume and release rate persists when GABAergic feedback inhibition is blocked. The initial rate of release in response to a step of light in control terminals (black, from Figure 1F2) is compared with measurements in which 100 M picrotoxin was injected in the front of the eye (77 ON terminals from three fish). The line fitted to the control has a slope of 20.09660.023, and the line fitted to the picrotoxin measurements has a slope of 20.13860.01 dependence peak release size (Figure 1F2). Note that picrotoxin also increased the amplitude of the exocytic response, as would be expected when feedback inhibition is reduced. doi:10.1371/journal.pbio.1001972.g002 Different Calcium Dynamics in Small and Large Terminals of Individual Bipolar Cells The OGB-5N signal in the smaller terminal (red) rose faster than that in the large (black), reached more than double the concentration, and then decayed more rapidly. Similar results were observed in eight cells generating purely graded responses. A correlation between the size of a terminal and the amplitude of its response was also observed when the temporal contrast of the stimulus was increased while keeping the mean luminance constant. Figure 1G1 shows averaged SyGCaMP2 responses to modulation of light intensity at 1 Hz (100% contrast). The power Many BCs in the retina of fish generate voltage spikes as well as graded responses [16,21,22], and terminal volume also affected the presynaptic calcium transient generated by these electrical responses [16,21–23]. In the example in Figure 3F, the small terminal (red) responded to each spike with a clear Ca2+ transient, October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org PLOS Biology \| www.plosbiology.org 2 Synaptic Filtering of Visual Signals Figure 1. Variations in terminal size, calcium signals, and vesicle release. (A) BCs transmit through multiple terminals. Left: published examples of zebrafish cone BCs [64] illustrate the range of presynaptic terminal sizes. Scalebar = 10 mm. (B) BC terminals in the IPL of a zebrafish (10 dpf) expressing sypHy under control of the ribeye promoter. Left: Raw image showing six strata of the IPL. Right: Overlay of ROIs defining terminals. PLOS Biology \| www.plosbiology.org 3 October 2014 \| Volume 12 \| Issue 10 \| e1001972 Figure 1. Variations in terminal size, calcium signals, and vesicle release. (A) BCs transmit through multiple terminals. Left: published examples of zebrafish cone BCs [64] illustrate the range of presynaptic terminal sizes. Scalebar = 10 mm. (B) BC terminals in the IPL of a zebrafish (10 dpf) expressing sypHy under control of the ribeye promoter. Left: Raw image showing six strata of the IPL. Right: Overlay of ROIs defining terminals. October 2014 \| Volume 12 \| Issue 10 \| e1001972 October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 3 Synaptic Filtering of Visual Signals Synaptic Filtering of Visual Signals Scale bar = 10 mm. (C) The distribution of the effective terminal radii. Black bars show estimates obtained in fish expressing sypHy (n = 5,061 terminals from seven fish), and the red bars shows the distribution measured in fish expressing synaptophysin-EGFP (n = 421 terminals from one fish). A Single-Compartment Model of Presynaptic Calcium Dynamics To explore how variations in the size of BC terminals might impact on transmission of visual signals we modeled this process in two stages, described in detail in Text S1. The output from the first stage was the dynamics of calcium in the terminal, and is shown schematically in Figure 4A. The second stage used these calcium October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 4 Synaptic Filtering of Visual Signals Figure 3. Different calcium signals in small and large terminals of the same cell. (A) A BC expressing GCaMP5. Large and small terminals are indicated by the blue and red arrows. Scalebar 10 mm. (B) Responses of the terminals in (A) to a stimulus modulated at 1 Hz. (C) Power spectrum of the responses from large and small terminals calculated for different stimulus frequencies. (D) Mixed BC filled with OGB-5N in a slice of goldfish retina. Scale bar 10 mm. (E) Spatially averaged Ca2+ signals in the small (red) and large (black) terminal during a 2 s depolarizing current step (10 pA). This BC did not generate spikes. (F) A comparison of Ca2+ signals in the small and large terminal of a ‘‘spiking’’ BC. Each spike caused a calcium transient that was larger and faster in the smaller terminal. (G–I) Time constants of the calcium signal rise (trise), decay (tdecay), and peak amplitude, evaluated in 16 pairs of small and large terminals. For small and large, trise = 0.2560.20 and 1.1660.64 s; tdecay = 1.4260.82 and 2.9162.05 s; amplitude = 1.2960.38 and 0.6860.21 mM. All these parameters were significantly different in small and large terminals (p,0.001; Wilcoxon ranked sum, n = 16 cells from nine adult retinae). The average radii of the small and large terminals were 1.160.3 and 5.261.2 mm, respectively. (J) Time constants of calcium decay were directly proportional to terminal radius determined using imaging (grey, n = 32 terminals) or capacitance measurements (black, n = 20 terminals) The linear fit was constrained to go through the origin Error bars show 1 standard deviation (SD) Synaptic Filtering of Visual Signals Figure 3. Different calcium signals in small and large terminals of the same cell. (A) A BC expressing GCaMP5. Large and small terminals are indicated by the blue and red arrows. Scalebar 10 mm. (B) Responses of the terminals in (A) to a stimulus modulated at 1 Hz. A Single-Compartment Model of Presynaptic Calcium Dynamics (C) Power spectrum of the responses from large and small terminals calculated for different stimulus frequencies. (D) Mixed BC filled with OGB-5N in a slice of goldfish retina. Scale bar 10 mm. (E) Spatially averaged Ca2+ signals in the small (red) and large (black) terminal during a 2 s depolarizing current step (10 pA). This BC did not generate spikes. (F) A comparison of Ca2+ signals in the small and large terminal of a ‘‘spiking’’ BC. Each spike caused a calcium transient that was larger and faster in the smaller terminal. (G–I) Time constants of the calcium signal rise (trise), decay (tdecay), and peak amplitude, evaluated in 16 pairs of small and large terminals. For small and large, trise = 0.2560.20 and 1.1660.64 s; tdecay = 1.4260.82 and 2.9162.05 s; amplitude = 1.2960.38 and 0.6860.21 mM. All these parameters were significantly different in small and large terminals (p,0.001; Wilcoxon ranked sum, n = 16 cells from nine adult retinae). The average radii of the small and large terminals were 1.160.3 and 5.261.2 mm, respectively. (J) Time constants of calcium decay were directly proportional to terminal radius determined using imaging (grey, n = 32 terminals) or capacitance measurements (black, n = 20 terminals). The linear fit was constrained to go through the origin. Error bars show 1 standard deviation (SD). doi:10.1371/journal.pbio.1001972.g003 October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 5 Synaptic Filtering of Visual Signals dynamics to predict the kinetics of vesicle release, as described in Figure 5B and below. analogous to the linear-nonlinear (LN) model that has been used to provide a description of responses in RGCs [7]. This model therefore identifies the rectifying I–V relation of the calcium current in BCs as the first major non-linearity in retinal processing. The time-course of the presynaptic calcium signal in response to a visual stimulus was calculated by convolving the time-course of calcium influx with the ‘‘presynaptic calcium filter’’ predicted by the one-compartment model, which has an impulse response decaying with time-constant tdecay (Figure 3E and 3F). The value of tdecay depended on the radius of the terminal, according to the linear relation measured experimentally in Figure 3J. To model the presynaptic calcium signal, the light stimulus was convolved with the impulse response of a BC to predict the voltage trajectory in the soma (Figure 4A, top). A Single-Compartment Model of Presynaptic Calcium Dynamics BCs in goldfish have bandpass characteristics with peak transmission varying between about 1–14 Hz [22], and we chose a value of 9.5 Hz [29]. The voltage response in the soma was then instantaneously mapped to the presynaptic calcium current using the I–V relation and density of L-type calcium channels measured in the terminal of BCs isolated from goldfish [30]. This I–V relation is an exponential function, changing e-fold in ,6.6 mV over the physiological range of membrane potentials [30]. These first two steps in the model are To test this model of calcium dynamics we compared its predictions with experimental measurements made with OGB-5N Figure 4. A simple model to predict global calcium changes in the presynaptic terminal. (A) The stimulus (here a 1 Hz square wave) was convolved with the photoreceptor impulse response to estimate membrane voltage (top). Subsequently, the current through L-type calcium channels was calculated based on the I–V relation and number of channels (middle). Convolution of the calcium current with the synaptic calcium impulse response, calculated from Figure 3G, yielded an estimate of global calcium concentration over time (bottom). (B, C) Measured (B), and modeled (C), global calcium changes in a small (red; radius = 1 mm) and large (black; radius = 3 mm) terminals responding to a 1 Hz square wave stimulus. Data in (B) from goldfish ‘‘mixed’’ BC (cf. Figure 3D–3J). doi:10.1371/journal.pbio.1001972.g004 Figure 4. A simple model to predict global calcium changes in the presynaptic terminal. (A) The stimulus (here a 1 Hz square wave) was convolved with the photoreceptor impulse response to estimate membrane voltage (top). Subsequently, the current through L-type calcium channels was calculated based on the I–V relation and number of channels (middle). Convolution of the calcium current with the synaptic calcium impulse response, calculated from Figure 3G, yielded an estimate of global calcium concentration over time (bottom). (B, C) Measured (B), and modeled (C), global calcium changes in a small (red; radius = 1 mm) and large (black; radius = 3 mm) terminals responding to a 1 Hz square wave stimulus. Data in (B) from goldfish ‘‘mixed’’ BC (cf. Figure 3D–3J). doi:10.1371/journal.pbio.1001972.g004 October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 6 Synaptic Filtering of Visual Signals Figure 5. A model of vesicle release through the ribbon. (A) Schematic showing three vesicle pools involved in the exocytic response triggered by calcium. A Single-Compartment Model of Presynaptic Calcium Dynamics (B) The model (black) closely reproduces the three phases of release measured by [45] (grey) upon maximal activation of calcium channels. (C1) Bulk calcium and (D1) vesicle release modeled for OFF terminals with different radii (1.2, 1.6, 2.8 mm) when a light step is turned off. (C2) The initial rate of rise of the calcium signal (calcium gain) varies as 1/r, while release gain falls linearly with r (D2). (E1) Calcium in response to a 1 H ti l i diff t i t i l (E ) P t th ti l f f ll ith 1/ ( f Fi 1E ) S l Fi S4 (E ) I t Figure 5. A model of vesicle release through the ribbon. (A) Schematic showing three vesicle pools involved in the exocytic respons triggered by calcium. (B) The model (black) closely reproduces the three phases of release measured by [45] (grey) upon maximal activation o calcium channels. (C1) Bulk calcium and (D1) vesicle release modeled for OFF terminals with different radii (1.2, 1.6, 2.8 mm) when a light step is turne off. (C2) The initial rate of rise of the calcium signal (calcium gain) varies as 1/r, while release gain falls linearly with r (D2). (E1) Calcium in response to 1 Hz square wave stimulus in different size terminals. (E2) Power at the stimulus frequency falls with 1/r (cf. Figure 1E2). See also Figure S4. (E2) Inse unlike the one-compartment model, the 3-D diffusion model (Figure S3) predicts a linear relation between power at the stimulus frequency an radius. doi:10.1371/journal.pbio.1001972.g005 Figure 5. A model of vesicle release through the ribbon. (A) Schematic showing three vesicle pools involved in the exocytic respon triggered by calcium. (B) The model (black) closely reproduces the three phases of release measured by [45] (grey) upon maximal activation calcium channels. (C1) Bulk calcium and (D1) vesicle release modeled for OFF terminals with different radii (1.2, 1.6, 2.8 mm) when a light step is turn off. (C2) The initial rate of rise of the calcium signal (calcium gain) varies as 1/r, while release gain falls linearly with r (D2). (E1) Calcium in response to 1 Hz square wave stimulus in different size terminals. (E2) Power at the stimulus frequency falls with 1/r (cf. Figure 1E2). See also Figure S4. Synaptic Filtering of Visual Signals in BCs from slices of goldfish retina. The calcium concentration in the smaller of two connected synaptic compartments rose higher and faster, and then decayed almost to baseline after each cycle of a 1 Hz stimulus, while in the larger terminals there was an accumulation of calcium and much smaller modulations in concentration (Figure 4B). The model could account for these differences by using the appropriate value of r while keeping all other variables constant (Figure 4C). of the model was the prediction of a secondary rise in the release rate beginning ,150 ms after stimulus onset. A second phase of release has also been observed experimentally by monitoring glutamate release from the BC terminal electrophysiologically in a second voltage-clamped ‘‘sniffer cell’’ [32]. In its simplest form, the model failed to predict the exact form of the relation between the power of the SyGCaMP2 signal and terminal radius: power varied linearly with r in vivo (Figure 1G2), but the model predicted that it would vary as 1/r (Figure 5E2). This discrepancy could be corrected with a 3-D diffusion model that captures local calcium differences during ongoing signaling (Figure 5E2, inset). We considered two extensions of this model. The first is the use of three dimensions to estimate changes in calcium concentration at the active zone: this provided a very similar prediction to the one-compartment model (Figure S3). The second extension was to add active conductances in the terminal leading to calcium spikes, but again this did not alter the main conclusions that we could draw about the impact of terminal volume on gain and kinetics (see below and Figure S6). We also explored predictions of the model to alterations in physiological parameters, including the possibility of a nonlinear relation between release rate and calcium concentration, and variations in the relative threshold for L-type calcium channel activation. These changes did not qualitatively alter the predicted effects of terminal volume on the gain and kinetics of signal transmission (Figure S4). The combination of experiment and modeling presented in Figures 1–5 converges on one basic idea: the volume of the presynaptic compartment is closely linked with the gain and kinetics of synaptic transmission by determining the amplitude and time-course of the presynaptic calcium transient. A Model of Synaptic Transmission through Bipolar Cells A Model of Synaptic Transmission through Bipolar Cells Having established that the model accounted adequately for variations in the presynaptic calcium signal, we extended it to explore the impact of terminal size on the kinetics of vesicle release. The following properties of exocytosis at the ribbon synapse of BCs were taken into account, all of which have been measured experimentally. Property 1. There are three anatomically distinct populations of vesicles in the synaptic terminal of BCs: the rapidly-releasable pool (RRP) docked at the active zone, the intermediate pool (IP) attached to the ribbon behind the active zone, and the reserve pool (RP) that is mobile across the whole terminal [31]. Based on the assumption of a constant ribbon density (see below), the sizes of the RRP and IP were proportional to terminal surface area, while RP size depended on volume (equation 4 in Text S1). Variations in Contrast Adaptation in Terminals of Different Volume The gain of signal transmission through the retina is not constant, but varies continuously according to the recent history of the stimulus [8]. Such plasticity has been studied particularly intensively in the context of adaptation after an increase in temporal contrast, which involves depression of excitatory synaptic transmission from BCs to RGCs [18,43,44], likely reflecting depletion of rapidly releasable vesicles within the terminal [45–47]. Might the size of BC terminals also impact on the process of contrast adaptation? Larger BC terminals can contain hundreds of thousands of vesicles, and these are more mobile than in conventional synapses, acting to support the continuous mode of transmission [31,34]. It might therefore be expected that larger terminals containing more vesicles are more resistant to depres- sion. Property 2. There are two kinetically distinct modes of neurotransmission [32]: one is fast and transient [33] and the other slow and sustained [34]. Property 3. Both modes of exocytosis can be driven by micromolar calcium concentrations, which can be achieved in the bulk cytoplasm [34–39]. Property 4. The spatial scale on which calcium entering through calcium channels triggers exocytosis is of the order of microns. The idea that microdomains of calcium control fast release of vesicles from BCs is supported by a large number of studies demonstrating that such release is effectively blocked by the slow calcium buffer ethylene glycol tetraacetic acid (EGTA) [33,35,36,40,41]. To quantify time-dependent changes in synaptic gain we used an ‘‘Adaptation Index’’ (AI), calculated as the ratio between the peak initial response to an increase in contrast and the later steady- state response. Using a stimulus of 100% contrast modulated at 5 Hz, and assuming a constant density of vesicles in terminals of different volume, the model predicted that the rate of vesicle release would depress more profoundly in smaller terminals (Figure 6A and 6B). This can be understood in terms the RRP and IP depleting faster and to a lower steady-state in small terminals compared to large (Figure 6A). Property 5. For simplicity we assumed that the number of ribbons, and therefore the number of L-type calcium channels, is proportional to terminal surface area. This assumption is tentatively supported by electron microscopy (EM) studies of goldfish ‘‘mixed’’ BCs [42]. This model closely described the different phases of exocytosis measured in isolated BCs stimulated by a voltage-clamp step (Figure 5A and 5B) [35]. Synaptic Filtering of Visual Signals This fundamental property of the synaptic compartment varies across BCs (Figure 1D) and will therefore contribute to variations in the gain of the visual signal transmitted to the inner retina, as well as the way gain varies as a function of frequency. A Single-Compartment Model of Presynaptic Calcium Dynamics (E2) Ins unlike the one-compartment model, the 3-D diffusion model (Figure S3) predicts a linear relation between power at the stimulus frequency a radius. doi:10.1371/journal.pbio.1001972.g005 Figure 5. A model of vesicle release through the ribbon. (A) Schematic showing three vesicle pools involved in the exocytic response triggered by calcium. (B) The model (black) closely reproduces the three phases of release measured by [45] (grey) upon maximal activation of calcium channels. (C1) Bulk calcium and (D1) vesicle release modeled for OFF terminals with different radii (1.2, 1.6, 2.8 mm) when a light step is turned off. (C2) The initial rate of rise of the calcium signal (calcium gain) varies as 1/r, while release gain falls linearly with r (D2). (E1) Calcium in response to a 1 Hz square wave stimulus in different size terminals. (E2) Power at the stimulus frequency falls with 1/r (cf. Figure 1E2). See also Figure S4. (E2) Inset: unlike the one-compartment model, the 3-D diffusion model (Figure S3) predicts a linear relation between power at the stimulus frequency and radius. doi:10.1371/journal.pbio.1001972.g005 October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 7 Synaptic Filtering of Visual Signals October 2014 \| Volume 12 \| Issue 10 \| e1001972 Variations in Contrast Adaptation in Terminals of Different Volume (A) Top, vesicle release modeled in small d) and large (black) terminals in response to a 5 Hz stimulus (100% contrast). Small terminals are predicted to exhibit stronger adaptation. Bottom, namics of three vesicle pools used in the model. RRP and IP deplete faster in small terminals while RP in small and large terminals remains near nstant. (B) Modeled adaptation index (Methods) decreases linearly with terminal radius. (C) Adaptation of synaptic output measured in vivo was ore profound in smaller terminals. Graph shows release dynamics of OFF terminals with r,1 mm (red) and r.1.2 mm (black) in response to a 5 Hz mulus (cf. (A)). (D) Adaptation index decreases linearly with terminal radius, as predicted by the model (n = 236 OFF terminals from seven fish, each is an average of 12 individual terminals). Spearman correlation = 20.86, critical value (p = 0.05) = 0.45. See also Figure S5. :10.1371/journal.pbio.1001972.g006 Figure 6. Contrast adaptation depends on terminal size: comparison of model and experiment. (A) Top, vesicle release modeled in small (red) and large (black) terminals in response to a 5 Hz stimulus (100% contrast). Small terminals are predicted to exhibit stronger adaptation. Bottom, dynamics of three vesicle pools used in the model. RRP and IP deplete faster in small terminals while RP in small and large terminals remains near constant. (B) Modeled adaptation index (Methods) decreases linearly with terminal radius. (C) Adaptation of synaptic output measured in vivo was more profound in smaller terminals. Graph shows release dynamics of OFF terminals with r,1 mm (red) and r.1.2 mm (black) in response to a 5 Hz stimulus (cf. (A)). (D) Adaptation index decreases linearly with terminal radius, as predicted by the model (n = 236 OFF terminals from seven fish, each bin is an average of 12 individual terminals). Spearman correlation = 20.86, critical value (p = 0.05) = 0.45. See also Figure S5. doi:10.1371/journal.pbio.1001972.g006 sensitization (Figure 6D). Sensitization of the BC output has been shown to result from reduced inhibition from amacrine cells [18,19,49], and so is not predicted by the simple one compartment model. geometry of the presynaptic compartment is one of the factors determining the kinetics of vesicle depletion and, therefore, adaptation. It has recently been demonstrated that there are two opposing forms of plasticity when the retina responds to an increase in temporal contrast: while some BCs and ganglion cells adapt, others become sensitized [18,48]. Variations in Contrast Adaptation in Terminals of Different Volume The balance between adaptation and sensitization was also found to vary as a function of terminal size [14,18]; on average, larger terminals tended to show less adaptation (Figure 6C) with the largest terminals exhibiting Variations in Contrast Adaptation in Terminals of Different Volume This model closely described the different phases of exocytosis measured in isolated BCs stimulated by a voltage-clamp step (Figure 5A and 5B) [35]. To assess the utility of the model, we compared its predictions with the experimental measurements of synaptic function illustrated in Figures 1E–1G. Variations in the initial rate of rise of presynaptic calcium signal (Figure 5C1,2), the initial rate of exocytosis (Figure 5D1,2), and the power of the calcium signal elicited by a 1 Hz stimulus could all be accounted for by variations in terminal radius, while keeping other variables constant. For instance, the initial rate of rise of calcium in response to a step stimulus decreased as 1/r while the rate of exocytosis was found to decrease linearly with r in vivo (Figure 1E2 and 1F2) and the model predicted the same (Figure 5C2 and 5D2). A notable feature To assess the utility of the model, we compared its predictions with the experimental measurements of synaptic function illustrated in Figures 1E–1G. Variations in the initial rate of rise of presynaptic calcium signal (Figure 5C1,2), the initial rate of exocytosis (Figure 5D1,2), and the power of the calcium signal elicited by a 1 Hz stimulus could all be accounted for by variations in terminal radius, while keeping other variables constant. For instance, the initial rate of rise of calcium in response to a step stimulus decreased as 1/r while the rate of exocytosis was found to decrease linearly with r in vivo (Figure 1E2 and 1F2) and the model predicted the same (Figure 5C2 and 5D2). A notable feature Assaying vesicle release in vivo using sypHy confirmed that smaller terminals displayed greater depression (Figure 6C), and AI decreased linearly with r (Figure 6D). The absolute values of AI measured using sypHy were, however, lower than those predicted by the model: in the smallest terminals, an AI of ,2.5 was measured using sypHy, while the model predicted values of ,6. This difference is likely to reflect the relatively low time-resolution of sypHy measurements, causing us to underestimate the initial peak release rate (cf. Figure 6A). Nonetheless, the model and experimental measurements together demonstrate that the October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 8 Synaptic Filtering of Visual Signals gure 6. Contrast adaptation depends on terminal size: comparison of model and experiment. Synaptic Filtering of Visual Signals Although the most obvious aspect of the SyGCaMP2 signal elicited by an increase in the variance of the stimulus was a maintained increase in presynaptic calcium, smaller fluctuations could also be detected (Figure 1G). The power of the fluctuations following a periodic stimulus was used to quantify the linear component of the synaptic calcium response [22], and the power of this linear component was ,3-fold larger in the smaller population of terminals (Figures 1G, 7C, and 7D). Further, when the contrast of the stimulus was varied between 10% and 100%, the power of the linear component was directly proportional to the power of the DC component (Figure 7E), as predicted by the model (Figure 7G). The proportionality constant for the small population of terminals was 2.4 times that of the large population (Figure 7E), which was also predicted by the model. The relatively slow response time of SyGCaMP2 (tdecay,200 ms [13]) prevented the imaging of synaptic responses following stimuli at frequencies greater than 3 Hz, but the results in Figure 7 demonstrate that smaller terminals generate relatively stronger linear responses than large terminals, and therefore encode fluctuating stimuli more effectively. Measurements of vesicle release with sypHy demonstrated strong rectification in the synaptic output because an increase in stimulus variance caused an increase in the mean rate of vesicle release even when the mean luminance was held constant, as shown in Figure 6C. This behavior is predicted by the model (Figure 6A), where it reflects the rectifying relation between membrane potential and the amplitude of the calcium current (Figure 4). To test this explanation by experiment, we imaged the presynaptic calcium signal using SyGCaMP2, while applying a square wave stimulus modulated at 3 Hz. Figure 7A compares the SyGCaMP2 signal averaged over two populations of terminals that generated a significant response to this stimulus: 66 with r, 1.5 mm and 119 with r.2.5 mm. In both populations the presynaptic calcium signal was a strongly rectifying function of light intensity, causing a steady increase in calcium that was graded with contrast (Figure 7B). These measurements identify voltage-dependent calcium channels in the synaptic terminal of BCs as being responsible for the first major non-linear transfor- mation of the visual signal as it is transmitted through the retina. Variations in Temporal Filtering in Terminals of Different Volume How do variations in filtering of the presynaptic calcium signal impact on the output from the synapse? Assaying vesicle release with SypHy demonstrated that smaller terminals respond to a step of light with higher relative release rates (Figure 1F), but the resolution of this reporter was too low to detect modulations at stimulus frequencies .1 Hz (i.e., the linear component of the output). Nonetheless, the model predicted that the modulation in release rate would be directly proportional to the steady rate once the terminal had adapted (Figure 7F), and sypHy can be used to assess variations in the steady-rate of release [14]. We therefore used steady-state measurements to compare temporal filtering in the output of small and large terminals. Discussion Many BCs transmit the visual signal through multiple terminals located in different strata of the IPL (Figure 1). Using a combination of in vivo imaging, slice electrophysiology, and modeling, we have found that variations in the size of these terminals will cause the signal in a single neuron to be transformed through different temporal filters as it is transmitted to the inner retina (Figures 1–4). The mechanistic link is the calcium signal that controls synaptic transmission: smaller terminals generate larger and faster calcium transients (Figures 1–5), increasing the gain of synaptic transmission and allowing higher frequencies to be transmitted (Figures 7 and 8). Diversity in the properties of synaptic transmission extend to time- dependent changes in gain: smaller terminals adapt more completely after an increase in stimulus contrast (Figure 6). These variations in synaptic properties will expand the number of processing channels that can operate in parallel through the limited number of neurons packed into a given region of the retinal network [55,56]. These results complement recent evidence for divergence of visual channels through individual BCs, obtained by direct electrical stimulation of these neurons while monitoring the effects in multiple ganglion cells [4,11,57]. Synaptic Filtering of Visual Signals A ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{ f=f o 2 z1 Q2 f=f o 2 s where A is a scaling factor, fo is the center frequency, and Q is a quality factor describing how much the response is damped. In small terminals, Q was estimated as 1.5760.37, while in large terminals it was 0.7860.17. Thus small terminals more obviously maintained the bandpass characteristics of the voltage signal measured in the cell body of BCs [22], while large terminals damped out the resonant frequencies This behavior can now be understood in terms of the impact of terminal volume on the dynamics of the calcium signal controlling neurotransmitter release. Although the most obvious aspect of the SyGCaMP2 signal elicited by an increase in the variance of the stimulus was a maintained increase in presynaptic calcium, smaller fluctuations could also be detected (Figure 1G). The power of the fluctuations following a periodic stimulus was used to quantify the linear component of the synaptic calcium response [22], and the power of this linear component was ,3-fold larger in the smaller population of terminals (Figures 1G, 7C, and 7D). Further, when the contrast of the stimulus was varied between 10% and 100%, the power of the linear component was directly proportional to the power of the DC component (Figure 7E), as predicted by the model (Figure 7G). The proportionality constant for the small population of terminals was 2.4 times that of the large population (Figure 7E), which was also predicted by the model. The relatively slow response time of SyGCaMP2 (tdecay,200 ms [13]) prevented the imaging of synaptic responses following stimuli at frequencies greater than 3 Hz, but the results in Figure 7 demonstrate that smaller terminals generate relatively stronger linear responses than large terminals, and therefore encode fluctuating stimuli more effectively. Synaptic Filtering of Visual Signals two stages: a linear transformation of the input feeding into a rectifying non-linearity [7,50,51]. How does this linear-nonlinear (LN) model map onto the retinal circuit [5]? Transformations of the visual input are roughly linear through the processes of phototransduction and transmission by the synapses of photore- ceptors, as far down the visual pathway as the cell body of BCs [22,43,52,53]. But what of the next neural compartment in the visual pathway—the synaptic terminal of BCs? large (r.1.5 mm) The amplitude of the response, reflecting the steady rate of vesicle release, is plotted as a function of frequency in Figure 8B. Small terminals transmitted frequencies between 5 and 10 Hz significantly better than large terminals. For instance, at a frequency of 8 Hz, the relative response of small terminals was ,2.4-fold that of large. If the power of the modulated response is proportional to the mean release rate, as suggested by the results in Figure 7E, these measurements can be considered an approximation of the ‘‘transfer function’’ of the visual system up to the point that BCs transmit the visual signal. We therefore described the measure- ments using an expression commonly used to describe the output of electrical circuits with some element of resonance [54]: p y y p Measurements of vesicle release with sypHy demonstrated strong rectification in the synaptic output because an increase in stimulus variance caused an increase in the mean rate of vesicle release even when the mean luminance was held constant, as shown in Figure 6C. This behavior is predicted by the model (Figure 6A), where it reflects the rectifying relation between membrane potential and the amplitude of the calcium current (Figure 4). To test this explanation by experiment, we imaged the presynaptic calcium signal using SyGCaMP2, while applying a square wave stimulus modulated at 3 Hz. Figure 7A compares the SyGCaMP2 signal averaged over two populations of terminals that generated a significant response to this stimulus: 66 with r, 1.5 mm and 119 with r.2.5 mm. In both populations the presynaptic calcium signal was a strongly rectifying function of light intensity, causing a steady increase in calcium that was graded with contrast (Figure 7B). These measurements identify voltage-dependent calcium channels in the synaptic terminal of BCs as being responsible for the first major non-linear transfor- mation of the visual signal as it is transmitted through the retina. PLOS Biology \| www.plosbiology.org Linear- and Non-linear Transformations of the Visual Signal: Impact of Terminal Volume An empirical description of the relation between variations in the intensity of light falling on the retina and the spike-rate of ganglion cells can often be obtained using a model comprised of October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 9 Synaptic Filtering of Visual Signals Different Temporal Channels through Individual Bipolar Cells The different temporal channels in the visual system were first distinguished by recording responses of ‘‘transient’’ and ‘‘sus- tained’’ ganglion cells in the retina [4]. These temporal channels partly reflect processing in the inner retina, where feedback inhibition and lateral inhibition from amacrine cells act directly on BC terminals to alter the gain and timing of the synaptic output [11,19,58]. Here we have described a fundamental and intrinsic property that will contribute further to the diversity of signals that BCs transmit—the geometry of the synaptic compartment [11,55,56]. Figure 8A shows averaged sypHy signals to stimuli of varying frequency, for two populations of terminals: small (r,1 mm) and October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 10 Synaptic Filtering of Visual Signals Figure 7. Linear and rectifying components of contrast response vary with terminal size. (A) SyGCaMP2 responses of BC terminals to stimuli of varying contrast (3 Hz). Average DF/F of all contrast responding terminals (both ON and OFF) with r,1.5 mm (n = 66 terminals, red) and r. 2.5 mm (n = 119 terminals, black). Note how these synaptic calcium responses are strongly rectifying. (B) Power of the DC component measured at 0.01 Hz for stimuli shown in (A). The DC component was larger in small terminals (cf. Figure 4C). (C) Power spectrum of the response to 100% contrast (3 Hz) for small (red) and large (black) terminals. (D) Power at 3 Hz varies with contrast. See also Figure S4. (E) Power of the linear and DC components are directly proportional, but the proportionality coefficient is larger for smaller terminals. The Pearson correlation coefficient was 0.99 for small terminals and 0.97 for large. (F) Modeled release in response to a 3 Hz stimulus (100% contrast). (G) The model predicts that the power of the calcium response at 3 Hz is directly proportional to the power of the DC component, as was observed experimentally in (E). (H) Power of the exocytic response modeled for a range of stimulus frequencies. Power at the stimulus frequency varies as 1/f for both small and large terminals. doi:10.1371/journal.pbio.1001972.g007 Figure 7. Linear and rectifying components of contrast response vary with terminal size. (A) SyGCaMP2 responses of BC terminals to stimuli of varying contrast (3 Hz). Average DF/F of all contrast responding terminals (both ON and OFF) with r,1.5 mm (n = 66 terminals, red) and r. Extrinsic Factors Affecting the Frequency Response of Bipolar Cell Synapses morphologically distinct BCs can be recognized in the vertebrate retina, and probably more than 20 in zebrafish [64,65], but we still do not fully understand how these differ in their response properties. The general thinking has been that one type of BC transmits one type of signal, but more recent work indicates that it might be more fruitful to focus on the synaptic terminal as the fundamental unit of signal transmission, which would allow for divergence of different signals from the one neuron [11]. The purpose of the model we have presented is to provide a basic mechanistic understanding for the impact of terminal volume on calcium dynamics and vesicle release from BCs. A more comprehensive model of signal transmission from BCs would not treat the neuron in isolation but would also consider the local circuits into which the terminals are connected. In particular we have neglected the effects of direct inhibitory feedback from amacrine cells [11,19,58]. It is, however, notable that when the GABAergic component of this feedback was blocked, the size- dependence of release rate was maintained (Figure 2), indicating that negative feedback was not the cause of this correlation. Implicit in this idea is the notion that the different synaptic outputs of a neuron are functionally isolated from each other, at least to some extent. Does the geometry of the terminal arborization allow this? Simultaneous measurements of calcium signals in connected terminals demonstrate that the answer is yes: connecting processes provide a sufficient diffusional barrier to allow calcium signals with different amplitudes and kinetics to remain local to individual terminals (Figure 3). Such isolation of presynaptic calcium signals would also allow for independent modulation of different synaptic compartments by amacrine cells. The potential number and diversity of these synaptic transforma- tions becomes even greater if one considers that individual amacrine cells provide negative feedback though many neurites with distinct biophysical and synaptic properties [66]. Such presynaptic heterogeneity has previously been observed in sensory pathways of insects, including cricket auditory afferents [67] and Drosophila olfactory receptor neurons [68]. The temporal tuning of BCs also reflects processes in the outer retina, especially cone [59] inputs with different contact morphol- ogies acting on dendritic glutamate receptors with different kinetics of recovery from desensitization [3,60,61]. Different Temporal Channels through Individual Bipolar Cells (A) Mean sypHy fluorescence signals in OFF terminals in response to full-field stimuli of different temporal frequencies measured in vivo (red, rsmall,1 mm, n = 10; black, rlarge.1.5 mm, n = 32). (B) Synaptic gain of large (black) and small (red) terminals as a function of stimulus frequency. Results are described by the function A ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1 f= 2 1 f= 2 s : Figure 8. Frequency tuning of BCs with different terminal size. (A) Mean sypHy fluorescence signals in OFF terminals in response to full-field stimuli of different temporal frequencies measured in vivo (red, rsmall,1 mm, n = 10; black, rlarge.1.5 mm, n = 32). (B) Synaptic gain of large (black) and small (red) terminals as a function of stimulus frequency. Results are described by the function Figure 8. Frequency tuning of BCs with different terminal size. (A) Mean sypHy fluorescence signals in OFF terminals in response to full-field stimuli of different temporal frequencies measured in vivo (red, rsmall,1 mm, n = 10; black, rlarge.1.5 mm, n = 32). (B) Synaptic gain of large (black) and small (red) terminals as a function of stimulus frequency. Results are described by the function A ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{ f=f o 2 z1 Q2 f=f o 2 s : For large terminals, A = 0.2260.02, fo = 4.9660.45, Q = 0.7860.17. For small terminals A = 0.1660.02, fo = 6.4360.27, Q = 1.5760.37. Asterisks mark significantly different responses at a given frequency, as evaluated by Mann-Whitney U test (p,0.05). doi:10.1371/journal.pbio.1001972.g008 A ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ 1{ f=f o 2 z1 Q2 f=f o 2 s : For large terminals, A = 0.2260.02, fo = 4.9660.45, Q = 0.7860.17. For small terminals A = 0.1660.02, fo = 6.4360.27, Q = 1.5760.37. Asterisks mark significantly different responses at a given frequency, as evaluated by Mann-Whitney U test (p,0.05). doi:10.1371/journal.pbio.1001972.g008 For large terminals, A = 0.2260.02, fo = 4.9660.45, Q = 0.7860.17. For small terminals A = 0.1660.02, fo = 6.4360.27, Q = 1.5760.37. Asterisks mark significantly different responses at a given frequency, as evaluated by Mann-Whitney U test (p,0.05). doi:10.1371/journal.pbio.1001972.g008 Extrinsic Factors Affecting the Frequency Response of Bipolar Cell Synapses The general picture that emerges is one in which amacrine cells modulate transmission of different frequency components in the visual input by acting on the background of at least two intrinsic properties varying between BCs: tuning of dendritic inputs summed at the cell body and the filter determining calcium dynamics in the synaptic terminal providing the output (Figure 3G) [62]. The key new idea that we propose here is that this synaptic filter varies between different outputs because of variations in an intrinsic property of the synaptic compartment—its volume. The Synaptic Compartment of Bipolar Cells as a Computational Unit In Vivo Imaging of Presynaptic Ca2+ Signals and Vesicle Fusion Different Temporal Channels through Individual Bipolar Cells 2.5 mm (n = 119 terminals, black). Note how these synaptic calcium responses are strongly rectifying. (B) Power of the DC component measured at 0.01 Hz for stimuli shown in (A). The DC component was larger in small terminals (cf. Figure 4C). (C) Power spectrum of the response to 100% contrast (3 Hz) for small (red) and large (black) terminals. (D) Power at 3 Hz varies with contrast. See also Figure S4. (E) Power of the linear and DC components are directly proportional, but the proportionality coefficient is larger for smaller terminals. The Pearson correlation coefficient was 0.99 for small terminals and 0.97 for large. (F) Modeled release in response to a 3 Hz stimulus (100% contrast). (G) The model predicts that the power of the calcium response at 3 Hz is directly proportional to the power of the DC component, as was observed experimentally in (E). (H) Power of the exocytic response modeled for a range of stimulus frequencies. Power at the stimulus frequency varies as 1/f for both small and large terminals. doi:10.1371/journal.pbio.1001972.g007 sizes. For instance, layer 6 of the IPL contains the highest density of large terminals, which leads to the testable prediction that RGCs extending dendrites in this layer will be more likely to exhibit low-pass characteristics. In contrast, layer 5 has smaller terminals than the average, suggesting that RGCs collecting inputs from this layer will be tuned to higher frequencies. In the future, it will be important to assess the extent to which RGCs tuned to different frequencies receive inputs from BC terminals of different sizes. Such a study will be technically demanding, requiring a detailed anatomical reconstruction of IPL circuitry. Nonetheless, Figure 1D provides the first evidence that RGCs with dendrites in different layers of the IPL will, on average, receive excitatory input from BC terminals of different October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 11 Synaptic Filtering of Visual Signals Figure 8. Frequency tuning of BCs with different terminal size. (A) Mean sypHy fluorescence signals in OFF terminals in response to full-field stimuli of different temporal frequencies measured in vivo (red, rsmall,1 mm, n = 10; black, rlarge.1.5 mm, n = 32). (B) Synaptic gain of large (black) and small (red) terminals as a function of stimulus frequency. Results are described by the function Figure 8. Frequency tuning of BCs with different terminal size. Calculation of Vesicle Release Rates V9exo, the fraction of total vesicles in the terminal released per second, was calculated from the sypHy signal according to the equation: V0 exo(t)~ 1 19Fmin dF dt zkendo(F{Fmin) ð1Þ ð1Þ where F is the average fluorescence intensity over the terminal at time t, Fmin is the intensity when the rate of vesicle release is at a minimum, and kendo is the rate-constant of vesicle retrieval. The calculation of this formula is described in [14,18]. Estimation of V9exo requires differentiation of the sypHy trace, which in turn amplifies noise, so Equation 1 was applied after smoothing with a series of single or double exponential functions to obtain ‘‘non- noisy’’ traces before calculation of V9exo. These fits are shown in Figures 1F and 5C. In Vivo Imaging of Presynaptic Ca2+ Signals and Vesicle Fusion It has long been recognized that the visual system separates signals encoding different aspects of a stimulus for transmission through different pathways or ‘‘channels.’’ The most fundamental of these parallel representations is the separation of ON and OFF signals, which begins in distinct types of BC [63]. The separation of signals with different speeds into transient and sustained pathways also begins in BCs [6]. More than ten types of All procedures were carried out according to the UK Animals (Scientific Procedures) Act 1986 and approved by the UK Home Office. Transgenic zebrafish expressing SyGCaMP2.0 and sypHy were maintained on a 14/10 h light/dark cycle at 28uC. From 24 hpf the larvae were maintained in fish medium (E2) containing 1- phenyl-2-thiourea at a final concentration of 200 mM (Sigma) to prevent pigment formation. Fish were imaged as described October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 12 Synaptic Filtering of Visual Signals previously [13]. Briefly, before experiments 8–10 dpf larvae were anesthetized in 0.016% Tricaine (Sigma) and immobilized in low melting point agarose. To prevent eye movement a-bungarotoxin (2 mg/ml) was injected into the extraocular space. Imaging was performed using a custom-built multiphoton microscope, equipped with a mode-locked Chameleon titanium-sapphire laser (Coherent) tuned to 920 nm and controlled using ScanImage v.3.6 software [69]. ,2 mm. Given an average radius of just above 1 micron per terminal, this resolution was therefore large enough to avoid underestimating the size of terminals traversed non-centrally by the optical plane, but small enough to avoid out-of-focus terminals contributing to the signal. Image Analysis Movies were processed using the SARFIA suite of analysis routines [17] running in Igor Pro 6 (Wavemetrics). We detected terminals on the basis of single images obtained by averaging many frames of the movie corresponding to total integration times of several tens of seconds. ROIs corresponding to terminals within these averaged images were defined using a filtering algorithm based on a Laplace operator followed by application of a threshold, as described in detail in [17]. This algorithm will define most or all of the ROIs that an experienced observer would recognize by eye. To prevent bias between ON and OFF terminals within a single field of view, the average image was obtained from movies in which light steps were applied, and/or light intensity modulated at 1 Hz or faster. Statistical Analysis All statistical analysis was performed in Igor Pro 7 (Wave- metrics). Differences in different parameters in large and small terminals, described in Figure 3, were analyzed using Wilcoxon signed-rank test. This test was chosen because some responses of large and small terminals were recorded from the same neuron. Sample sizes were not determined a priori. Analysis of the response dynamic was automatic and no knowledge of terminal size was used until the last moment. All animals demonstrating robust response to light were included in the analysis. Figure 1B represents biological replicate, representative from more than five fields of view. No lack of reproducibility was found. All error bars in figures show 6 1 SEM, unless stated otherwise in the legend. The retina was imaged through an Olympus LUMPlanFI 406 water immersion (0.8 NA) objective. Green emission from sypHy and SyGCaMP2 was collected both through the objective and through an oil condenser (ND 1.4, Olympus), filtered through GFP filters (530/50 nm, Chroma Technology), and detected with GaAsP photomultipliers (Hamamatsu). Images (1286128 pixels) were acquired every 0.128 seconds, resulting in a sampling frequency of 7.8 Hz. Full-field light stimuli were delivered using an amber LED (590 nm) filtered through a 600/10 BP filter and projected through a light guide onto the surface of the bath, close to the eye of the fish. The mean intensity of light stimuli was ,26105 photons/mm2/s, which is in the low photopic range. Importantly, optical measurements in the live retina necessarily give rise to a background activation of photoreceptors. This is due to direct laser activation of photopigment, but usually more importantly, due to indirect activation of photopigments from the emission light of excited fluophores [70]. As a result, rods are typically saturated, and cones partially adapted, giving rise to a low-photopic background light-level. Indeed, we observed similar effects in our in vivo experiments (Figure S1F). However, this background illumination was weaker than typically observed in e.g., rabbit [70], presumably as the zebrafish retina is cone dominated. Nevertheless, following activation of the laser scan- ning, we typically waited 30 seconds before commencing with additional visual stimulation to ensure the retina adapted to the background levels attributed to the laser. Slice Electrophysiology and Imaging Slices of goldfish retina were perfused with extracellular solution containing (in mM) 120 NaCl, 2.5 CaCl2, 2 KCl, 1 MgCl2, 0.1 CaCl2, 4 HEPES, 10 glucose (pH 7.7, 255 mOsm). Experiments were carried out at room temperature and slices visualized under oblique infrared illumination through a 606objective (NA 0.9) on an upright microscope. Whole cell recordings were obtained from ‘‘large’’ terminals (diameter: 4–12 mm) or from the soma of ‘‘mixed’’ BCs using 8–12 MV patch electrodes [71]. For this, we targeted large terminals in layers 5/6. The intracellular solution contained (in mM) 104 Kgluconate, 8 KCl, 2 MgCl2, 4 HEPES, 0.5 EGTA, 2 MgATP, 1 NaGTP, 1 NacGMP (pH 7.4, 250 Osm), and 100 mM of the hexapotassium salt of the low affinity Ca2+ indicator OGB-5N. Recordings were left for 1–5 min to allow time for the Ca2+-indicator to diffuse into adjacent small terminals (diameter 1–4 mm) see also [20]. Series resistance ranged from 8– 15 MV while input resistance was .1 GV at 270 mV. Record- ings were corrected for junction potentials (calculated as 2 11.95 mV). Supporting Information Figure S1 Size estimation does not depend on the stimulus condition (related to Figure 1). (A) Two images of the same field of view: the left obtained from an average of 45 s in the dark, and the right averaged over a 45 s period in which the mean light intensity was in the low photopic range, including 45 s of temporal contrast applied at 1 Hz (100% contrast). Scale bar 10 m. (B) OFF terminals have brightest fluorescence during contrast presentation and dimmest fluorescence when exposed to steady light. Black bars show parts of the movie used for image averaging. (C) Distribution of terminal sizes estimated from averages shown in (A) (at steady light, red and temporal contrast, black). (D) Cumulative distributions, calculated from (C). (E) Terminal sizes estimated from terminals during steady and flickering light stimulation (c.f. (A)). Each point represents size estimation of an individual terminal. All points are scatted around a line through the origin with slope of 1, suggesting that size estimation is not affected significantly by the terminal activity. (F) Example of n = 589 OFF cells responding to laser and visual stimulation (arrows). Top: average of all responses, bottom: individual responses. Error in (standard error of the mean) SEM. (G) Responses of OFF terminals of different sizes to light decrement. Bins are the same as in Figure 1E. (I) Distributions of terminal radii calculatEed from individual layers. (TIF) Figure S5 Comparison of terminal sizes and adaptation dynamics in different fish (related to Figure 5). (A) Average terminal sizes were similar in each of six different fish. (B) Adaptation to temporal contrast (100%, 5 Hz) in a single fish. Smaller terminals (red) respond with higher gain and adapt more profoundly than large (black), in a manner similar to the behavior averaged over 6 fish (Figure 5C). (C) Same as Figure 5C, but on a longer time scale. Contrast facilitation is more pronounced in larger terminals. Figure S6 Active voltage spikes should boost high frequency components (related to Figure 6). (A) A ‘‘chirp’’ stimulus modulating at 100% contrast ramping from 0.1 to 20 Hz and back down again over a period of 10 s (top) was convolved with the same impulse response used in Figure 3 to yield a prediction of the generator potential. (B) Addition of Brownian motion noise (standard deviation [SD] = 1.4 mV) was used to yield an estimate of membrane voltage. Estimation of Terminals Size by Two-Photon Imaging Estimation of Terminals Size by Two Photon Imaging The point-spread function of the used microscope in XY dimension was 0.5 mm and terminals appeared larger when we increased intensity of the 2-photon laser. We therefore tested whether terminals might appear smaller when less active, but found that this was not the case (as described in Figure S1 and Text S1). Average terminal sizes were similar between different fish (Figure S5A) and the effects of terminal size on adaptation kinetics could be observed in individual fish (Figure S5B) as well as in data averaged over multiple fish (Figure 6C). However, the distribution of estimated terminal sizes in SyGCaMP2 was shifted to larger values, compared to sypHy or Synaptophysin-EGFP fish (Figure S1H). This is likely explained by the greater overall brightness of SyGCaMP2. The z-resolution of the microscope was OGB-5N was imaged at 40 Hz using an electron multiplying charge coupled device (EM CCD) camera (Hamamatsu C9100). Subsequent image analysis was performed using ImageJ, Igor Pro, and Matlab. OGB-5N signals were quantified as changes in fluorescence relative to background fluorescence at each pixel (DF/ Fo) and converted to estimates of absolute Ca2+ concentration: OGB-5N was imaged at 40 Hz using an electron multiplying charge coupled device (EM CCD) camera (Hamamatsu C9100). Subsequent image analysis was performed using ImageJ, Igor Pro, and Matlab. OGB-5N signals were quantified as changes in fluorescence relative to background fluorescence at each pixel (DF/ Fo) and converted to estimates of absolute Ca2+ concentration: October 2014 \| Volume 12 \| Issue 10 \| e1001972 13 PLOS Biology \| www.plosbiology.org Synaptic Filtering of Visual Signals Figure S4 Modulation of release with changed ICa threshold and calcium dependence of release: predic- tions of the model (related to Figure 4). (A, B, left) Modeled calcium (A) and release (B) in response to a 3 Hz flickering stimulus from an r = 1 micron compartment with different thresholds for activation of the L-type calcium current (Vrest always = 244 mV). The threshold was increased (light green) and decreased (dark green) from the value used in the main model (red) by 3 mV in each case. Right: modulation amplitude of calcium (A) and release (B) quantified for the three threshold conditions in different size compartments. Changing the threshold had only minimal effect on the overall size dependence of calcium and release modulation. Supporting Information (A–C) Predicted calcium levels at different distances from a hotspot in a 5 mm (black) and a 1 mm (red) radius spherical compartment, shown at three different time-scales. (D) Estimated release rates driven by calcium as shown in (C). (E) Concentration of unbound ‘‘fixed’’ (grey) and ‘‘diffusible’’ (green) buffers under normal buffering conditions during step depolarisation of a 1 micron radius terminal (c.f. red in (B)). The dotted line indicates unbound buffer concentration at the channel mouth (‘‘hotspot’’), which the solid line indicates concentration at the center of the compartment. The coloration indicates the possible range of unbound buffer concentration at different locations within the compartment. (F) Corresponding calcium concentration at the hotspot (dotted) and globally (solid). (G, H) as (E, F) but with 10 times elevated ‘‘fixed’’ buffer concentration. Elevating the fixed buffer has only small effects on the kinetics of calcium free calcium concentration, but does affect peak calcium concentration at the hotspot. Estimation of Terminals Size by Two-Photon Imaging (C, D) Steady state modulation of modeled calcium (C) and release (D) in an r = 1 micron compartment in response to an ongoing 3 Hz stimulus. Changing the Hill coefficient for calcium dependence of release from 1 (linear = in main model, red) to 3 (cooperative, light blue) systematically increases the modulation amplitude of release (D, left) across all frequencies tested (D, right). (TIF) Ca2z ~Kd: F{Fmin Fmax{F ð2Þ ð2Þ Fmax/Fmin of OGB-5N is ,14.7. Since resting Ca2+ in bipolar terminals (,50 nM [30]) is much lower than the Kd of OGB-5N (20 mM) we assumed that Frest = Fmin. Supporting Information Two separate predictions were drawn from the model at this point: graded (black) and spiking (red). A threshold was added to the ‘‘membrane voltage’’ trace to predict spikes, which occurred with an exponential refractory period of 300 ms. Spike amplitude was fixed at 20 mV, with a half width of 3 ms. (C) Bulk calcium and (D) release was calculated as before (Figure 4) from the graded and spiking voltage traces. (E) Average release rates of 100 graded (black) and 100 spiking (red) model BCs. Note that the mean frequency response of the spiking system is highly reminiscent of the generator frequency response (A), while the graded system imposes a powerful low pass filter on the signal. Figure S2 Recording ‘‘mixed’’ BCs from the soma (related to Figure 2). To ensure that the size dependence of current evoked calcium signals measured in different terminals belonging to the same ‘‘mixed’’ BC was not dependent on the position of the micropipette, we repeated experiments shown in Figure 2D–2F but this time targeted the soma of individual cells rather than the large terminal. The size dependence persisted in somatal recordings. One example of n = 4 is shown (c.f. Figure 2D–2F). Figure S3 A 3-D diffusion model of Ca2+ in the bipolar cell synapse (related to Figure 3). (A–C) Predicted calcium levels at different distances from a hotspot in a 5 mm (black) and a 1 mm (red) radius spherical compartment, shown at three different time-scales. (D) Estimated release rates driven by calcium as shown in (C). (E) Concentration of unbound ‘‘fixed’’ (grey) and ‘‘diffusible’’ (green) buffers under normal buffering conditions during step depolarisation of a 1 micron radius terminal (c.f. red in (B)). The dotted line indicates unbound buffer concentration at the channel mouth (‘‘hotspot’’), which the solid line indicates concentration at the center of the compartment. The coloration indicates the possible range of unbound buffer concentration at different locations within the compartment. (F) Corresponding calcium concentration at the hotspot (dotted) and globally (solid). (G, H) as (E, F) but with 10 times elevated ‘‘fixed’’ buffer concentration. Elevating the fixed buffer has only small effects on the kinetics of calcium free calcium concentration, but does affect peak calcium concentration at the hotspot. ( ) Figure S3 A 3-D diffusion model of Ca2+ in the bipolar cell synapse (related to Figure 3). Table S1 List of parameters used in single compart- ment model. (DOCX) Table S2 List of parameters used in 3-D model. (DOCX) References Abbott LF, Regehr WG (2004) Synaptic computation. Nature ott LF, Regehr WG (2004) Synaptic computation. Nature 431: 796–8 11. Asari H, Meister M (2012) Divergence of visual channels in the inner retina. Nat Neurosci 15: 1581–1589. 38. Jackman SL, Choi SY, Thoreson WB, Rabl K, Bartoletti TM, Kramer RH (2009) Role of the synaptic ribbon in transmitting the cone light response. Nat Neurosci 12: 303–310. 12. Mintz I, Sabatini B, Regehr W (1995) Calcium control of transmitter release at a cerebellar synapse. Neuron 15: 675–688. 39. Rieke F, Schwartz EA (1996) Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse. J Physiol 493: 1–8. y p 13. Dreosti E, Odermatt B, Dorostkar M, Lagnado L (2009) A genetically encoded reporter of synaptic activity in vivo. Nat Methods 6: 883–889. 40. Burrone J, Neves G, Gomis A, Cooke A, Lagnado L (2002) Endogenous calcium buffers regulate fast exocytosis in the synaptic terminal of retinal bipolar cells. Neuron 33: 101–112. 14. Odermatt B, Nikolaev A, Lagnado L (2012) Encoding of luminance and contrast by linear and nonlinear synapses in the retina. Neuron 73: 758–773. 15. Granseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51: 773–786. 41. Coggins M, Zenisek D (2009) Evidence that exocytosis is driven by calcium entry through multiple calcium channels in goldfish retinal bipolar cells. J Neur- ophysiol 101: 2601–2619. 16. Dreosti E, Esposti F, Baden T, Lagnado L (2011) In vivo evidence that retinal bipolar cells generate spikes modulated by light. Nat Neurosci 14: 951–952. 42. von Gersdorff H, Vardi E, Matthews G, Sterling P (1996) Evidence that vesicles on the synaptic ribbon of retinal bipolar neurons can be rapidly released. Neuron 16: 1221–1227. 17. Dorostkar MM, Dreosti E, Odermatt B, Lagnado L (2010) Computational processing of optical measurements of neuronal and synaptic activity in networks. J Neurosci Methods 188: 141–150. 43. Baccus SA, Meister M (2002) Fast and slow contrast adaptation in retinal circuitry. Neuron 36: 909–919. 18. Nikolaev A, Leung KM, Odermatt B, Lagnado L (2013) Synaptic mechanisms of adaptation and sensitization in the retina. Nat Neurosci 16: 934–941. 44. Manookin MB, Demb JB (2006) Presynaptic mechanism for slow contrast adaptation in mammalian retinal ganglion cells. Neuron 50: 453–464. 19. Acknowledgments We thank Jamie Johnston for criticisms of the manuscript and all members of the lab for discussions. Text S1 Supplemental information. (1) Variations in estimates of terminal size were not correlated with variations in terminal brightness. (2) Modeling presynaptic calcium dynamics and vesicle release. (3) A 3-D model predicting calcium spread at the channel mouth. (4) The effect of changing L-type calcium channel threshold and calcium dependency of release: predictions of the model. (5) Encoding of high frequency components using spikes. (6) Supplemental references. Table S2 List of parameters used in 3-D model. (DOCX) Data S1 All raw data files as well as averages and statistical parameters presented in the manuscript. Formats provided include raw-text (Ascii) and Excel (xls). In addition, we provide the original Igor-Pro files (Wavemetrics), which contain both the raw data and the original figure formatting. Data referring to particular panels are located in the Data S1 All raw data files as well as averages and statistical parameters presented in the manuscript. Formats provided include raw-text (Ascii) and Excel (xls). In addition, we provide the original Igor-Pro files (Wavemetrics), which contain both the raw data and the original figure formatting. Data referring to particular panels are located in the p (TIF) October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org PLOS Biology \| www.plosbiology.org 14 Synaptic Filtering of Visual Signals respective folders. For any further information, please contact Tom Baden at thomas.baden@uni-tuebingen.de. (ZIP) References Vigh J, Vickers E, von Gersdorff H (2011) Light-evoked lateral GABAergic inhibition at single bipolar cell synaptic terminals is driven by distinct retinal microcircuits. J Neurosci 31: 15884–15893. 45. Burrone J, Lagnado L (2000) Synaptic depression and the kinetics of exocytosis in retinal bipolar cells. J Neurosci 20: 568–578. 20. Kim MH, Vickers E, von Gersdorff H (2012) Patch-clamp capacitance measurements and Ca(2)(+) imaging at single nerve terminals in retinal slices. J Vis Exp pii: 3345. 46. Jarsky T, Cembrowski M, Logan SM, Kath WL, Riecke H, et al. (2011) A synaptic mechanism for retinal adaptation to luminance and contrast. J Neurosci 31: 11003–11015. 47. Ozuysal Y, Baccus SA (2012) Linking the computational structure of variance adaptation to biophysical mechanisms. Neuron 73: 1002–1015. 21. Protti DA, Flores-Herr N, von Gersdorff, H (2000) Light evokes Ca2+ spikes in the axon terminal of a retinal bipolar cell. Neuron 25: 215–227. 22. Baden T, Esposti F, Nikolaev A, Lagnado L (2011) Spikes in retinal bipolar cells phase-lock to visual stimuli with millisecond precision. Curr Biol 21: 1859–1869. 48. Kastner DB, Baccus SA (2011) Coordinated dynamic encoding in the retina using opposing forms of plasticity. Nat Neurosci 14: 1317–1322. using opposing forms of plasticity. Nat Neurosci 14: 1317–1322. 49. Li GL, Vigh J, von Gersdorff H (2007) Short-term depression at the reciprocal synapses between a retinal bipolar cell terminal and amacrine cells. J Neurosci 27: 7377–7385. 23. Saszik S, DeVries SH (2012) A mammalian retinal bipolar cell uses both graded changes in membrane voltage and all-or-nothing na+ spikes to encode light. J Neurosci 32: 297–307. 24. Lagnado L, Cervetto L, McNaughton PA (1992) Calcium homeostasis in the outer segments of retinal rods from the tiger salamander. J Pphysiol 455: 111– 142. 50. Schwartz GW, Okawa H, Dunn FA, Morgan JL, Kerschensteiner D, et al. (2012) The spatial structure of a nonlinear receptive field. Nat Neurosci 15: 1572–1580. 25. Sabatini BL, Oertner TG, Svoboda K (2002) The life cycle of Ca(2+) ions in dendritic spines. Neuron 33: 439–452. 51. Bolinger D, Gollisch T (2012) Closed-loop measurements of iso-response stimuli reveal dynamic nonlinear stimulus integration in the retina. Neuron 73: 333– 346. 26. Crank J (1975) The mathematics of diffusion. 2nd edition. Oxford: Clarendon Press. 52. Rieke F (2001) Temporal contrast adaptation in salamander bipolar cells. J Neurosci 21: 9445–9454. 27. Author Contributions The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: TB AN FE ED BO LL. Performed the experiments: TB AN FE ED BO. Analyzed the data: TB AN FE ED BO LL. Wrote the paper: TB AN LL. The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: TB AN FE ED BO LL. Performed the experiments: TB AN FE ED BO. Analyzed the data: TB AN FE ED BO LL. Wrote the paper: TB AN LL. References 30. Burrone J, Lagnado L (1997) Electrical resonance and Ca2+ influx in the synaptic terminal of depolarizing bipolar cells from the goldfish retina. J Physiol 505: 571–584. 1. Masland RH (2001) The fundamental plan of the retina. Nat Neurosci 4: 877– 886. 2. Roska B, Werblin F (2001) Vertical interactions across ten parallel, stacked representations in the mammalian retina. Nature 410: 583–587. 31. Holt M, Cooke A, Neef A, Lagnado L (2004) High mobility of vesicles supports continuous exocytosis at a ribbon synapse. Curr Biol 14: 173–183. 3. Wa¨ssle H (2004) Parallel processing in the mammalian retina. Nat Rev Neurosci 5: 747–757. 32. von Gersdorff H, Sakaba T, Berglund K, Tachibana M (1998) Submil- lisecond kinetics of glutamate release from a sensory synapse. Neuron 21: 1177– 1188. 4. Masland RH (2012) The neuronal organization of the retina. Neuron 76: 266– 280. 33. Mennerick S, Matthews G (1996) Ultrafast exocytosis elicited by calcium current in synaptic terminals of retinal bipolar neurons. Neuron 17: 1241–1249. 5. Euler T, Haverkamp S, Schubert T, Baden T (2014). Retinal bipolar cells: elementary building blocks of vision. Nat Rev Neurosci 15: 507–519. . Awatramani GB, Slaughter MM (2000) Origin of transient an 34. Lagnado L, Gomis A, Job C (1996) Continuous vesicle cycling in the synaptic terminal of retinal bipolar cells. Neuron 17: 957–967. 6. Awatramani GB, Slaughter MM (2000) Origin of transient and sustained responses in ganglion cells of the retina. J Neurosci 20: 7087–7095. responses in ganglion cells of the retina. J Neurosci 20: 7087–7095 p 35. Neves G, Lagnado L (1999) The kinetics of exocytosis and endocytosis in the synaptic terminal of goldfish retinal bipolar cells. J Physiol 515: 181–202. 7. Gollisch T, Meister M (2010) Eye smarter than scientists believed: neural computations in circuits of the retina. Neuron 65: 150–164. computations in circuits of the retina. Neuron 65: 150–164. 8. Hosoya T, Baccus SA, Meister M (2005) Dynamic predictive coding by the retina. Nature 436: 71–77. 36. Beaumont V, Llobet A, Lagnado L (2005) Expansion of calcium microdomains regulates fast exocytosis at a ribbon synapse. Proc Natl Acad Sci U S A 102: 10700–10705. 9. Taylor W, Smith R (2011) Trigger features and excitation in the retina. Curr Opin Neurobiol 21: 672–678. 37. Thoreson WB, Rabl K, Townes-Anderson E, Heidelberger R (2004) A highly Ca2+-sensitive pool of vesicles contributes to linearity at the rod photoreceptor ribbon synapse. Neuron 42: 595–605. p 10. Synaptic Filtering of Visual Signals 55. Perge JA, Koch K, Miller R, Sterling P, Balasubramanian V (2009) How the optic nerve allocates space, energy capacity, and information. J Neurosci 29: 7917–7928. 64. Connaughton V, Graham D, Nelson R (2004) Identification and morphological classification of horizontal, bipolar, and amacrine cells within the zebrafish retina. J Comp Neurol 477: 371–385. 56. Balasubramanian V, Sterling P (2009) Receptive fields and functional architecture in the retina. J Physiol 587: 2753–2767. J p 65. Li YN, Tsujimura T, Kawamura S, Dowling JE (2012) Bipolar cell- photoreceptor connectivity in the zebrafish (Danio rerio) retina. J Comp Neurol 520: 3786–3802. 57. Msghina M, Millar AG, Charlton MP, Govind CK, Atwood HL (1999) Calcium entry related to active zones and differences in transmitter release at phasic and tonic synapses. J Neurosci 19: 8419–8434. 66. Grimes WN, Zhang J, Graydon CW, Kachar B, Diamond JS (2010) Retinal parallel processors: more than 100 independent microcircuits operate within a single interneuron. Neuron 65: 873–885. 58. Baccus SA (2007) Timing and computation in inner retinal circuitry. Annu Rev Physiol 69: 271–290. g 67. Baden T, Hedwig B (2010) Primary afferent depolarization and frequency processing in auditory afferents. J Neurosci 30: 14862. 59. Baden T, Euler T, Weckstrom M, Lagnado L (2013) Spikes and ribbon synapses in early vision. Trends Neurosci 36: 480–488. 68. Gaudry Q, Hong EJ, Kain J, de Bivort BL, Wilson RI (2013) Asymmetric neurotransmitter release enables rapid odour lateralization in Drosophila. Nature 493: 424–428. 60. DeVries SH, Schwartz EA (1999) Kainate receptors mediate synaptic transmission between cones and ‘Off’ bipolar cells in a mammalian retina. Nature 397: 157–160. 69. Pologruto TA, Sabatini BL, Svoboda K (2003) ScanImage: flexible software for operating laser scanning microscopes. Biomed Eng OnLIne 2: 9. 61. DeVries SH, Li W, Saszik S (2006) Parallel processing in two transmitter microenvironments at the cone photoreceptor synapse. Neuron 50: 735–748. 70. Euler T, Hausselt SE, Margolis DJ, Breuninger T, Castell X, et al. (2009) Eyecup scope–optical recordings of light stimulus-evoked fluorescence signals in the retina. Pflugers Arch 457: 1393–1414. 62. Robinson J, Schmitt EA, Harosi FI, Reece RJ, Dowling JE (1993) Zebrafish ultraviolet visual pigment: absorption spectrum, sequence, and localization. Proc Natl Acad Sci U S A 90: 6009–6012. g 71. Palmer MJ, Hull C, Vigh J, von Gersdorff H (2003) Synaptic cleft acidification and modulation of short-term depression by exocytosed protons in retinal bipolar cells. J Neurosci 23: 11332–11341. 63. References Allbritton NL, Meyer T, Stryer L (1992) Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate. Science 258: 1812–1815. 53. Schwartz G, Rieke F (2011) Perspectives on: information and coding in mammalian sensory physiology: nonlinear spatial encoding by retinal ganglion cells: when 1+1 not equal 2. J Gen Physiol 138: 283–290. 28. Kim MH, Li GL, vanGersdorff H (2013) Single calcium channels and exocytosis in sensory synapses. J Physiol 591: 3167–3178. cells: when 1+1 not equal 2. J Gen Physiol 138: 283–290. cells: when 1+1 not equal 2. J Gen Physiol 138: 283–290. in sensory synapses. J Physiol 591: 3167–3178. 29. Schnapf JL, Nunn BJ, Meister M, Baylor DA (1990) Visual transduction in cones of the monkey Macaca fascicularis. J Physiol 427: 681–713. 54. Wickert M (2013) Signals and systems for dummies. Hoboken (New Jersey): Wiley. October 2014 \| Volume 12 \| Issue 10 \| e1001972 October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org 15 Synaptic Filtering of Visual Signals Werblin FS, Dowling JE (1969) Organization of the retina of the mudpuppy, Necturus maculosus. II. Intracellular recording. J Neurophysiol 32: 339–355. October 2014 \| Volume 12 \| Issue 10 \| e1001972 PLOS Biology \| www.plosbiology.org PLOS Biology \| www.plosbiology.org 16

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